CA2147355A1

CA2147355A1 - Dna encoding atp-dependent fructose 6-phosphate 1-phosphotransferase originating from plant, recombinant vector containing the same and method for changing sugar content in plant cells under low temperature

Info

Publication number: CA2147355A1
Application number: CA002147355A
Authority: CA
Inventors: Toru Hiyoshi; Toshiki Mine; Keisuke Kasaoka; Robert Huw Tyson; Anthony Miles John Page
Original assignee: Individual
Current assignee: Japan Tobacco Inc
Priority date: 1993-08-19
Filing date: 1994-08-16
Publication date: 1995-02-23
Also published as: JP3437577B2; WO1995005457A1; US5824862A; EP0677581A4; EP0677581A1

Abstract

The invention discloses a DNA coding for a cold-resistant PFK, a recombinant vector capable of expressing a cold-resistant PFK in a host cell, and a method of changing the sugar content of a plant cell by using the vector at low temperature. The invention provides a DNA coding for a plant-derived ATP -dependent fructose-6-phosphate 1-phosphotransferase, a recombinant vector containing the DNA and capable of expressing a plant-derived ATP-dependent fructose-6-phosphate 1-phosphotransferase in a plant cell, and a method of changing the sugar content of a plant cell at low temperature by transforming a plant with the vector.

Description

SPECIFICATION
DNA Encoding ATP-Dependent Fructose 6-phosphate 1-phosphotransferase Originating from Plant, Recombinant Vector Containing the Same and Method for Changing Sugar Content in Plant Cells under Low Temperature TECHNICAL FIELD
This invention relates to a DNA encoding ATP-dependent fructose 6-phosphate l-phosphotransferase (EC

2.7.1.11) (hereinafter referred to as "PFK"), a recombinant vector containing the same and a method for changing sugar content in plant cells under low temperature using the recombinant vector.
BACKGROUND ART
PFK is an enzyme which catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate, a key regulatory step in the glycolytic pathway.
It is well-known that when plant tissues are exposed to a low temperature, the content of sugars such as sucrose, glucose, fructose and the like is generally increased. For example, this phenomenon is observed in potato tubers. Accumulation of reducing sugars such as glucose and fructose in potato tubers during storage at low temperature is undesirable in industry because they cause excess browning of potato chips during processing.
As for the cause of the accumulation of the reducing sugars, there are various hypotheses. However, it is 21g7355 thought that the activity of the glycolytic pathway is greatly reduced at low temperatures, so that the flow of the breakdown products of starch to sucrose is increased, thereby increasing the accumulation of the reducing sugars. PFK is thought to be one of the key enzymes of the glycolytic pathway, and it is well-known that PFK is cold labile. Therefore, it is thought that the drastic reduction of the activity of the glycolytic pathway at low temperature is caused by the drastic reduction of the activity of PFK.
PFK genes have been isolated from prokaryotes such as Escherichia coli, thermophilic bacteria, Bacillus subtilis and mycoplasma, and from eukaryotic tissues such as human muscle, human liver, rabbit muscle and mouse liver. However, the isolation of a plant PFK gene has not been reported, and the isolation of a gene encoding cold stable PFK has also not been reported. Therefore, it has been difficult hitherto to develop a potato variety resistant to Cold Induced Sweetening by introducing PFK genes. Nor has it been possible to reduce the activity of the glycolytic pathway in plant tissues by expressing an anti-sense PFK gene, thereby developing a plant having a high sugar content, which has a new taste.
European Patent Publication 0 438 904 (Japanese Laid-open Patent Application (Kokai) No. 4-341126) discloses an example of introducing a PFK gene into a plant. In the invention described in this publication, the E. coli PFK gene is expressed in potato and rice and it is shown that the amount of several intermediates of the pathways of the carbohydrate metabolism are changed.
In particular, it is shown that the sucrose content in the tubers immediately after harvest is significantly decreased. However, in the invention of the publication, the amount of glucose and fructose in the tubers during storage at low temperature, which is an industrial problem, is not mentioned. In view of the fact that E.
coli PFK is an enzyme which is cold labile (Kruger, N.J.
(1989) Biochemical Society Transaction 629th Meeting, London Vol. 17, 760-761), it is expected that a potato variety resistant to Cold Induced Sweetening cannot be obtained by introducing the E. coli PFK gene into potato and expressing it in the tubers. To attain this objective, a gene encoding PFK which is cold stable is necessary. However, a gene for a cold stable PFK has not yet been isolated.
In order to store potato tubers for a long time, storage at low temperature after harvest is very important for suppressing diseases, sprouting and aging.
However, when tubers stored at low temperature are directly used to make potato chips or French fries, browning of the products occurs during processing and the product values are greatly decreased (especially in potato chips). It is known that this browning is caused by the Maillard reaction between amino acids and reducing sugars contained in potato tubers, which occurs during processing in a hot cooking oil (Schallenberger, R.S. et al., (1959) J. Agric. Fd Chem., 7, 274). It is known that the amount of the reducing sugars (glucose and fructose) in the tubers is increased when the tubers are stored at a low temperature, and a high correlation is observed between the amount of glucose and fructose in the tubers and the degree of browning. Thus, it is thought that the increase in the amount of glucose and fructose during storage at a low temperature is the main cause of browning in the processing of the potato (Gray, D and Hughes, J.C. (1978) The Potato Crop (ed. P.M.
Harris), Chapman & Hall, London, pp.504-544).
At present, processors of potato chips use tubers stored at a low temperature of about 8C (the temperature varies depending on the varieties) and treatment with a sprout suppressant. However, reducing sugars accumulate to an unacceptable degree, so that they use the potato tubers after decreasing the amount of the reducing sugars by treatments called blanching or reconditioning before the processing. Since these treatments are costly and troublesome, if a new variety of potato were developed, in which the reducing sugars did not accumulate at the storage temperature currently employed, the cost of processing would be decreased. Further, if a variety of potato were developed, in which the reducing sugars -hardly accumulated at a temperature as low as 2 - 4C, not only the labor and cost for the pretreatments such as blanching and reconditioning can be saved, but also germination, which is the cause of aging and loss of dry weight, can be avoided, so that long-term storage without using a sprout suppressant can be possible. Residues of such chemicals in tubers are problematic from the point of view of health hazards. Thus, development of a potato variety resistant to Cold Induced Sweetening is strongly desired by the processors.
Although the mechanism of the accumulation of glucose and fructose during storage at low temperature is complicated and involves various physiological changes in the tubers, it is thought that one of the major causes is the drastic reduction in the activity of PFK at low temperature. PFK is said to be a key regulatory enzyme of the glycolytic pathway (Dixon, W. L. and ap Rees, T.
(1980) Phytochem., 19, 1653; Dixon, W.L. et al., (1981) Phytochem., 20, 969; Pollock, C.J. and ap Rees, T. (1975) Phytochem., I4, 613). In tubers during storage at low temperature, it is thought that reducing sugars are supplied by breakdown products of starch through F6P.
The biological reactions using F6P as a substrate are roughly divided into two. One is the reaction catalyzed by PFK, by which F6P enters the glycolytic pathway, and the other is the reaction catalyzed by sucrose-6-phosphate synthase (hereinafter referred to as "SPS", EC

21~735S

2.4.1.14), by which F6P enters the sucrose synthesis pathway. Thus, these two enzymes compete for F6P. In potato tubers stored at room temperature after harvest, the amount of glucose and fructose that accumulate is usually very small as long as sprouting and aging do not occur. It is thought that this is because of higher PFK
activity than SPS activity, so that F6P preferentially enters the glycolytic pathway. However, at low temperature, the enzyme activity of PFK is drastically decreased and SPS activity is higher than PFK activity, so that F6P preferentially flows into the sucrose synthesis pathway rather than into the glycolytic pathway. The sucrose is finally converted to glucose and fructose by invertase ( EC 3. 2.1. 26) and glucose and fructose accumulate. Thus, the decrease in the activity of the glycolytic pathway due to the decrease in PFK
activity is thought to be a cause of the increase in the amount of glucose and fructose in potato tubers during storage at low temperature. This hypothesis was supported by Hammond et al (Planta 180, 613-616, 1990) who reported that cold stable PFKs exist in the tubers of a variety resistant to Cold Induced Sweetening, whereas PFKs are not cold stable in the usual processing variety in which the reducing sugar content increases during storage at low temperature. Thus, it is suggested that the stability of PFK to low temperature in the tubers is the main factor which determines the amount of glucose , and fructose during storage at low temperatures.
If a DNA encoding cold stable PFK is obtained, the activity of the glycolytic pathway in the tubers during storage at low temperature can be promoted by introducing the DNA into potato, so that a potato maintaining low amounts of sugars under low temperature in tubers can be developed. In this way, processors can save the cost of blanching and reconditioning currently carried out for decreasing the content of reducing sugars.
DISCLOSURE OF THE INVENTION
Accordingly, an object of the present invention is to provide a DNA encoding a cold stable PFK. Another object of the present invention is to provide a recombinant vector which expresses the cold stable PFK in a host cell. Still another object of the present invention is to provide a method for changing the sugar content in plant cells under low temperature by transforming the plant with the recombinant vector.
To attain the above-described objects, the present inventors tried to isolate and analyze a cDNA (containing coding region and non-coding region) encoding one of the isozymes of PFK originating from a plant tissue having cold stable PFK, particularly from tubers of potato variety Brodick, and succeeded in accomplishing this.
Further, it was confirmed that the isolated gene expresses cold stable PFK in E. coli and in potato tubers, and that the glucose content in tubers of the potato in which the gene is expressed and which were stored at a low temperature is decreased and so the color of the potato chips produced from the tubers is improved.
Further, the inventors succeeded in isolating PFK genes of various plants using the potato PFK gene as a probe, and hence identified amino acid sequences which are specific to and commonly contained in various plant PFKs.
That is, the present invention provides a DNA
encoding PFK originating from a plant. The present invention also provides a recombinant vector comprising the DNA according to the present invention, which can express PFK originating from a plant in a host cell. The present invention still further provides a method for changing the sugar content in plant cells under low temperature, which comprises transforming said plant with said recombinant vector. The present invention further provides DNAs encoding the amino acid sequences identified as Sequence ID Nos. 11, 14, 21 and 22 in the Sequence Listing. The present invention still further provides a method for detecting a plant PFK gene by hybridizing sample DNAs with the DNA identified as Sequence ID Nos. 11 - 22 in the Sequence Listing or a part thereof, or a DNA containing said DNA identified as Sequence ID Nos. 11 - 22. The present invention still further provides a method for amplifying a plant PFK gene by the polymerase chain reaction (PCR) by using as a primer the DNA identified as Sequence ID Nos. 11 - 22 or 21g7355 a part thereof, or a DNA containing said DNA identified as Sequence ID Nos. 11 - 22.
By the present invention, a DNA encoding a cold stable PFK and a recombinant vector containing the same were first provided. By introducing the recombinant vector according to the present invention to a plant and expressing the same in the plant by a genetic engineering method, the glucose content in tuber tissue stored at a low temperature can be decreased when compared with tubers of non-transformed plants. Therefore, the present invention can be used for developing a potato resistant to Cold Induced Sweetening. Further, since the DNA
according to the present invention originated from a plant, it can be used for the isolation of other plant PFK genes, whereas the PFKs of other organisms are difficult to use as probes for the isolation of plant PFKs. Further, if various plant PFK genes are isolated, the PFK activity in plant cells can be reduced by antisense RNAs. Thus, for example, glycolysis can be modified, or respiration can be reduced, so that sweet fruits and vegetables containing more sugar can be prepared.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the induction of the PFK activity with time in E. coli No. 58 transformed with the recombinant vector of the present invention and of a control E. coli No. 1, after adding IPTG;

Fig. 2 shows the results of immunotitration of the PFK activity of E. coli No. 58 transformed with the recombinant vector according to the present invention;
Fig. 3 shows the results of the analyses by SDS-PAGE
and Western blot of the PFK purified from E. coli No. 58 transformed with the recombinant vector according to the present invention;
Fig. 4 shows an expression vector containing cold stable PFK-d gene;
Fig. 5 shows the results of Northern analyses of RNAs from stored tubers of lines B75 and B40; and Fig. 6 shows the results of Western analyses of proteins from stored tubers of lines B75 and B40.
BEST MODE FOR CARRYING OUT THE INVENTION
As described above, the DNA according to the present invention encodes a plant PFK. The DNAs encoding PFKs of potato (Solanum tuberosum L.), flaveria (Flaveria brownii), rice (Oryza sativa), maize (Zea mays) and radish (Raphanus sativus), which are examples of the DNA
of the present invention, encode the amino acid sequences identified as Sequence ID Nos. 2, 4, 6, 8 and 10 in the Sequence Listing described below, respectively. Examples of such DNAs are the DNAs identified as Sequence ID Nos.
1, 3, 5, 7 and 9 in the Sequence Listing, which were actually cloned and the DNA sequences were actually determined in the examples described below. However, the DNA according to the present invention is not limited thereto (It should be noted that the amino acid sequences of Sequence ID Nos. 2, 4, 6, 8 and 10 are the same as the amino acid sequences shown in Sequence ID Nos. 1, 3, 5, 7 and 9, respectively). In particular, although the DNA
having the DNA sequence shown in Sequence ID Nos. 1, 3, 5, 7 and 9 are cDNAs, since the amino acid sequences of plant PFKs and the DNA sequences encoding the PFKs were determined by the present invention, the genomic DNA
encoding the amino acid sequences identified as Sequence ID Nos. 2, 4, 6, 8 and 10 can be easily prepared by the PCR method using the genomic DNA as a template.
Therefore, it is construed that such genomic DNAs (which may contain introns) are within the scope of the present invention.
The plant PFK encoded by the above-described DNA
coding for the amino acid sequence identified as Sequence ID No. 2 or by the above-described DNA having the DNA
sequence shown in Sequence ID No. 1 has a Q10 value (described below) at 5C of not more than 2.4 as described concretely in the examples below. If the Q10 value of a PFK is not more than 2.4 at 5C, it can be said that the PFK is cold stable.
The DNA according to the present invention may be obtained by, for example, the following method.
Firstly, to isolate poly(A)+ RNA corresponding to PFK from a plant tissue, it is preferred to isolate the total RNAs which have not been decomposed. The preferred starting material is a plant tissue which is known to contain cold stable PFK, such as potato variety Brodick (which is resistant to Cold Induced Sweetening).
The total RNAs may be isolated from potato tubers by, for example, sodium dodecyl sulfate (SDS)/phenol method. PFK poly(A)+ RNA may be obtained from the total RNAs by using Dynabeads mRNA purification kit (DYNAL).
Since it is not easy to directly isolate the PFK poly(A)+
RNA by this treatment, it is preferred to prepare cDNAs using the obtained poly(A)+ RNAs as templates and to prepare a cDNA library containing the cDNAs. More particularly, a cDNA library may be prepared by synthesizing double-stranded cDNAs by the method of Gubler and Hoffman (Gene, 25:263, 1983) or the like, ligating the cDNAs to appropriate vectors via an adaptor DNA by a DNA ligase, and by transforming the host microorganisms with the thus prepared vectors. In cases where the host microorganism is E. coli, it is preferred to use a vector of the pUC series or A phage series.
Double-stranded cDNAs, prepared from poly(A)+ RNAs from tubers of potato variety Brodick, may be inserted using a DNA ligase into the Eco RI site of ~gtlO phage vector via an adaptor DNA containing Eco RI and Not I sites.
Phage particles may then be prepared, thereby obtaining a cDNA library. A cDNA clone corresponding to the PFK
poly(A)+ RNA is then identified in the thus prepared cDNA
library.

The identification of a PFK cDNA clone may be carried out by the plaque hybridization method using synthesized oligonucleotides based on the partially determined amino acid sequence of the purified plant PFK, or a DNA having a part of the DNA sequence of the PFK
gene, which may be amplified by the polymerase chain reaction (PCR) using the above-mentioned oligonucleotides as primers and using the plant genomic DNA or cDNA as a template.
Then the transformants containing the DNA encoding the PFK originated from potato tubers are cultured in a large scale by the plate lysate method or the like. The desired phage DNA is then purified by a conventional method such as Sambrook's method (Molecular Cloning: A
laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, 1989). The purified phage DNA is then digested with restriction enzyme Not I and subjected to agarose gel electrophoresis or the like to obtain the cDNA encoding PFK. Further, once the potato PFK gene is isolated, PFK genes can easily be isolated from various plants using the isolated potato PFK gene or a part thereof as a probe or using a part thereof as a primer for conducting PCR.
As described in examples below, cDNAs of PFK genes of various plants were cloned and their nucleotide sequences and amino acid sequences deduced therefrom were determined. By comparing the amino acid sequences of the various plants, 13 amino acid sequences having not less than 5 amino acid residues, which are common in plant PFKs, were identified. Therefore, PFK genes of various plants can be detected or amplified using oligonucleotides encoding these sequences or a part thereof, or nucleotides containing the region encoding these sequences, as a probe or a primer for PCR. The DNAs encoding the amino acid sequences shown in Table 7 can be easily synthesized chemically. Among these, sequences (1), (4), (11) and (12) in Table 7 (i.e., Sequence ID Nos. 11, 14, 21 and 22) are common in PFKs of plants but not found in PFKs of organisms other than plants. Thus, these are specific for plants. Therefore, by utilizing these sequences, plant PFKs can be detected or amplified avoiding the possible contamination by PFK
from other organisms. As probes, oligonucleotides of at least 15 bases up to the full length of the gene are preferred. Methods for labelling the oligonucleotides with a radioactive marker, a fluorescent marker or the like are well-known in the art. As primers for PCR, oligonucleotides of 15 - 30 bases are preferred.
The PFK cDNA thus obtained may be used for transforming microorganisms, plants and animals after preparing a recombinant vector with a vector for microorganisms, plants or animals, thereby increasing the PFK activity in the transformed microorganism, plant or animal tissue. The PFK cDNA may also be used for inhibiting the intrinsic PFK activity of a plant by inserting the PFK cDNA in a reverse direction in a vector and expressing the cDNA in a plant tissue such as potato tuber.
Examples of the microorganisms include bacteria belonging to the genus Escherichia such as E. coli and yeasts belonging to the genus Saccharomyces such as baker's yeast. Examples of the plants include dicotyledons which can be transformed by Aqrobacterium-Ri/Ti plasmid system, such as those belonging to the family Solanaceae such as potato, tobacco, and tomato;
those belonging to the family Cucurbitaceae such as melon and cucumber; those belonging to the family Cruciferae such as radish and rapeseed; and fruits such as grape and oranges. Examples of the plants which can be transformed by PEG-calcium phosphate method, electroporation method, particle bombardment method or the like include monocotyledons such as those belonging to the family Gramineae represented by rice, corn and the like.
Examples of the animals include various cultured cells (e.g., BALB/c-3T9 and the like) of human, mouse and the like. The vectors which may be used for these hosts will now be exemplified below.
Examples of the vectors for bacteria include various plasmid vectors (e.g., pBR322, pUC series vectors and the like) and phage vectors (e.g., lgtlO, lgtll, ~ZAP and the like) which are described in references (e.g., Molecular Cloning: A Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, 1989). Examples of the vectors for yeast and animal cells include those described in references (e.g., Molecular Cloning: A
Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, 1989). Examples of the vectors for plants lnclude various plasmid vectors and those originated from Ti plasmid such as binary vectors (e.g., pGA482, pBinl9 and the like) (An G. et al., (1986) Plant Physiol. 81, 301; Bevan, M., (1984) Nucleic Acids Research 12, 8711). In cases where a plant vector originated from Ti plasmid is used, the obtained recombinant DNA is introduced into a bacterium belonging to the genus Aqrobacterium such as Aqrobacterium tumefaciens (e.g., LBA4404), and the resulting transformed bacterium is infected to a plant tissue or callus by, for example, culturing the tissue or callus with the bacterium, thereby introducing the cDNA to the host plant (Komari, T. (1989) Plant Science, 60, 223:
Visser, R.G.F. et al., (1989) Plant Molecular Biology 12, 329, etc). Plant individuals containing the recombinant DNA may be obtained by regenerating an organ or whole plant by a known tissue culture method.
EXAMPLES
The present invention will now be described more concretely by way of examples. It should be noted that the present invention is not restricted to the following examples.
1: Puri~ication of Potato PFK
PFK was purified from 5 kg of tubers of potato variety Record or Maris Piper by a modification of the method of Kruger et al (Arch. Bioichem. Biophys. 267, 690-700, 1988), in which PFK was fractionated and purified by sodium dodecyl sulfate polyacrylamide gel (containing 4M urea) electrophoresis (SDS-PAGE) rather than by Mono Q column chromatography, after the ATP
agarose column chromatography. More particularly, the fraction containing PFK activity was concentrated to about 0.5 ml by ultrafiltration and equivolume of a sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 4M urea, 0.001% Bromphenol Blue) was added. The resulting mixture was heated at 65C for 10 minutes and the resultant was subjected to electrophoresis. The composition of the gel was the same as that employed in the method of Kruger et al (Arch.
Bioichem. Biophys. 267, 690-700, 1988). After the electrophoresis, the gel was stained with Coomassie Briliant Blue R-250 (CBB) and the portion of the gel containing the PFK polypeptide having a molecular weight of about 53 kDa was cut out with a razor blade. The polypeptide was eluted from the gel by using an electroelution apparatus (Model 422 Electroeluter, commercially available from BIO-RAD), and the resultant was dialyzed against a solution containing 50 mM ammonium bicarbonate and 0.001% SDS, followed by drying under vacuum to obtain a final purified sample.
2: Determination of Amino Acid Sequence of Potato PFK
The purified PFK sample was digested with V8 protease (originated from Staphylococcus aureus) in accordance with the method of Kruger et al (Arch.
Bioichem. Biophys. 267, 690-700, 1988), and the digestion product was fractionated by SDS-PAGE according to the method of Kruger et al (Arch. Bioichem. Biophys. 267, 690-700, 1988). The fractionated polypeptides were transferred to a PVDF membrane (Millipore) and stained with CBB. The stained band was cut out together with the PVDF membrane using a razor blade and the obtained band was subjected to determination of the amino acid sequence of the N-terminal region. For determining the amino acid sequence of the N-terminal region of the purified PFK, the above-described final purified PFK sample was used as it is. The analysis was carried out by the solid phase or gas phase sequencing method (This analysis was entrusted to The Department of Biochemistry, Leeds University and The Department of Biochemistry, University of Cambridge). From both samples, the amino acid sequence of the N-terminal region of the purified PFK was determined, which is shown in Table 1. The determined amino acid sequence was compared with amino acid sequences of known proteins by using a database (The Swiss Prot data bank (Release 23)), but no amino acid 21 ~ 7355 sequences having a significant homology were found.

Table 1 Determined N-terminal Amino Acid Sequence of PFK from Potato Tubers from N-terminal hr Glu Ser Asn Iyr Gln Met Lys Val Val Lys Gly Asp Tyr Gly Tyr Val Leu Glu Asp Val ? ? Leu Val Glu Asp Ala

3. Synthesis of DNA Encoding Determined Amino Acid Sequence of Potato PFK
Based on the determined amino acid sequence, the oligonucleotides having the base sequences shown in Table 2 were prepared by a DNA synthesizer (Applied Biosystems) according to the manual of the synthesizer, and the synthesized oligonucleotides were used as primers of PCR
described below.

Table 2 DNA Primers Synthesized Based on Determined Amino Acid Sequence Primer Name DNA Sequence (5' ~ 3') Degeneracy N10:ACGGAAAGTAATTATCAAATG 256 A GTC C C G
cT

N20RI:TCCTCGAGIACGTAICCGTA 64 T A A A A
T

*A: adenine, T: thymine, G: guanine, C: cytosine, I:
inosine

4. Preparation of Poly(A)+ RNA
From tubers of potato variety Brodick stored at 5C

for 4 months, or from sprouts of potato variety Record or Brodick, the total RNAs were isolated by the SDS-phenol method. Poly(A)+ RNAs were purified from the total RNAs using a Dynabeads mRNA purification kit (DYNAL) according to the manual attached to the kit.

5. Synthesis of cDNA
Using the thus isolated poly(A)+ RNAs as templates and oligo dT(12-18) or random hexanucleotide as a primer, and using RNase H-free reverse transcriptase (BRL), single-stranded cDNAs were firstly prepared. Then double-stranded cDNAs were prepared using a cDNA
synthesis kit (Amersham). The single-stranded cDNAs synthesized here were used as the templates in the PCR
described below, and the double-stranded cDNAs thus prepared were used for the preparation of the cDNA
library described below. The method for synthesizing the cDNAs was in accordance with the manual attached to the reagent or the kit.

6. Preparation of ~gtlO cDNA Library Using the double-stranded cDNAs prepared from the poly(A)+ RNAs from tubers of variety Brodick, which were synthesized using oligo dT (12-18) as a primer, or using the double-stranded cDNAs prepared from the poly(A)+ RNAs from the sprouts of tubers of variety Record, which were synthesized using the random hexanucleotide, ~gtlO cDNA
libraries were prepared. The libraries were prepared by using a ~gtlO cloning kit (Amersham) according to the manual attached to the kit. However, the adaptor included in the kit was not used and an Eco RI/Not I
adaptor (Pharmacia LKB) was used therefor.

7. Isolation of cDNA Encoding N-terminal Region of Purified PFK

214735~

PCR was performed using 500 pmol each of N10 and N20RI (see Table 2) synthesized by a DNA synthesizer based on the amino acid sequence shown in Table 1 as primers, and using 1.0 ~g of the single-stranded cDNA
originated from the genomic DNA of variety Record or 0.1 ~g of the single-stranded cDNA originated from the poly(A)+ RNAs from tuber sprouts of variety Brodick as a template. The buffer which was supplied with the Taq polymerase (Ampli Taq, Perkin-Elmer Cetus) was used according to the manual attached thereto. To the reaction mixture, 2.5 U of the enzyme and 20 nmol each of the nucleotides were added to the reaction mixture and the total volume of the reaction mixture was 100 ~1. A
cycle of 94C, 1 minute (denaturing)/50C, 2 minutes (annealing)/72C, 2 minutes (extension) was repeated 35 times and then the reaction was further continued at 72C
for 10 minutes. An aliquot of the reaction mixture was sampled and analyzed by 4% agarose gel electrophoresis.
A PCR product having 59 base pairs was detected in both cases where either of the DNAs was used as the template.
These DNAs of 59 base pairs were subcloned into plasmid vector pCR1000 (Invitrogen) according to the manual attached to the kit. As a result, a number of E. coli colonies having recombinant plasmids were obtained.
Plasmids were recovered by a conventional method from 7 clones (i.e., 4 clones originated from Record genomic DNA
and 3 clones originated from Brodick cDNA) having the plasmid containing the insert of 59 base pairs. The DNA
sequences of the PCR products contained in the 7 clones were determined by the dideoxy chain termination method.
The DNA sequences were determined using SEQUENASE Var2 (U.S. Biochemical Corp) according to the manual attached thereto. Among the 7 clones, 6 clones had the same DNA
sequence (23 base pairs) in the region between the PCR
primers. A DNA having the 23 base pairs was synthesized by a DNA synthesizer, and was named PFK23 (Table 3).
PFK23 was used as a primer in the PCR described below and as a probe for screening the cDNA library.
Table 3 DNA Sequence of PFK23 (5' ~ 3') ATGAAGGTGGTGAAAGGAGATTA

8. Isolation of PFK cDNA with Partial Length The lgtlO cDNA library (150,000 pfu) originated from tuber sprouts of variety Record was amplified by the plate lysate method and the lDNA was purified. PCR was performed using 1.0 ~g of the obtained ADNA as a template, and using 100 pmol each of PFK23 and 11232 (Table 4) which has the DNA sequence of lgtlO. The reaction conditions of the PCR were the same as described above except that the temperature in annealing was 60C.

As a result, a PCR product having about 600 base pairs was obtained. The obtained PCR product was subcloned in the above-mentioned plasmid vector pCR1000, and one of the obtained recombinant plasmids was named pPFKO1. The DNA sequence of the PCR product inserted in plasmid pPFKO1 was determined by the method described above. The amino acid sequence was deduced from the DNA sequence.
As a result, a part of the amino acid sequence had a significant homology with the amino acid sequence of known PFKs. Further, in the 5'-terminal region, the DNA
sequence encoded the amino acid sequence determined by the analysis of the purified PFK mentioned above.

9. Isolation of PFK cDNA with Full Length The DNA fragment having about 600 base pairs obtained by digesting the plasmid pPFKO1 with restriction enzyme Not I was labelled with a radioactive isotope 32p.
Using the labelled DNA as a probe, the lgtlO cDNA
library (about 400,000 pfu) originated from tubers of variety Brodick was screened by the plaque hybridization method. As a result, 57 independent positive plaques were obtained. From these plaques, 24 plaques were randomly selected and were subjected to secondary screening using the above-mentioned DNA fragment having about 600 base pairs and PFK23 as the probes. As a result, 11 clones were positive to both labelled probes.
After third screening, plate lysates were prepared from these independent 11 clones. PCR was performed using 10 ~1 of the lysate as a template and 50 pmol each of the synthetic DNAs A1232 and ~1231 shown in Table 4 as primers. The PCR was performed in the same conditions as described above except that the annealing temperature was 60C. By analysis of the PCR product by 0.8% agarose gel electrophoresis, it was estimated that the length of the cDNA fragment per se excluding the length of the ~DNA
was about 1700 - 2200 base pairs. Potato tuber poly(A)+
RNA was analyzed by the conventional Northern blotting method using as a probe the above-mentioned DNA fragment of about 600 base pairs labelled with radioactive isotope 32p. As a result, a poly(A)+ RNA having about 2000 -2300 base pairs was detected, which is almost coincident with the results of the above-described PCR. The recombinant plasmid vectors prepared by subcloning the DNA insert cut out by restriction enzyme Not I from the 11 AgtlO clones into the Not I site of a plasmid vector pBluescript SK II(-) (Stratagene) were named pPFK16, pPFK17, pPFK19, pPFK26, pPFK28, pPFK29, pPFK31, pPFK32, pPFK33, pPFK34 and pPFK35, respectively.
Table 4 DNA Sequence of Primers Originated from ~gtlO
used in PCR (5~ ~ 3~) ~1232: CTTATGAGTATTTCTTCCAGGGTA
~1231: AGCAAGTTCAGCCTCGTTAAG

The total DNA sequence (1978 base pairs) of the cDNA
insert in pPFK32 was determined by the method described above, and is shown as Sequence ID No. 1 in the Sequence Listing together with the deduced amino acid sequence encoded thereby. The DNA contained a region of 1455 base pairs translated into 485 amino acids containing the determined amino acid sequence of the N-terminal of the purified PFK (i.e., the third amino acid, threonine to the 26th amino acid, leucine (although the 24th and 25th amino acid of the purified PFK could not be determined, as shown in Table 1) shown in Sequence ID No. 1 in the Sequence Listing). In the deduced amino acid sequence, an initiation codon encoding methionine exists upstream of the N-terminal of the purified potato tuber PFK
polypeptide by two amino acids. The deduced molecular weight is 53.8 kDa which is almost coincident with the molecular weight (53 kDa) of the potato tuber PFK-d polypeptide, which was deduced by Kruger et al (Arch.
Bioichem. Biophys. 267, 690-700, 1988). The reason why the codon "ATG" of 133rd to 135th nucleotides is the initiation codon of PFK is that termination codons in different reading frames exist upstream of this ATG
(e.g., TGA: 15th - 17th, TAA: 26th - 28th, and TGA: 55th - 57th), so that even if an ATG codon exists upstream of the first base C of the isolated cDNA, the codon cannot be the initiation codon of PFK. Further, although an ATG
codon exists at 36th - 38th nucleotides, a termination codon TGA exists at 90th - 92nd nucleotides in the same reading frame, so that the ATG codon cannot be the initiation codon of PFK.
<Characterization of PFK Encoded by Isolated cDNA>

~1 17355

10. Expression of Plant PFK Gene in E. coli To prove that the isolated gene is the gene encoding PFK having an enzyme activity, it is necessary to actually express the gene. Therefore, the inventors tried to express the isolated gene by introducing it into E. coli as follows.
First, PCR was performed using 250 ng of plasmid pPFK32 as a template and 30 pg each of PFK32 and PFK32R
(Table 5) to which Eco RI site and Pst I site had been introduced, respectively, as primers. The reaction conditions were the same as described above except that the annealing temperature was 30C, 35C or 40C, the number of cycles was five, and Pfu DNA po lymerase (Stratagene) was used as the DNA polymerase. The obtained PCR product was fractionated by 0.8~ agarose gel electrophoresis and the desired band corresponding to about 1800 base pairs was cut out. The DNA in the cut out band was recovered from the gel by a conventional method. The DNA thus obtained, having about 1800 base pairs, was digested with restriction enzymes Eco RI and Pst I, and the resultant was ligated with plasmid vector pKK223-2 (Pharmacia LKB) which had been digested with restriction enzymes Eco RI and Pst I. The resulting recombinant plasmid (pKK32) was introduced into E coli XL1-Blue (Biotechniques, 5, 376-378, 1987) according to a conventional method and the E. coli was cultured overnight at 37C on Luria-Bertani (hereinafter referred - 21g7355 to as "LB") agar medium containing antibiotic carbenicillin (50 ~g/ml). From a number of emerged colonies, 110 colonies were selected. The DNA fragment having about 2000 base pairs, which was cut out from the plasmid pPFK32 by restriction enzyme Not I, was labelled with a radioactive isotope 32p and colony hybridization was performed using the labelled DNA as a probe. As a result, 66 positive colonies were obtained. These E.
coli clones, which were confirmed to contain the potato PFK gene, could hardly grow on the LB agar medium containing 1 mM of isopropyl~-D-thiogalactopyranoside (hereinafter referred to as "IPTG") presumably because the metabolism is confused by the PFK of which activity is controlled in a different manner from that of the E.
coli PFK. Thus, the PFK gene introduced into E. coli was expressed as follows. Firstly, the E. coli cells were cultured with shaking at 37C on LB liquid medium containing 50 ~g/ml of carbenicillin until the absorbance at 600 nm reached 0.3 - 0.7. Then IPTG (1 mM) was added and the cells were recovered after culturing the cells with shaking for a prescribed time. The cells were then lyzed by the method of Tabita et al (Anal.
Biochem. 84, 462-472, 1978) using toluene, and the PFK
activity was measured by the method of Kruger et al (Arch. Bioichem. Biophys. 267, 690-700, 1988). E. coli No. 58 showed a PFK activity which is about 7 times higher than that of the control E. coli No. 1 (into which 214735s pKK223-2 was introduced) (Fig. 1).
Table 5 DNA Sequences of PCR Primers Containing Introduced Eco RI site and Pst I site (5' ~3') *****
PFK32: TATATATTTGGAATTCATGGGTACTGAG
Eco RI

PFK32R: CAAAAGACCCTGCAGCCACACAG
Pst I
*: mismatched region To determine whether the high PFK activity of E.
coli No. 58 was due to the plant PFK activity or to the PFK activity of E. coli XL1-Blue, immunotitration was performed according to the method of Kruger et al (Arch.
Bioichem. Biophys. 267, 690-700, 1988).
In the immunotitration, the high PFK activity of E.
coli No. 58 was not substantially reduced by anti-E. coli PFK antibody, but was significantly reduced by anti-potato PFK-c antibody (presented by Dr. Kruger, the Department of Plant Sciences, University of Oxford) (Fig.
2). It has been confirmed by Western blot analysis and by immunotitration that the anti-potato PFK-c antibody strongly reacts with potato PFK-c and PFK-d, but does not substantially react with E. coli PFK.
These results established that the protein expressed in E. coli No. 58 after induction by IPTG was potato PFK, and that the cDNA inserted in the plasmid pPFK32 was a 214735~
.

potato PFK gene.

11. Comparison of Amino Acid Sequence between Potato PFK
and PFKs of Other Organisms The amino acid sequence deduced from the DNA
sequence of the cDNA contained in pPFK32, which encodes potato PFK, was compared with amino acid sequences of known PFKs using a database (The Swiss Prot data bank (Release 23)). Although the amino acid sequence has not more than 30 percent identity with the reported PFKs, the homologous regions are concentrated in the binding sites of the substrate, coenzyme and of the regulatory substance which characterize PFK (Evans, P.R and Hudson, P.J. (1979) Nature, 279, 500 - 504) and in the vicinity thereof. The region showing significant homology is approximately 98th - 321st amino acids, and little significant homology with the reported PFKs of other organisms is found in the N-terminal and C-terminal regions of 1st - 97th amino acids and 322 - 485 amino acids, respectively. The reported eukaryotic PFKs have a duplicate structure in which a single polypeptide chain contains both a catalytic site to which F6P binds and a regulatory site to which fructose-2,6-bisphosphate binds, the regulatory site has a similar amino acid sequence to the catalytic site. It was shown that the amino acid sequence deduced from the DNA sequence of the plant PFK
gene isolated by the present invention does not have such a structure. Further, there are as many as several tens of amino acids, including some with putative F6P binding sites, which are commonly conserved in the PFKs of other organisms but are not conserved in the potato PFK. These facts suggest that the plant PFK has a structure and function different from those of the reported PFKs.
It is said that there are two kinds of PFKs in plants, that is, plastidic PFK and cytoplasmic type PFK.
The PFK encoded by the potato PFK cDNA according to the present invention has a N-terminal longer than the purified protein by two amino acids (Met, Gly). It is not clear whether this difference was caused by degradation during the purification or by processing performed in the cells. However, it was shown that the PFK does not have a transit peptide taking part in the transport of a polypeptide to plastids such as the amyloplast and chloroplast, so that the PFK is cytoplasmic.

12. Purification of Potato PFK Expressed in E. coli Potato PFK was purified from E. coli No. 58, which was confirmed to express potato PFK by the following method, and its stability under low temperatures was examined. About 0.3 g of cells were suspended in an extraction buffer (100 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 1 mM EDTA, 14 mM 2-mercaptoethanol, 1 mM PMSF, 1 ~M
leupeptin and 1 ~M pepstatin) and disrupted by ultrasonication. The resultant was centrifuged at 50,000 x g for 30 minutes and the supernatant was recovered.

- 214735s The supernatant was then applied to a Cibacron Blue agarose (type 3000-CL, Sigma) column (16 x 55 mm) which had been equilibrated with buffer A prepared by adding glycerol to the extraction buffer to 10% (v/v). By this operation, most of the E. coli PFK was strongly bound to the gel and most of the potato PFK was not bound to the gel, so that the latter was recovered in the flow through fraction. The thus recovered solution was then applied to a Reactive Red 120-agarose (type 3000-Cl, Sigma) column (16 x 110 mm) equilibrated with buffer A to adsorb the PFK on the gel. After washing the column with 25 ml of buffer A, PFK was eluted by 150 ml of buffer A having a linear gradient of 0 - 1.0 M KCl. The fractions containing PFK activity were combined and concentrated to 3 ml by ultrafiltration (Amicon PM10 membrane). The resultant was desalted by a Biogel P-6 column (Bio-Rad) equilibrated with buffer A. Finally, the desalted sample was applied to a Mono Q column (0.5 x 50 mm, Pharmacia LKB) equilibrated with buffer A. After washing the column with 5 ml of buffer A, PFK was eluted by 150 ml of buffer A having a linear gradient of 0 - 1.0 M KCl. The fractions having PFK activity were combined to obtain a purified sample. After fractionating the purified sample by urea/SDS-PAGE, the gel was stained by CBB. On the other hand, after fractionating the purified sample by -urea/SDS-PAGE, the polypeptide was transferred to a nitrocellulose filter and was subjected to Western blot 214735~

analysis using the anti-potato PFK-c antibody. In both analyses, a polypeptide with a molecular weight of 53 kDa was detected (Fig. 3). Thus, it was proved that the isolated PFK encodes PFK-d.

13. Confirmation of Stability at Low Temperature of Potato PFK Expressed in E. coli Using the purified PFK thus obtained, the low temperature stability of the enzyme activity was examined in the temperature range of 0 - 25C by the method of Hammond et al (Planta 180, 613-616, 1988). As an indicator expressing the stability of the enzyme at low temperatures, Q10 value was used. Taking the temperature along the X axis and taking the logarithm of the enzyme activity along the Y axis, an equation of dy/dx = O.llogQ10 is satisfied, wherein dy/dx means the slope in the above-mentioned coordinates. The PFK expressed in E.
coli, which originated from potato variety Brodick, had a Q10 value at 0C of as low as 2.42, which is not much larger than the Q10 value at room temperature, that is 1.66 (Table 6). The PFK encoded by the cDNA inserted in the plasmid pPFK32 had a Q10 value at 5C of 2.24. The Q10 value at 5C of E. coli PFK-l is 2.89 (Kruger, N.J.
(1989) Biochemical Society Transaction 629th Meeting, London Vol. 17, 760-761), and the Q10 values at 2 - 6C
of the PFKs in the tubers of potato variety Record, which undergoes Cold Induced Sweetening, are 3.10 (PFK III) and .

4.20 (PFK IV), respectively (Hammond, J.B.W. et al.
Planta (1990) 180, 613-616). Thus, it was proved that the PFK encoded by the potato PFK gene isolated in the present invention has a significantly higher cold stability than the E. coli PFK and the PFKs of the potato variety which undergoes Cold Induced Sweetening. On the other hand, ap Rees et al reported that the Q10 value at 2 - 10C of SPS, which is the rate-determining enzyme in the sucrose synthesis pathway and which competes with PFK
for the substrate F6P, is 2. 25 (Plants and Temperature (ed. Long, S.P. and Woodward, F.I.) Society of Experimental Biology Seminor Series No. 42, Cambridge, U.K.; Cambridge University Press, pp.377-393). Thus, the PFK encoded by the DNA according to the present invention has cold stability comparable to that of SPS (Table 6).
Therefore, by expressing the PFK gene according to the present invention under the control of a promoter which is strongly expressed in the tubers stored at a low temperature, a potato variety having low sucrose content in the tubers stored at low temperature can be developed, so that a potato variety resistant to Cold Induced Sweetening (i.e., the content of glucose and fructose is low) can be prepared.

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14: Isolation of PFK Genes from Various Plants Isolation of PFK genes from various plants was tried using the isolated potato PFK cDNA. Messenger RNAs were isolated from a callus originated from rice (variety:
Tsukinohikari) immature embryo, a callus originated from maize endosperm and from green leaves of Flaveria and green leaves of radish by the method described for potato, and cDNA libraries were prepared by the following method. That is, the cDNA libraries of rice, Flaveria and radish were prepared using TimeSaver cDNA Synthesis Kit (commercially available from Pharmacia), 1 ZAP II
Cloning Kit (commercially available from Stratagene) and Gigapack II Gold (commercially available from Stratagene) in accordance with the manuals attached to the commercial products. The cDNA library of maize was prepared using cDNA Synthesis Kit (commercially available from Amersham) and A gtlO cDNA Cloning Kit (commercially available from Amersham) in accordance with the manuals attached to the commercial products.
From the prepared cDNA libraries, ~ phages in which PFK cDNAs were incorporated were isolated by the plaque hybridization method. In this operation, the DNA
fragment of about 2 kbp obtained by digesting the above-mentioned plasmid pPFK32 with Not I was used as a probe after labelling the fragment with radioactive 32p. From all of the cDNA libraries, positive plaques which react with this probe were obtained. As for the maize PFK

214735~

cDNA, A DNA purified by the plate lysate method was digested with Eco RI and the insert containing the PFK
cDNA was subcloned into a plasmid pBluescript SK II(-) (commercially available from Stratagene). Thereafter, the DNA sequence was determined by the method described for potato PFK. As for cDNAs of other plants, the inserts were subcloned into a plasmid pBluescript SK(-) (commercially available from Stratagene) using the helper phage (ExAssist helper phage (M13)) and E. coli (SOLR
strain) contained in the A ZAP II Cloning Kit (commercialy available from Stratagene) in accordance with the manual attached to the kit. Thereafter, the DNA
sequence was determined by the method described for potato PFK. The nucleotide sequences of the cDNAs of Flaveria, rice, maize and radish PFKs are shown in Sequence ID Nos. 3, 5, 7 and 9, respectively, and the deduced amino acid sequences encoded by the cDNAs are shown in Sequence ID Nos. 4, 6, 8 and 10, respectively.
All of the amino acid sequences of the plant PFKs showed very high homology with the amino acid sequence of the potato PFK-d (Sequence ID No. 2), while their homologies with the reported PFKs of other organisms such as bacteria, mammals and yeasts were significantly lower.
The plants from which PFK genes were isolated include a wide variety of plants. That is, they include both monocotyledons (rice and maize) and dicotyledons (Flaveria, potato and radish), and include the families 21~73SS

Gramineae (rice, maize), Compositae (Flaveria), Solanaceae (potato) and Cruciferae (radish). Thus, it was proved that the potato PFK cDNA shown in Sequence ID
No. 1 can be used for isolating PFK genes of a wide variety of plants.
By comparing the amino acid sequences of the PFKs of the various plants, 13 amino acid sequences (having not less than 5 amino acid residues) which are common to all of these plants were discovered (Table 7). These amino acid sequences include those specific to plants (Sequence ID Nos. 11, 14, 21 and 22) which do not exist in the reported PFKs of other organisms. Particularly, the amino acid sequence shown in Sequence ID No. 22 is thought to correspond to the amino acid sequence (in the vicinity of Leu Gly His Val Gln Arg Gly Gly) defining the binding site of the substrate F6P and vicinity thereof (Evans, P.R. and Hudson, P.J. (1979) Nature, 279, 500-504). The amino acid sequence of the binding site and the vicinity thereof is very well conserved in other organisms (Heinisch, J. et al., (1989) Gene, 78, 309-321). However, plants do not have this amino acid sequence but have the amino acid sequence shown in Sequence ID No. 22 as a consensus sequence.
Thus, it was proved that the cDNA of potato PFK
having the nucleotide sequence shown in Sequence ID No. 1 is useful as a probe for isolating the PFK genes of other plants. Further, by comparison of the amino acid sequences of PFKs of various plants, amino acid sequences which are thought to commonly exist in plants were identified (Table 7). These amino acid sequences are useful, for example, for synthethizing primers for isolating a PFK gene from a plant by the PCR method.
Still further, amino acid sequences which are thought to commonly exist in PFKs of plants but not exist in PFKs of other organisms were identified. That is, by the present invention, the amino acid sequences characteristic to plants (i.e., amino acid sequences shown in Sequence ID
Nos. 11, 14, 21 and 22) were first identified.

Table 7 Consensus Amino Acid Sequences in PFKs of Various Plants (Numbering corresponds to the amino acid residue number in Sequence ID No. 1) 1) Arg Ala Gly Pro Arg 2) Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn 3) Gly Tyr Arg Gly Phe 4) Ile Val Asp Ser Ile Gln 5) Pro Lys Thr Ile Asp Asn Asp Ile (6) Phe Gly Phe Asp Thr Ala Val Glu (7) Ala Gln Arg Ala Ile Asn (8) Val Lys Leu Met Gly Arg 9) Ser Gly Phe Ile Ala (10) Ala Glu Gly Ala Gly Gln 11) Asp Ala Ser Gly Asn (12) Leu Lys Tyr Ile Asp Pro Thr Tyr Met (13) Cys Leu Ile Pro Glu

15: Construction of Plasmid Vector fo~ Transforming 21g7355 Plants To introduce the cold stable PFK gene of potato variety Brodick into a plant, a plasmid pPFK(35S) shown in Fig. 4 was prepared. The method for preparing the plasmid pPFK(35S) will now be described in detail.
Firstly, a polylinker containing recognition sites of Hind III, Not I, Bql II, Bam HI, Eco RI, Sma I, Pst I, Sst I, Bcl I, Bql II, Not I and Eco RI in the order mentioned (commercially available from Agricultural Genetics Company, Cambridge, United Kingdom) was ligated to pUC19 preliminarily digested with Hind III and Eco RI.
The obtained plasmid was named pUCl9(PL). The polyadenylation signal sequence (about 0.3 kbp) of nopaline synthetase gene, which was obtained by digesting a plasmid pAPT9 (commercially available from Agricultural Genetics Company, Cambridge, United Kingdom), was ligated to pUCl9(PL) preliminarily digested with Sst I and Bcl I.
The obtained plasmid was named pUC19 (nos term). Then a DNA fragment of about 2.3 kbp containing a patatin promoter sequence was excised from a plasmid pBI240.7 (Bevan et al., Nucleic Acid Research 14: 4625-4638, 1986) with Bql II and Bam HI, and this DNA fragment was ligated to pUC19 (nos term) preliminarily digested with Bql II
and Bam HI. The obtained plasmid was named pUCl9(pat/nos term). Thereafter, the cold stable PFK gene (about 1.8 kbp) was excised from the above-described plasmid pKK32 with Eco RI and Pst I and ligated to pUCl9(pat/nos term) preliminarily digested with Eco RI and Pst I. The obtained plasmid was named pPFK(pat). The plasmid pPFK(pat) was digested with Eco RI and the resultant was blunted with Klenow fragment of E. coli DNA polymerase I.
Finally, the thus obtained plasmid was digested with Sst I and the excised cold stable PFK gene of about 1.8 kbp was ligated to a plasmid pROK2 (originating from pBinl9:
Baulcombe, D. et al., (1986), Nature, 321, 446-449) preliminarily digested with Sma I and Sst I. The thus obtained plasmid was named pPFK(35S) (Fig. 4).

16: Transformation of Potato The vector plasmid pPFK(35S) shown in Fig. 4 was introduced into Aqrobacterium tumefaciens LBA4404 by the electroporation method (Shen, W-J and Forde, B.G. (1989) Nucleic Acid Research 17, 8385), and the obtained strain was named LBA4404(35S/PFKd). The strain LBA4404(35S/PFKd) was used to transform potato variety Bintje as an example of plants. The method for the transformation will now be described in detail. Virus-free sterilized plants of potato variety Bintje were purchased from Scottish Agricultural Services Agency, Edinburgh, United Kingdom. From the purchased in vitro plants, a plurality of single nodes were aseptically cut out and each single node was cultured on a solid medium containing inorganic salts of Lismaier and Skoog's medium (hereinafter referred to as "LS medium") (Lismaier, E.
and Skoog, F. (1965) Physiol. Plant, 18, 100-127), 30 g/l 21~7355 .

of sucrose and 8 g/l of agar. The stems and leaves of the plants were used for the transformation described below. That is, sections (having a length of 0.5 - 2.0 cm) of the stems or sections (having a length of about 0.6 - 1.0 cm in the longitudinal direction and about 0.5 - 1.0 cm in the transverse direction) of the leaves were cultured together with LBA4404(35S/PFKd) in a liquid medium containing the inorganic salts of LS medium and 30 g/l of glucose at 25C for 48 hours. The population density of LBA4404(35S/PFKd) at the beginning of this culture was adjusted to about 108 cells/ml. After this culture, sections of the stems or leaves were washed with sterilized water containing 250 mg/l of cefotaxime several times and placed on KS1 medium reported by Kasaoka et al (Japanese Laid-open Patent Application No.
6-133783). Calli resistant to kanamycin emerged about 20 days after the beginning of this culture. By continuing the culture for another 10 - 30 days, plants were regenerated from the calli. The kanamycin-resistant plants were cut into single nodes and each of the single nodes was cultured on a solid medium containing inorganic salts of LS medium, 30 g/l of sucrose, 250 mg/l of cefotaxime, 100 mg/l of kanamycin and 8g/l of agar to grow kanamycin-resistant plants. The plants were transplanted to pots and cultivated in a green house. As a control, non-transformed potato variety Bintje was used. The control plants were aseptically grown in test tubes in the same manner as the transformed plants and then cultivated in pots in a green house. When the transformed plants and control plants completely wilted naturally after about 4 months from the transplantation to the pots, potato tubers were harvested. The harvested tubers were stored in a cold storeroom (5 - 8.5C or 15C). These tubers were used for the various analyses described below.

17: Generation of Cold Stable PFK in Transformed Potato DNAs were extracted from the non-transformed line B40 and transformed line B75 having resistance to kanamycin by CTAB method (Doyle, J.J. and Doyle, J.L.
(1987) Phytochemical Bulletin, 19, 11-15), and were subjected to Southern analysis in accordance with the method of Sambrook et al (Molecular Cloning: A Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, 1989). More particularly, the DNAs were digested with Eco RI and the resulting DNA fragments were blotted on a nylon membrane. The blots were then reacted with a probe labelled with radioactive 32p. As the probe, nos terminator region (280 bp) or the 3' flanking region (235 bp) of the cold stable PFK gene, which is excised from the above-mentioned plasmid pKK32 with Bql I and Pst I, was used. It was confirmed that in line B75, 5 copies of the gene per tetraploid genome were introduced when any of the DNA fragments was used as the probe (data not shown).

RNAs were purified from the tubers (stored at 15C
for 2 months) of line 40 or line B75 cultivated in pots in a green house, and the purified RNAs were subjected to Nothern analysis according to the method of Sambrook et al (Molecular Cloning: A Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, 1989). As the sample, 25 ~g of the total RNAs was used. As the probe, the 3' flanking region (235 bp) of the cold stable PFK DNA, excised by digesting the above-described plasmid pKK32 with Bql I and Pst I, was used after labelling the DNA with radioactive 32p. The results are shown in Fig.
5. Very strong signals were observed for the line B75 but no PFK mRNA was detected for the non-transformed line B40. From the experiences of the present inventors, it is difficult to detect the PFK mRNA in potato tubers by Nothern analysis unless a purified mRNA, not the total RNAs, is used. Presumably an extremely large amount of PFK mRNA is expressed in tubers of line B75, which never occurs in normal potatoes, and such excess production of PFK mRNA is thought to be the transcript of the introduced cold stable PFK gene.
From the tubers of line B75 or line B40, which were stored at 15C for 2 months after harvest, crude extracts were prepared according to the method of Kruger et al (Kruger, N.J. et al., (1989) Arch. Biochem. Biophys. 267, 690 - 700) and subjected to Western analysis. As the antibody, the above-mentioned anti-potato PFK-c antibody was used. The results are shown in Fig. 6. Among the polypeptides which reacted specifically or non-specifically with the antibody employed, the polypeptide which was different in amount was PFK-d alone. The intensity of the bands in the Western analysis suggest that, in tubers of line B75, polypeptide PFK-d is present at 3-4 times the level in the control line B40. This is in general agreement with the results of the Northern analysis described above. However, the amount of polypeptide PFK-d in line B75 was smaller than expected from PFK poly(A)+RNA detected. This is presumably because the amount of protein expressed from poly(A)+RNA
derived from the foreign PFK gene is not commensulate with the protein expressed from poly(A)+RNA derived from the endogenous PFK genes.
From tubers (stored at 15C for two months after harvest) of lines B75 and B40, crude extracts were prepared according to the method of Kruger et al (Kruger, N.J. et al., (1989) Arch. Biochem. Biophys. 267, 690 -700) and PFK activities were determined. One tuber was used for each measurement, and the average of three measurements is shown in Table 8. As shown in Table 8, it was confirmed that line B75 had a total PFK activity 1.3 times higher than that of line B40. The total PFK
activities means the total of the activities of PFK I, II, III and IV constituted by various combinations of four polypeptides PFK-a, PFK-b, PFK-c and PFK-d reported by Kruger et al (Kruger, N.J. et al., (1989) Arch.
Biochem. Biophys. 267, 690 - 700). By the above-described expression experiments in E. coli, it was confirmed that an active enzyme can be constructed by PFK-d alone. Assuming that the increase in the PFK
activity observed in the tubers of line B75 is due to the expression of the introduced PFK-d gene, the result that the total PFK activity was increased is consistent with the result of the Western analysis.
A crude extract was prepared from a tuber (stored at 15C for 2 months after harvest), and PFK IV containing PFK-d was partially purified from the crude extract by using a Reactive Red 120 Agarose column (commercially available from Sigma) and a MonoQ column (commercially available from Pharmacia). The PFK activity of the partially purified PFK IV was measured at various low temperatures. The procedure of the above-described operation will now be described in detail. Firstly, a tuber having a weight of about 20 g of line B75 (stored at 15C for 2 months) was sliced to a thickness of about 2 - 5 mm and frozen in liquid nitrogen. The frozen slices were stored at -70C until use. All of the following operations were carried out at 4C. In 40 ml of extraction buffer (the above-mentioned buffer A to which 2 mM benzamidine, 1 mM PMSF, 1 ~M leupeptin, 1 ~M
pepstatin and 1% (w/v) insoluble polyvinylpyrrolidone were added), the frozen sample was sufficiently 21g7355 pulverized in a mortar using a pestle. The resulting extract was filtered through Miracloth and the filtrate was centrifuged at 20,000 x g for 30 minutes. The supernatant was recovered and filtered (0.45 ~m) and was applied to a Reactive Red 120 Agarose (Type 3000-CL, commercially available from Sigma) column (16 x 110 mm) to adsorb PFK. The procedure of purification from this point was the same as the above-described purification procedure of the PFK expressed in E. coli. By the MonoQ
column chromatography, PFK was divided into 4 peaks and the fractions containing PFK IV were collected. The PFK
activity was measured at various temperatures and Q10 values were determined. The results are shown in Table 9. The partially purified PFK IV was very stable at low temperatures, and the Q10 values were within the range between 1.9 and 2.0 at any temperature from 0 - 25C.
The Q10 values of the PFKs purified from E. coli No. 58 strain are shown in Table 6. By comparing the results shown in Tables 6 and 9, it is seen that the PFK IV
expressed in line B75 was more stable than the PFK
expressed in E. coli, in spite of the fact that the same gene was introduced into both of them. Further, although the PFK gene introduced into line B75 was isolated from potato variety Brodick, it was more stable to cold temperature than PFK IV of the potato variety Brodick reported by Hammond et al (Hammond, J.B.W. et al., (l990) Planta., 180, 613-616). Based on the fact that 2/3 to 3/4 of the PFK-d expressed in line B75 originated from the introduced gene judging from the Western analysis, and that PFK IV mainly contains PFK-d as reported by Kruger et al (Kruger, N.J. et al., (1989) Arch. Biochem.
Biophys. 267, 690 - 700), it is thought that the cold stability of PFK IV of line B75 is brought about by the introduced gene.
Thus, the gene which was confirmed to express in E.
coli was introduced into potato using the vector plasmid shown in Fig. 4. As a result, it was confirmed that the introduced gene was expressed in line B75 and that PFK IV
of line B75 had cold stability.
Table 8 Total PFK Activity of Tubers of Line B75 Line PFK Activity (nmol/min/g, FW) B40 (control) 108.4 B75 141.4 Table 9 Q10 Value of PFK IV Partially Purified from Tubers of Line B75 Temperature (C) 2 5 7 10 15 20 25 Q10 Values 1.91 1.92 1.92 1.93 1.94 1.95 1.96

18: Change in Sugar Content of Potato Tubers Stored at Low Temperature, to Which Cold Stable PFK Gene was Introduced, and Change in Color of Potato Chips Produced from the Tubers The sugar content of tubers of lines B75 and B40 stored at a low temperature (5 - 8.5C), and the color of potato chips produced from the tubers were examined. The results are shown in Table 10. The glucose content was determined by using a commercially available test paper for measuring glucose in urine (Testape, commercially available from Shionogi & Co., Ltd.). More particularly, a spatula was inserted into a tuber to form a groove with a depth of about 5 mm. The test paper was inserted into this groove, and the glucose content was measured by comparing the color of the test paper with the color scale attached to the container of the test paper. The scores 0, +, ++, +++ and ++++ shown in the color scale attached to the container correspond to glucose 214735~

concentrations of 0~, 0.1%, 0.25~, 0.5% and 2~ or more, respectively. In Table 10, scores 0, +, ++, +++ and ++++
are expressed as values 0, 1.0, 2.0, 3.0 and 4.0, respectively. The values shown in Table 10 are averages of the measured values of 5 tubers. The smaller the value, the smaller the glucose content in the tuber. As shown in Table 10, it was confirmed that the glucose content in the tuber of line B75 stored at a low temperature for 4 weeks was smaller than that of line B40.
It is known that there is a strong correlation between the glucose content of a tuber and the color of the potato chips produced therefrom (Gray, D and Hughes, J.C. (1978), The Potato Crop (ed. P.M. Harris), Chapman &
Hall, London, pp.504-544). Thus, potato chips were produced from tubers of lines B75 and B40 stored at a low temperature (for 2 weeks, 4 weeks and 12 weeks) and the degree of browning was compared. More particularly, the tubers were sliced using a commercially available slicer and the slices were fried in an edible oil (mixture of rapeseed oil and soybean oil) at 180C for about 3 to 4 minutes until bubbles were no longer formed. Three potato chips from each of 5 tubers, totally 15 chips, per one line per one test were produced and the color of each potato chip was compared with color cards for potato chips (prepared by The Institute for Storage and Processing of Agricultural Produce, Wageningen, The 21g735~

-Netherlands). The degree of browning is expressed in terms of the score described in the color card. The lower the score, the darker the fly color. The results are shown in Table 10. Each score is the average of 15 potato chips. The potato chips made from tubers of line B75 exhibited a lower degree of browning than those produced from line B40 irrespective of the duration of storage.
As described above, it was proved that the glucose content in the tubers stored at a low temperature can be decreased and, in turn, the degree of browning of potato chips can be decreased by transforming a potato plant using the vector plasmid shown in Fig. 4 and expressing the cold stable PFK in the tubers.
Table 10 Glucose Content in Tubers of Line B75 in Which Cold Stable PFK was Expressed and Color of Potato Chips Produced from the Tubers Glucose Content Potato Chip Color (color score) (color card score) Duration of Storage Duration of Storage after Harvest after Harvest (weeks) (weeks) B40 (control) 1.0 2.67.0 4.0 2.6 2.3 B75 1.0 1.2 7.0 5.5 3.7 3.5 SEQUENCE LISTING
SEQUENCE ID No.: 1 SEQUENCE LENGTH: 1978 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
ORIGINAL SOURCE
ORGANISM: Solanum tuberosum L.
VARIETY: Brodick TISSUE TYPE: tuber IMMEDIATE SOURCE
LIBRARY: ~ gtlO cDNA library originated from mRNA from tuber stored at low temperature CLONE: pPFK32 SEQUENCE DESCRIPTION
CTCTTTTCTT GGGTTGACTC AAATTTAACA TATATATGTA TT'lITl"l'GTT TTTGTGATTC 60 Met Gly Thr Glu Ser Asn Tyr Gln Met Lys Val Val Lys Gly Asp Tyr Gly Tyr Val Leu Glu Asp Val Pro His Leu Thr Asp Tyr Ile Pro Asp Leu Pro Thr Tyr Asp Asn Pro Leu Arg Ser Asn Pro Ala Tyr Ser Val Val Lys Gln Tyr Phe Val Asp Met Asp Asp Thr Val Pro 21~7355 :

~ CAA AAG GTT GTT GTT CAC AAG GAC AGT CCC AGA GGG GTG CAT TTC CGG 363 Gln Lys Val Val Val His Lys Asp Ser Pro Arg Gly Val His Phe Arg Arg Ala Gly Pro Arg Gln Lys Val Tyr Phe Ser Ser Asp Asp Val Arg Ala Cys Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn Thr Val Ile Arg Glu lle Val His Ser Leu Asp Tyr Met Tyr Gly Val Asn Lys Val Phe Gly Ile Asp Gly Gly Tyr Arg Gly Phe Tyr Ser Lys Asn Ile Ile Asn Leu Thr Pro Lys Thr Val Asn Asp Ile His Lys Arg Gly Gly Thr Ile Leu Gly Ser Ser Arg Gly Gly His Asp Thr Thr Lys Ile Val Asp Ser Ile Gln Asp Arg Glu Ile Asn Gln Val Tyr Ile Ile Gly Gly Asp Gly Thr Gln Lys Gly Ala Ala Val Ile Tyr Glu Glu Ile Arg Arg , 21~735S

Arg Gly Leu Lys Val Ile Val Ala Gly Ile Pro Lys Thr Ile Asp Asn ~ 210 215 220 Asp Ile Pro Val lle Asp Lys Ser Phe Gly Phe Asp Thr Ala Val Glu Glu Ala Gln Arg Ala Ile Asn Ala Ala His Val Glu Ala Glu Ser Ala Glu Asn Gly Ile Gly Val Val Lys Leu Met Gly Arg Tyr Ser Gly Phe Ile Ala Met Tyr Ala Thr Leu Ala Ser Arg Asp Val Asp Leu Cys Leu Ile Pro Glu Ser Pro Phe Tyr Leu Glu Gly Asp Gly Gly Leu Phe Glu Tyr Ile Glu Lys Arg Leu Lys Glu Asn Gly His Met Val Ile Val Ile Ala Glu Gly Ala Gly Gln Glu Leu Leu Ala Glu Glu Asn Ala His Ala - Lys Asn Glu Gln Asp Ala Ser Gly Asn Lys Leu Leu Gln Asp Val Gly Leu Trp Ile Ser Gln Lys Ile Arg Asp His Phe Ala Thr Lys Thr Lys Met Pro Ile Thr Leu Lys Tyr lle Asp Pro Thr Tyr Met Ile Arg Ala Val Pro Ser Asn Ala Ser Asp Asn Val Tyr Cys Thr Leu Leu Ala Gln Ser Cys Val His Gly Ala Met Ala Gly Tyr Thr Gly Phe Thr Ser Gly Leu Val Asn Gly Arg Gln Thr Tyr Ile Pro Phe Asn Arg Ile Thr Glu Lys Gln Asn Met Val Val lle Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr Asn Gln Pro Ser Phe Leu Arg Val Lys Asp Ile Glu Glu lle Lys Lys Glu Glu Gln Pro Gln Thr Gln Leu Leu Asp Gly Asp Asn Asn Val His Glu Asn Ser Gly His SEQUENCE ID. No.: 2 SEQUENCE LENGTH: 485 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Met Gly Thr Glu Ser Asn Tyr Gln Met Lys Val Val Lys Gly Asp Tyr Gly Tyr Val leu Glu Asp Val Pro His Leu Thr Asp Tyr Ile Pro Asp Leu Pro Thr Tyr Asp Asn Pro Leu Arg Ser Asn Pro Ala Tyr Ser Val Val Lys Gln Tyr Phe Val Asp Met Asp Asp Thr Val Pro Gln Lys Val Val Val His Lys Asp Ser Pro Arg Gly Val His Phe Arg Arg Ala Gly Pro Arg Gln Lys Val Tyr Phe Ser Ser Asp Asp Val Arg Ala Cys Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn Thr Val Ile Arg Glu Ile Val His Ser Leu Asp Tyr Met Tyr Gly Val Asn Lys Val Phe Gly Ile Asp Gly Gly Tyr Arg Gly Phe Tyr Ser Lys Asn Ile Ile Asn Leu Thr Pro Lys Thr Val Asn Asp Ile His Lys Arg Gly Gly Thr Ile Leu Gly Ser Ser Arg Gly Gly His Asp Thr Thr Lys Ile Val Asp Ser Ile Gln Asp Arg Glu Ile Asn Gln Val Tyr Ile Ile Gly Gly Asp Gly Thr Gln Lys Gly Ala Ala Val Ile Tyr Glu Glu Ile Arg Arg Arg Gly Leu Lys Val Ile Val Ala Gly Ile Pro Lys Thr Ile Asp Asn Asp Ile Pro Val Ile Asp Lys Ser Phe Gly Phe Asp Thr Ala Val Glu Glu Ala Gln Arg Ala Ile Asn Ala Ala His Val Glu Ala Glu Ser Ala Glu Asn Gly Ile Gly Val Val Lys Leu Met Gly Arg Tyr Ser Gly Phe Ile Ala Met Tyr Ala Thr Leu Ala Ser Arg Asp Val Asp Leu Cys Leu Ile Pro Glu Ser Pro Phe Tyr Leu Glu Gly Asp Gly Gly Leu Phe Glu Tyr Ile Glu Lys Arg Leu Lys Glu Asn Gly His Met Val Ile Val Ile Ala Glu Gly Ala Gly Gln Glu Leu Leu Ala Glu Glu Asn Ala His Ala Lys Asn Glu Gln Asp Ala Ser Gly Asn Lys Leu Leu Gln Asp Val Gly Leu Trp Ile Ser Gln Lys Ile Arg Asp His Phe Ala Thr Lys Thr Lys Met Pro Ile Thr Leu Lys Tyr Ile Asp Pro Thr Tyr Met Ile Arg Ala Val Pro Ser Asn Ala Ser Asp Asn Val Tyr Cys Thr Leu Leu Ala Gln Ser Cys Val His Gly Ala Met Ala Gly Tyr Thr Gly Phe Thr Ser Gly Leu Val Asn 214735~

Gly Arg Gln Thr Tyr Ile Pro Phe Asn Arg Ile Thr Glu Lys Gln Asn Met Val Val Ile Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr Asn Gln Pro Ser Phe Leu Arg Val Lys Asp Ile Glu Glu Ile Lys Lys Glu Glu Gln Pro Gln Thr Gln Leu Leu Asp Gly Asp Asn Asn Val His Glu Asn Ser Gly His SEQUENGE ID No. :3 SEQUENCE LENGTH : 1778 SEQUENCE TYPE : nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE :cDNA to mRNA
ORIGINAL SOURCE
ORGANISM : Flaveria brownii TISSUE TYPE: leaf IMMEDIATE SOURCE
LIBRARY : ~ ZAP II cDNA library originated from mRNA from green leaves CLONE : pPFK-FB1 SEQUENCE DESCRIPTION

Met Asp Asn Asn Ile Ser Cys Glu Met Lys Val 214735~

Glu Thr Gly Asp Ala Gly Tyr Val Leu Glu Asp Val Pro His Ile Thr Asp Tyr Ile Pro Asn Leu Pro Thr Tyr Pro Asn Pro Leu Arg Ser Asn Pro Ala Tyr Ser Val Val Lys Gln Tyr Phe Val Asp Ala Asp Asp Thr Val Pro Gln Lys Val Val Val His Lys Asp Gly Pro Arg Gly lle His Phe Arg Arg Ala Gly Pro Arg Gln Arg Val Tyr Phe Ala Pro Asp Glu Val His Ala Ala Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn Thr Val Ile Arg Glu Ile Val Cys Ala Leu Tyr His Met Tyr Gly Val Thr Lys Val Leu Gly Ile Asp Gly Gly Tyr Arg Gly Phe Tyr Ser Lys Asn Thr Ile Thr Leu Thr Pro Lys Val Val Asn Asp Ile His Lys Arg Gly Gly Thr Ile Ile Gly Thr $er Arg Gly Gly His Asp Lys Pro Lys 21~73SS

lle Val Asp Ser lle Gln Asp Arg Gly lle Asn Gln Val Tyr lle lle Gly Gly Asp Gly Thr Gln Lys Gly Ala Ala Val lle Tyr Gln Glu Val Arg Arg Arg Gly Leu Lys Ala Val Val Ala Gly lle Pro Lys Thr lle Asp Asn Asp lle Pro Val lle Asp Lys Ser Phe Gly Phe Asp Thr Ala Val Glu Glu Ala Gln Arg Ala lle Asn Ala Ala His Val Glu Ala Glu Ser Ala Glu Asn Gly lle Gly Val Val Lys Leu Met Gly Arg Tyr Ser Gly Phe lle Ala Met Tyr Ala Thr Leu Ala Ser Arg Asp Val Asp Leu Cys Leu lle Pro Glu Ser Pro Phe Tyr l,eu Glu Gly Glu Gly Gly Leu Leu Glu Tyr Val Glu Lys Arg Leu Lys Asp Asp Gly His Met Val lle Val Val Ala Glu Gly Ala Gly Gln Glu Leu Leu Ala Ala Glu Asn Leu 21~735S

Lys Thr Ser Thr Ala Lys Asp Ala Ser Gly Asn Lys Leu Leu His Asp Val Gly Leu Trp lle Ser Asp Lys Ile Lys Ala His Phe Ala Lys Ile Pro Pro Met Pro Ile Thr Leu Lys Tyr Ile Asp Pro Thr Tyr Met Ile Arg Ala Val Pro Ser Asn Ala Ser Asp Asn Val Tyr Cys Thr Leu Leu Ala Gln Ser Cys Val His Gly Val Met Ala Gly Tyr Thr Gly Phe Thr Ser Gly Leu Val Asn Gly Arg Gln Thr Tyr Ile Pro Phe Asn Arg Ile Thr Glu Lys Gln Asn Asn Val Val Ile Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr Asn Gln Pro Ser Phe Leu Arg Pro Gln Asp Val Ile Glu Val Gln Lys Gln Glu Glu Pro Pro Ser Gln Leu Leu Asp Gly ` ~ 21g7355 Asp Ser Ser Lys Pro Asn Asp Ile SEQUENCE ID. No.: 4 SEQUENCE LENGTH : 484 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Met Asp Asn Asn Ile Ser Cys Glu Met Lys Val Glu Thr Gly Asp Ala Gly Tyr Val Leu Glu Asp Val Pro His Ile Thr Asp Tyr Ile Pro Asn Leu Pro Thr Tyr Pro Asn Pro Leu Arg Ser Asn Pro Ala Tyr Ser Val Val Lys Gln Tyr Phe Val Asp Ala Asp Asp Thr Val Pro Gln Lys Val Val Val His Lys Asp Gly Pro Arg Gly Ile His Phe Arg Arg Ala Gly Pro Arg Gln Arg Val Tyr Phe Ala Pro Asp Glu Val His Ala Ala Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn Thr Val Ile Arg Glu Ile Val Cys Ala Leu Tyr His Met Tyr Gly Val Thr Lys Val Leu Gly 115 120 ` 125 Ile Asp Gly Gly Tyr Arg Gly Phe Tyr Ser Lys Asn Thr Ile Thr Leu -Thr Pro Lys Val Val Asn Asp Ile His Lys Arg Gly Gly Thr Ile Ile Gly Thr Ser Arg Gly Gly His Asp Lys Pro Lys Ile Val Asp Ser Ile Gln Asp Arg Gly Ile Asn Gln Val Tyr lle Ile Gly Gly Asp Gly Thr Gln Lys Gly Ala Ala Val Ile Tyr Gln Glu Val Arg Arg Arg Gly Leu Lys Ala Val Val Ala Gly Ile Pro Lys Thr Ile Asp Asn Asp Ile Pro Val Ile Asp Lys Ser Phe Gly Phe Asp Thr Ala Val Glu Glu Ala Gln Arg Ala Ile Asn Ala Ala His Val Glu Ala Glu Ser Ala Glu Asn Gly Ile Gly Val Val Lys Leu Met Gly Arg Tyr Ser Gly Phe Ile Ala Met Tyr Ala Thr Leu Ala Ser Arg Asp Val Asp Leu Cys Leu Ile Pro Glu Ser Pro Phe Tyr Leu Glu Gly Glu Gly Gly Leu Leu Glu Tyr Val Glu Lys Arg Leu Lys Asp Asp Gly His Met Val Ile Val Val Ala Glu Gly Ala Gly Gln Glu Leu Leu Ala Ala Glu Asn Leu Lys Thr Ser Thr Ala Lys Asp Ala Ser Gly Asn Lys Leu Leu His Asp Val Gly Leu Trp Ile Ser Asp Lys Ile Lys Ala His Phe Ala Lys Ile Pro Pro Met Pro Ile -Thr Leu Lys Tyr Ile Asp Pro Thr Tyr Met lle Arg Ala Val Pro Ser Asn Ala Ser Asp Asn Val Tyr Cys Thr Leu Leu Ala Gln Ser Cys Val His Gly Val Met Ala Gly Tyr Thr Gly Phe Thr Ser Gly Leu Val Asn Gly Arg Gln Thr Tyr lle Pro Phe Asn Arg lle Thr Glu Lys Gln Asn Asn Val Val Ile Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr Asn Gln Pro Ser Phe Leu Arg Pro Gln Asp Val Ile Glu Val Gln Lys Gln Glu Glu Pro Pro Ser Gln Leu Leu Asp Gly Asp Ser Ser Lys Pro Asn Asp lle 21~7355 SEQUENCE ID No.: 5 SEQUENCE LENGTH: 1623 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
ORIGINAL SOURCE
ORGANISM: Oryza sativa L.
VARIETY: Tsukinohikari TISSUE TYPE: callus originated from immature embryo IMMEDIATE SOURCE
LIBRARY: ~ ZAP II cDNA library originated from mRNA from callus CLONE: pPFK-OSl SEQUENCE DESCRIPTION

Val Val Ala His Met Arg His Val Leu Asp Leu Pro Thr Tyr Ser Asn Pro Leu Gln Asp Asn Pro Ala Tyr Ser Val Val Lys Gln Tyr Phe Val Asn Pro Asp Asp Thr Val Cys Gln Lys Ala Ile Val His Lys Asp Gly Pro Arg Gly Asn His Phe Arg Arg Ala Gly Pro Arg Gln Arg Val Phe Phe Glu Ser Asp Glu Val His Ala Cys Ile Val Thr Cys Gly Gly Leu -Cys Pro Gly Leu Asn Thr Val Ile Arg Glu Ile Val Cys Gly Leu Asn Asp Met Tyr Gly Val Ser Arg Val Leu Gly Ile Gln Gly Gly Tyr Arg Gly Phe Tyr Ala Cys Asn Thr Ile Asp Leu Ser Pro Lys Ser Val Asn Asp lle His Lys Arg Gly Gly Thr Val Leu Gly Thr Ser Arg Gly Gly His Asp Thr Met Lys lle Val Asp Ser lle Gln Asp Arg Gly Ile Asn Gln Val Tyr Val Ile Gly Gly Asp Gly Thr Gln Arg Gly Ala Gly Val lle Phe Glu Glu lle Arg Arg Arg Gly Leu Lys Val Ala Val Ala Gly Ile Pro Lys Thr lle Asp Asn Asp lle Pro Val lle Asp Arg Ser Phe Gly Phe Asp Thr Ala Val Glu Glu Ala Gln Arg Ala Ile Asn Ala Ala His Val Glu Ala Gly Ser Ala Glu Asn Gly Ile Gly Leu Val Lys Leu Met Gly Arg His Ser Gly Phe Ile Ala His Tyr Ala Thr Leu Ala Ser Arg Asp Val Asp Cys Cys Leu Ile Pro Glu Ser Pro Phe Tyr Leu Glu Gly Glu Gly Gly Leu Phe Arg Tyr Leu Glu Lys Arg Leu Lys Glu Asn Gly His Met Val Ile Val Val Ala Glu Gly Ala Gly Gln Lys Leu Ile Asn Glu Thr Lys Glu Ser Met Gly Lys Asp Ala Ser Gly Asn Ser Ile Leu Leu Asp Val Gly Leu Trp Leu Ser Gln Lys Ile Lys Glu His Phe Lys Lys Ile Lys Thr Thr Ile Asn Leu Lys Tyr Ile Asp Pro Thr Tyr AT& ATA CGT GCC ATT CCT AGC AAT GCA TCT GAC AAT GTG TAT TGC ACA 1106 Met Ile Arg Ala Ile Pro Ser Asn Ala Ser Asp Asn Val Tyr Cys Thr ~ 355 360 365 Leu Leu Ala His Arg Val Val His Gly Ala Met Ala Gly Tyr Thr GLy 21~735S

Phe Thr Val Gly Gln Val Asn Gly Arg His Cys Tyr Ile Pro Phe Tyr Arg Ile Thr Glu 1ys Gln Asn Lys Val Ser Ile Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr Asn Gln Pro Ser Phe Leu Ser Lys Lys Asp Val Glu Asp Ala Lys Met Glu Glu Glu Arg Ala Ser Lys Phe Phe Asp Gly Pro Pro Pro Asn Pro Lys Val Glu Asp Lys Val Ala Ser Asn Gly Lys Ala Val Lys SEQUENCE ID. No.: 6 SEQUENCE LENGTH; 479 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Val Val Ala His Met Arg His Val Leu Asp Leu Pro Thr Tyr Ser Asn Pro Leu Gln Asp Asn Pro Ala Tyr Ser Val Val Lys Gln Tyr Phe Val Asn Pro Asp Asp Thr Val Cys Gln Lys Ala lle Val His Lys Asp Gly Pro Arg Gly Asn His Phe Arg Arg Ala Gly Pro Arg Gln Arg Val Phe Phe Glu Ser Asp Glu Val His Ala Cys lle Val Thr Cys Gly Gly Leu 80ys Pro Gly Leu Asn Thr Val Ile Arg Glu Ile Val Cys Gly Leu Asn 95sp Met Tyr Gly Val Ser Arg Val Leu Gly lle Gln Gly Gly Tyr Arg Gly Phe Tyr Ala Cys Asn Thr Ile Asp Leu Ser Pro Lys Ser Val Asn Asp Ile His Lys Arg Gly Gly Thr Val Leu Gly Thr Ser Arg Gly Gly His Asp Thr Met Lys Ile Val Asp Ser Ile Gln Asp Arg Gly Ile Asn 145 150 155 160ln Val Tyr Val Ile Gly Gly Asp Gly Thr Gln Arg Gly Ala Gly Val 165 170 175le Phe Glu Glu Ile Arg Arg Arg Gly Leu Lys Val Ala Val Ala Gly 180 ~ 185 190 Ile Pro Lys Thr Ile Asp Asn Asp Ile Pro Val Ile Asp Arg Ser Phe Gly Phe Asp Thr Ala Yal Glu Glu Ala Gln Arg Ala Ile Asn Ala Ala His Val Glu Ala Gly Ser Ala Glu Asn Gly Ile Gly Leu Val Lys Leu 225 230 235 240et Gly Arg His Ser Gly Phe Ile Ala His Tyr Ala Thr Leu Ala Ser 245 250 255rg Asp Val Asp Cys Cys Leu Ile Pro Glu Ser Pro Phe Tyr Leu Glu 21~7355 Gly Glu Gly Gly Leu Phe Arg Tyr Leu Glu Lys Arg Leu Lys Glu Asn Gly His Met Val Ile Val Val Ala Glu Gly Ala Gly Gln Lys Leu Ile Asn Glu Thr Lys Glu Ser Met Gly Lys Asp Ala Ser Gly Asn Ser Ile Leu Leu Asp Val Gly Leu Trp Leu Ser Gln Lys Ile Lys Glu His Phe Lys Lys Ile Lys Thr Thr Ile Asn Leu Lys Tyr Ile Asp Pro Thr Tyr Met lle Arg Ala Ile Pro Ser Asn Ala Ser Asp Asn Val Tyr Cys Thr Leu Leu Ala His Arg Val Val His Gly Ala Met Ala Gly Tyr Thr GLy Phe Thr Val Gly Gln Val Asn Gly Arg His Cys Tyr Ile Pro Phe Tyr Arg Ile Thr Glu Lys Gln Asn Lys Val Ser Ile Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr Asn Gln Pro Ser Phe Leu Ser Lys Lys Asp Val Glu Asp Ala Lys Met Glu Glu Glu Arg Ala Ser Lys Phe Phe Asp Gly Pro Pro Pro Asn Pro Lys Val Glu Asp Lys Val Ala Ser Asn Gly Lys Ala Val Lys SEQUENCE ID No.: 7 SEQUENCE LENGTH: 2048 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
ORIGINAL SOURCE
ORGANISM: Zea mays L.
TISSUE TYPE: callus originated from endosperm IMMEDIATE SOURCE
LIBRARY: ~ gtlO cDNA library originated from mRNA from callus CLONE: pPFK-ZMl SEQUENCE DESCRIPTION

Met Ala Val Ala Phe Lys Ala Ser Thr Ser Ser Val Thr Gln Gln His Trp Ser Ser Pro Thr Lys Asp Gln Cys Gln Tyr Gly Phe Thr His Leu Ser Arg Gln Lys Cys Arg Lys Arg Ala Leu Cys Val Thr Ala Ile Ser Gly Lys Leu Asp Leu Asp Phe Thr Asp Pro Ser Trp Asn Gln Lys Tyr Gln Glu Asp Trp Asn Arg Arg Phe Ser Leu Pro His Ile Asn Asp Ile 214735~

Tyr Asp ~eu Glu Pro Arg Arg Thr Thr Phe Ser Leu Lys Lys Asn Arg Ile Pro Leu Gly Asp Gly Asp Gly Ser Ser Thr Asp Met Trp Asn Gly Tyr Val Asn Lys Asn Asp Arg Ala Leu Leu Lys Val Ile Lys Tyr Ala Ser Pro Thr Ser Ala Gly Ala Glu Cys Ile Asp Pro Asp Cys Ser Trp Val Glu His Trp Val His Arg Ala Gly Pro Arg Lys Glu Ile Tyr Tys Glu Pro Glu Glu Val Lys Ala Ala Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn Asp Val Ile Arg Gln Ile Val Phe Thr Leu Glu Thr Tyr Gly ~al Lys Asn Ile Val Gly Ile Pro Phe Gly Tyr Arg Gly Phe Phe Glu Lys Gly Leu Lys Glu Met Pro Leu Ser Arg Asp Val Val Glu Asn Ile Asn Leu Ser Gly Gly Ser Phe Leu Gly Val Ser Arg Gly Gly ~ GCT AAA ACT AGT GAG ATT GTA GAT AGC ATA CAA GCC AGA AGA ATT GAC 888 Ala Lys Thr Ser Glu Ile Val Asp Ser Ile Gln Ala Arg Arg Ile Asp Met Leu Phe Val Ile Gly Gly Asn Gly Ser His Ala Gly Ala Asn Ala Ile His Glu Glu Cys Arg Lys Arg Lys Leu Lys Val Ser Val Val Ala Val Pro Lys Thr Ile Asp Asn Asp Ile Leu Phe Met Asp Lys Thr Phe Gly Phe Asp Thr Ala Val Glu Lys Ala Gln Arg Ala Ile Asn Ser Ala Tyr Ile Glu Ala Arg Ser Ala Tyr His Gly Ile Gly Leu Val Lys Leu Met Gly Arg Ser Ser Gly Phe Ile Ala Met His Ala Ser Leu Ser Ser Gly Gln Ile Asp Val Cys Leu Ile Pro Glu Val Ser Phe Thr Leu Asp Gly Glu His Gly Val Leu Arg His Leu Glu His Leu Leu Asn Thr Lys ` _ 2117355 Gly Phe Cys Val Val Cys Val Ala Glu Gly Ala Gly Gln Asp Leu Leu Gln Lys Ser Asn Ala Thr Asp Ala Ser Gly Asn Val Ile Leu Ser Asp Phe Gly Val His Met Gln Gln Lys Ile Lys Lys His Phe Lys Asp Ile Gly Val Pro Ala Asp Leu Lys Tyr Ile Asp Pro Thr Tyr Met Val Arg ~ 435 440 445 Ala Cys Arg Ala Asn Ala Ser Asp Ala lle Leu Cys Thr Val Leu Gly Gln Asn Ala Val His Gly Ala Phe Ala Gly Phe Ser Gly Ile Thr Ser Gly Val Cys Asn Thr His Tyr Val Tyr Leu Pro Ile Thr Glu Val Ile Thr Thr Pro Lys His Val Asn Pro Asn Ser Arg Met Trp His Arg Cys Leu Thr Ser Thr Gly Gln Pro Asp Phe His 21~7355 `

_ SEQUENCE ID. No.: 8 SEQUENCE LENGTH: 522 MOLECULE TYPE: an~ino acid SEQUENCE DESCRIPTION
Met Ala Val Ala Phe Lys Ala Ser Thr Ser Ser Val Thr Gln Gln His Trp Ser Ser Pro Thr Lys Asp Gln Cys Gln Tyr Gly Phe Thr His Leu Ser Arg Gln Lys Cys Arg Lys Arg Ala Leu Cys Val Thr Ala Ile Ser : 35 40 45 Gly Lys Leu Asp Leu Asp Phe Thr Asp Pro Ser Trp Asn Gln Lys Tyr Gln Glu Asp Trp Asn Arg Arg Phe Ser Leu Pro His Ile Asn Asp Ile Tyr Asp Leu Glu Pro Arg Arg Thr Thr Phe Ser Leu Lys Lys Asn Arg Ile Pro Leu Gly Asp Gly Asp Gly Ser Ser Thr Asp Met Trp Asn Gly Tyr Val Asn Lys Asn Asp Arg Ala Leu Leu Lys Val Ile Lys Tyr Ala Ser Pro Thr Ser Ala Gly Ala Glu Cys Ile Asp Pro Asp Cys Sçr Trp Val Glu His Trp Val His Arg Ala Gly Pro Arg Lys Glu Ile Tyr Tys Glu Pro Glu Glu Val Lys Ala Ala Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn Asp Val Ile Arg Gln Ile Val Phe Thr Leu Glu Thr Tyr Gly Val Lys Asn Ile Val Gly Ile Pro Phe Gly Tyr Arg Gly Phe Phe Glu Lys Gly Leu Lys Glu Met Pro Leu Ser Arg Asp Val Val Glu Asn Ile Asn Leu Ser Gly Gly Ser Phe Leu Gly Val Ser Arg Gly Gly Ala Lys Thr Ser Glu Ile Val Asp Ser Ile Gln Ala Arg Arg Ile Asp Met Leu Phe Val Ile Gly Gly Asn Gly Ser His Ala Gly Ala Asn Ala Ile His Glu Glu Cys Arg Lys Arg Lys Leu Lys Val Ser Val Val Ala Val Pro Lys Thr Ile Asp Asn Asp Ile Leu Phe Met Asp Lys Thr Phe Gly Phe Asp Thr Ala Val Glu Lys Ala Gln Arg Ala Ile Asn Ser Ala Tyr Ile Glu Ala Arg Ser Ala Tyr His Gly Ile Gly Leu Val Lys Leu Met Gly Arg Ser Ser Gly Phe Ile Ala Met His Ala Ser Leu Ser Ser Gly Gln Ile Asp Val Cys Leu Ile Pro Glu Val Ser Phe Thr Leu Asp Gly Glu His Gly Val Leu Arg His Leu Glu His Leu Leu Asn Thr Lys Gly Phe Cys Val Val Cys Val Ala Glu Gly Ala Gly Gln Asp Leu Leu ln Lys Ser Asn Ala Thr Asp Ala Ser Gly Asn Val Ile Leu Ser Asp 405 410 415he Gly Val His Met Gln Gln Lys Ile Lys Lys His Phe Lys Asp Ile Gly Val Pro Ala Asp Leu Lys Tyr Ile Asp Pro Thr Tyr Met Val Arg Ala Cys Arg Ala Asn Ala Ser Asp Ala Ile Leu Cys Thr Val Leu Gly Gln Asn Ala Val His Gly Ala Phe Ala Gly Phe Ser Gly Ile Thr Ser 465 470 475 480ly Val Cys Asn Thr His Tyr Val Tyr Leu Pro Ile Thr Glu Val Ile 485 490 495hr Thr Pro Lys His Val Asn Pro Asn Ser Arg Met Trp His Arg Cys 500 505 510eu Thr Ser Thr Gly Gln Pro Asp Phe His SEQUENCE ID No.: 9 SEQUENCE LENGTH: 1558 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
ORIGINAL SOURCE
ORGANISM: Raphanus sativus L.
TISSUE TYPE: leaf IMMEDIATE SOURCE
LIBRARY: ~ ZAP II cDNA library originated from mRNA from green leaf CLONE: pPFK-RSl SEQUENCE DESCRIPTION

Ser Val Val Lys Gln Tyr Phe Val Asp Glu Asp Asp Thr Val Pro Gln Lys Ile Val Val His Pro Asp Ser Pro Arg Gly Thr His Phe Arg Arg Ala Gly Pro Arg Gln Arg Val Tyr Phe Asp Ser Asp Asp Val Val Ala Cys Ile Val Thr Cys Gly Gly Leu Cyc Pro Gly Leu Asn Thr Val Ile Arg Glu Ile Val Cys Gly Leu Ser Tyr Met Tyr Gly Val Lys Lys Ile Leu Gly Ile Glu Gly Gly Tyr Arg Gly Phe Tyr Ala Arg Asn Thr Ile Asp Leu Asp Leu Lys Thr Val Asn Asp Ile His Lys Arg Gly Gly Thr Ile Leu Gly Thr Ser Arg Gly Gly His Asp Thr Thr Lys Ile Val Asp Ser Ile Gln Asp Arg Gly Ile Asn Gln Val Tyr Ile Ile Gly Gly Asp Gly Ser Gln Lys Gly Ala Ala Val Ile Phe Glu Glu Ile Arg Arg Arg Gly Leu Lys Val Ala Val Ala Gly Ile Pro Lys Thr Ile Asp Asn Asp Ile Pro Ile Ile Asp Arg Ser Phe Gly Phe Asp Thr Ala Val Glu Glu Ala Gln Arg Ala Ile Asn Ala Ala His Val Glu Ala Thr Ser Phe Glu Asn Gly Ile Gly Leu Val Lys Leu Met Gly Arg Tyr Ser Gly Phe Ile Ala Met Tyr Ala Thr Leu Ala Ser Arg Asp Val Asp Cys Cys Leu Ile Pro Glu Ser Pro Phe Phe Leu Glu Gly Lys Gly Gly Leu Phe Glu Phe ` 240 245 250 Ile Gly Lys Arg Leu Lys Glu Ile Gly His Met Val Ile Val Ile Ala Glu Gly Ala Gly Gln Asp Leu Leu Ala Glu Ser Asn Glu Gln Ser Thr Thr Leu Lys Asp Ala Ser Gly Asn Lys Leu Leu Gln Asp Val Gly Leu Trp Ile Ser Gln Arg Ile Lys Asp His Phe Ala Lys Lys Met Thr Leu Asn Leu Lys Tyr Ile Asp Pro Thr Tyr Met Ile Arg Ala Val Pro Ser Asn Ala Ser Asp Asn Val Cys Cys Thr Leu Leu Ala Gln Ser Ala Val His Gly Val Met Ala Gly Tyr Asn Gly Phe Thr Val Gly Leu Val Asn Gly Arg His Thr Tyr Ile Pro Phe Tyr Arg Ile Thr Glu Lys Gln Asn Lys Val Val Ile Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr 21~7355 Asn Gln Pro Ser Phe Met Lys His Asp Asp His His Glu Pro Asn His Ser Gly Gly Glu Ala Gly Ala Met Asn Trp SEQUENCE ID. No.: 10 SEQUENCE LENGTH: 421 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Ser Val Val Lys Gln Tyr Phe Val Asp Glu Asp Asp Thr Val Pro Gln Lys Ile Val Val His Pro Asp Ser Pro Arg Gly Thr His Phe Arg Arg Ala Gly Pro Arg Gln Arg Val Tyr Phe Asp Ser Asp Asp Val Val Ala Cys lle Val Thr Cys Gly Gly Leu Cyc Pro Gly Leu Asn Thr Val Ile Arg Glu lle Val Cys Gly Leu Ser Tyr Met Tyr Gly Val Lys Lys Ile Leu Gly lle Glu Gly Gly Tyr Arg Gly Phe Tyr Ala Arg Asn Thr lle Asp Leu Asp Leu Lys Thr Val Asn Asp lle His Lys Arg Gly Gly Thr Ile Leu Gly Thr Ser Arg Gly Gly His Asp Thr Thr Lys lle Val Asp Ser Ile Gln Asp Arg Gly Ile Asn Gln Val Tyr Ile Ile Gly Gly Asp Gly Ser Gln Lys Gly Ala Ala Val Ile Phe Glu Glu Ile Arg Arg Arg Gly Leu Lys Val Ala Val Ala Gly Ile Pro Lys Thr Ile Asp Asn Asp Ile Pro Ile Ile Asp Arg Ser Phe Gly Phe Asp Thr Ala Val Glu Glu Ala Gln Arg Ala Ile Asn Ala Ala His Val Glu Ala Thr Ser Phe Glu Asn Gly Ile Gly Leu Val Lys Leu Met Gly Arg Tyr Ser Gly Phe Ile Ala Met Tyr Ala Thr Leu Ala Ser Arg Asp Val Asp Cys Cys Leu Ile Pro Glu Ser Pro Phe Phe Leu Glu Gly Lys Gly Gly Leu Phe Glu Phe Ile Gly Lys Arg Leu Lys Glu Ile Gly His Met Val Ile Val Ile Ala Glu Gly Ala Gly Gln Asp Leu Leu Ala Glu Ser Asn Glu Gln Ser Thr Thr Leu Lys Asp Ala Ser Gly Asn Lys Leu Leu Gln Asp Val Gly Leu Trp Ile Ser Gln Arg Ile Lys Asp His Phe Ala Lys Lys Met Thr Leu Asn Leu Lys Tyr Ile Asp Pro Thr Tyr Met Ile Arg Ala Val Pro Ser 21g7355 ~ Asn Ala Ser Asp Asn Val Cys Cys Thr Leu Leu Ala Gln Ser Ala Val His Gly Val Met Ala Gly Tyr Asn Gly Phe Thr Val Gly Leu Val Asn Gly Arg His Thr Tyr lle Pro Phe Tyr Arg Ile Thr Glu Lys Gln Asn Lys Val Val lle Thr Asp Arg Met Trp Ala Arg Leu Leu Ser Ser Thr Asn Gln Pro Ser Phe Met Lys His Asp Asp His His Glu Pro Asn His Ser Gly Gly Glu Ala Gly Ala Met Asn Trp SEQUENCE ID. No.: 11 SEQUENCE LENGTH: 5 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Arg Ala Gly Pro Arg SEQUENCE ID. No.: 12 SEQUENCE LENGTH: 11 MOLECULE TYPE: a~ino acid SEQUENCE DESCRIPTION
Ile Val Thr Cys Gly Gly Leu Cys Pro Gly Leu Asn SEQUENCE ID. No.: 13 SEQUENCE LENGTH: 6 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Gly Tyr Arg Gly Phe Tyr SEQUENCE ID. No.: 14 SEQUENCE LENGTH: 6 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Ile Val Asp Ser Ile Gln SEQUENCE ID. No.: 15 SEQUENCE LENGTH: 8 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Pro Lys Thr Ile Asp Asn Asp Ile SEQUENCE ID. No.: 16 SEQUENCE LENGTH: 8 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Phe Gly Phe Asp Thr Ala Val Glu SEQUENCE ID. No.: 17 SEQUENCE LENGTH: 6 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Ala Gln Arg Ala lle Asn SEQUENCE ID. No.: 18 SEQUENCE LENGTH: 6 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Val Lys Leu Met Gly Arg SEQUENCE ID. No.: 19 SEQUENCE LENGTH: 5 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Ser Gly Phe lle Ala SEQUENCE ID. No.: 20 SEQUENCE LENGTH: 6 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Ala Glu Gly Ala Gly Gln SEQUENCE ID. No.: 21 SEQUENCE LENGTH: 5 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Asp Ala Ser Gly Asn SEQUENCE ID. No.: 22 SEQUENCE LENGTH: 9 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Leu Lys Tyr lle Asp Pro Thr Tyr Met SEQUENCE ID. No.: 23 SEQUENCE LENGTH: 5 MOLECULE TYPE: amino acid SEQUENCE DESCRIPTION
Cys Leu lle Pro Glu

Claims

1. A DNA encoding ATP dependent fructose 6-phosphate 1-phosphotransferase originating from a plant.

2. The DNA according to claim 1, that encodes ATP
dependent fructose 6-phosphate 1-phosphotransferase originating from a plant, Q10 value at 5°C of which is not more than 2.4.

3. The DNA according to claim 1, which encodes the amino acid sequence identified as Sequence ID No. 2 in the Sequence Listing.

4. The DNA according to claim 3, which has the DNA
sequence identified as Sequence ID No. 1 in the Sequence Listing.

5. The DNA according to claim 1, which encodes the amino acid sequence identified as Sequence ID No. 4 in the Sequence Listing.

6. The DNA according to claim 5, which has the DNA
sequence identified as Sequence ID No. 3 in the Sequence Listing.

7. The DNA according to claim 1, which encodes the amino acid sequence identified as Sequence ID No. 6 in the Sequence Listing.

8. The DNA according to claim 7, which has the DNA
sequence identified as Sequence ID No. 5 in the Sequence Listing.

9. The DNA according to claim 1, which encodes the amino acid sequence identified as Sequence ID No. 8 in the Sequence Listing.

10. The DNA according to claim 9, which has the DNA
sequence identified as Sequence ID No. 7 in the Sequence Listing.

11. The DNA according to claim 1, which encodes the amino acid sequence identified as Sequence ID No. 10 in the Sequence Listing.

12. The DNA according to claim 11, which has the DNA
sequence identified as Sequence ID No. 9 in the Sequence Listing.

13. A DNA encoding the amino acid sequence identified as Sequence ID No. 11 in the Sequence Listing.

14. A DNA encoding the amino acid sequence identified as Sequence ID No. 14 in the Sequence Listing.

15. A DNA encoding the amino acid sequence identified as Sequence ID No. 21 in the Sequence Listing.

16. A DNA encoding the amino acid sequence identified as Sequence ID No. 22 in the Sequence Listing.

17. A method for detecting a plant PFK gene by hybridizing any one of the DNA encoding the amino acid sequence identified as Sequence ID No. 11 - 22 or a part thereof, or a DNA containing said DNA encoding the amino acid sequence identified as Sequence ID No. 11 - 22 with a sample DNA.

18. A method for amplifying a plant PFK gene comprising amplifying said gene by PCR by using any one of the DNA
encoding the amino acid sequence identified as Sequence ID No. 11 - 22 or a part thereof, or a DNA containing said DNA encoding the amino acid sequence identified as Sequence ID No. 11 - 22 as a primer for the PCR.

19. A recombinant vector comprising the DNA according to any one of claims 1 to 12, which can express ATP
dependent fructose 6-phosphate 1-phosphotransferase originating from a plant in a host cell.

20. A method for changing sugar content in plant cells under low temperature, which comprises transforming said plant with said recombinant vector according to claim 19.

21. The method according to claim 20, wherein said plant is potato.