Low temperature expression cDNAs encoding fructan synthesizing enzymes and method of isolating the same

The present invention relates to low temperature expression cDNAs encoding fructan synthesizing enzymes and a method of isolating the same. In detail, this invention relates to cDNAs encoding enzymes effective for synthesizing a fructan which is useful for improving cold resistance of plants and can serve as a fructo-oligosaccharide useful for improving human's health. In particular, this invention relates to the provision of a novel sucrose:fructan 6-fructosyltransferase cDNA isolated from a winter wheat (PI173438) having high freezing tolerance and a novel invertase cDNA isolated from the same winter wheat (PI173438).

It has been known that wintering crops such as wheats and forage grasses will acquire a freezing tolerance induced during a cold acclimation from autumn to winter. Further, it has been found that these wintering crops have fructan synthesizing enzymes which will express under a low temperature during cold acclimation, and as a result, polysaccharide fructan will be synthesized and accumulated as an energy source within vacuoles of these crops. Since wintering crops are often planted in severely cold areas, it is desired that the fructan be accumulated as large amount as possible.

Moreover, it has been found that the fructan can serve not only as an energy source for a plant to live through a severe winter, but also can serve to prevent a plant from freezing and to adjust the osmotic pressure of a plant so as to prevent it from desiccation.

Recently, some new wheat varieties have been developed in some northern countries, but it has been proved difficult to obtain a new wheat variety having more improved capabilities than those of existing ones, if we only use a conventional hybridization technique. For this reason, there has been a demand that research and development of a new wheat variety be conducted by means of gene engineering.

On the other hand, fructan can serve as a fructo-oligosaccharide which has been proved useful for preventing a carious tooth and for increasing intestinal bifid bacteria (useful for improving human health). In fact, a cDNA encoding fructan synthesizing enzyme (it may also be referred to as fructan synthesizing cDNA) is useful for producing the fructo-oligosaccharide at an industrial level.

In detail, enzymes capable of synthesizing fructan may be classified into several species and can have several isozymes in view of different reaction stages and different combined states. In more detail, sucrose:fructan 6-fructosyltransferase is mainly useful for synthesizing a trisaccharide or a tetraccharide using sucrose as starting substance and for extending a degree of fructose polymer using fructan. Invertase can usually serve as an enzyme for decomposing the sucrose (disaccharide). Further, invertase of plants possessing a fructan accumulation ability (such plants include wheat and forage grasses) may be used to synthesize a trisaccharide in the presence of a high concentration sucrose.

It is reported that the sucrose:fructan 6-fructosyltransferase cDNA may be isolated from barley, while the invertase cDNA may be isolated from several other kinds of plants. However, since these cDNAs are all isolated under a condition suitable for cDNAs to express at the room temperature, it is not sure whether they can express at a low temperature and hence they may not have a function as an enzyme under a low temperature environment. As a result, the conventional fructan 6-fructosyltransferase cDNA and the conventional invertase cDNA can not be used to improve the cold resistance of a plant.

It is an object of the present invention to provide cDNAs encoding fructan synthesizing enzymes capable of expressing under a low temperature environment, which cDNAs are isolated from a winter wheat (PI173438) having a high freezing tolerance.

It is another object of the present invention to provide cDNAs encoding fructan synthesizing enzymes useful for industrially producing fructo-oligosaccharides which are useful for preventing a carious tooth and for increasing intestinal bifid bacteria.

It is a further object of the present invention to provide a method for isolating the above cDNAs capable of expressing in a low temperature environment.

According to one aspect of the present invention, there is provided a wheat-derived sucrose:fructan 6-fructosyltransferase cDNA, characterized in that said cDNA has a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.1 in FIG. 1 . In detail, said cDNA comprises 1848 nucleotides/616 amino acids and has 93% identity (on amino acid sequence level) with barley-derived sucrose:fructan 6-fructosyltransferase cDNA. In more detail, said cDNA encodes a fructan synthesizing enzyme in a low temperature environment and can confer a cold resistance to a plant upon being introduced into the plant. In further detail, said cDNA encodes an enzyme having a function of synthesizing a fructo-oligosaccharide useful for improving human's health.

According to one aspect of the present invention, there is provided a wheat-derived invertase cDNA, characterized in that said cDNA has a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.2 in FIG. 2 . In detail, said cDNA comprises 1986 nucleotides/662 amino acids and has 55% identity (on amino acid sequence level) with sugarcane-derived invertase cDNA. In more detail, said cDNA encodes a fructan synthesizing enzyme in a low temperature environment and can confer a cold resistance to a plant upon being introduced into the plant. In further detail, said cDNA encodes an enzyme having a function of synthesizing a fructo-oligosaccharide useful for improving human's health.

According to a further aspect of the present invention, there is provided a method of isolating a wheat-derived sucrose:fructan 6-fructosyltransferase cDNA having a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.1 in FIG. 1 and a wheat-derived invertase cDNA having a nucleotide sequence corresponding to an amino acid sequence listed as SEQ.ID. No.2 in FIG. 2, said method comprising the steps of: extracting mRNA from winter wheat variety PI173438 (having a high freezing tolerance) that has undergone a sufficient cold acclimation; preparing cDNA and a cDNA library based on said mRNA; analyzing nucleotide sequences of a number of plant-derived sucrose:fructan 6-fructosyltransferase cDNAs and plant-derived invertase cDNAs which have all been published by EMBL/Genebank/DDBJDNA Databank; designing a pair of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers with reference to highly conserved nucleotide sequence portions of the sucrose:fructan 6-fructosyltransferase cDNAs and the invertase cDNAs; conducting PCR (polymerase chain reaction) using the pair of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers and using said cDNA as a template, thereby amplifying fragments of sucrose:fructan 6-fructosyltransferase cDNA and invertase cDNA and obtaining amplified DNA fragments; and using said amplified DNA fragments as probes for screening said cDNA library by a hybridization assay, to isolate recombinant plaques containing full length of cDNA.

In the method of the present invention, one of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers has the following nucleotide sequence:

(Forward): 5′ A-T-G-A-A-T/C-G-A-T/C-C-C-N-A-A-T/C-G-G (SEQ.ID. No.3)

and the other has the following nucleotide sequence:

(Reverse): 5′ C-C-N-G-T-N-G-C-A/G-T-T-A/G-T-T-A-/G-A-A (SEQ.ID. No.4).

The above objects and features of the present invention will become better understood from the following description with reference to the accompanying drawings.

FIG. 1 shows an amino acid sequence of SEQ.ID No.1.

FIG. 2 shows an amino acid sequence of SEQ.ID No.2.

The cDNAs of the present invention are those encoding fructan synthesizing enzymes capable of expressing at low temperature environment.

The isolation method for isolating the above cDNAs of the present invention may be carried out in the following manner.

Specifically, mRNA is extracted from winter wheat PI173438 (having a high freezing tolerance) that has undergone a cold acclimation under natural conditions in Sapporo City Japan until November 22. This mRNA is then used to prepare cDNA and a cDNA library.

Next, nucleotide sequences of a number of plant-derived sucrose:fructan 6-fructosyltransferase cDNAs and invertase cDNAs which have all been published by EMBL/Genebank/DDBJDNA Databank are closely analyzed, and a pair of cDNA-specific degenerated primers (capable of simultaneously amplifying both the sucrose:fructan 6-fructosyltransferase cDNA and invertase cDNA) are designed with reference to highly conserved nucleotide sequence portions.

The pair of designed (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers are used in a PCR (polymerase chain reaction) using the above-mentioned cDNA as the template for amplifying the expected sucrose:fructan 6-fructosyltransferase cDNA fragments and invertase cDNA fragments (both are approximately 1500 bp), and the amplified fragments are isolated.

The amplified fragments are used as probes for screening the cDNA library by a hybridization assay, to isolate recombinant plaques containing full length of cDNA. The nucleotide sequences of the isolated plagues were analyzed and demonstrated to be two different isolated cDNAs which are sucrose:fructan 6-fructosyltransferase cDNA fragments and invertase cDNA fragments, all are novel in plants.

An example of the method for isolating the cDNAs of the present invention was carried out in the following steps 1)-3).

1) Preparation of cDNA and cDNA Library from Freezing Tolerance Winter Wheat Variety PI173438

mRNA was extracted by a common method from the crown portion of winter wheat ( Triticum astivum L.) PI173438 (having high freezing tolerance) that had been seeded in a container in late September and had then undergone a cold acclimation under natural conditions until November 22. A portion (5 μg) of the obtained mRNA was used to synthesize cDNA utilizing a cDNA Synthesis Kit (STRATAGENE Co.). After attaching adaptors to both ends of the cDNA, it was incorporated into a ZAP Expression Vector (STRATAGENE Co.), thereby obtaining a cDNA library of approximately 6×10 6 pfu.

2) PCR Reaction Using a Pair of (Sucrose:fructan 6-fructosyltransferase and Invertase) cDNA-specific Degenerated Primers and Using the cDNA as a Template

One of the pair of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers having the following nucleotide sequence:

(Forward): 5′ A-T-G-A-A-T/C-G-A-T/C-C-C-N-A-A-T/C-G-G (SEQ.ID. No.3)

the other of the pair of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers, having the following nucleotide sequence:

(Reverse): 5′ C-C-N-G-T-N-G-C-A/G-T-T-A/G-T-T-A-/G-A-A (SEQ.ID. No.4),

both of which were synthesized based on highly conserved regions of the nucleotide sequences of known sucrose:fructan 6-fructosyltransferase cDNA and invertase cDNA (published by EMBL/Genebank/DDBJDNA Databank), were used in a PCR using the cDNA (synthesized in the manner described in the above) as the template.

The PCR reaction was performed in a final volume of 50 μl. In detail, 1 μl of Taq DNA polymerase (5 units/μl) by Nippon Gene Co., 5 μl of 10×PCR buffer (containing MgCl 2 ), 5 μl of dNTP solution (10 mM), 2 μl of each primer (12 μM) and about 10 ng of the cDNA synthesized in the above, were mixed and then brought to a total of 50 μl with distilled water. The PCR condition and number of reaction cycles are shown in Table 1 below.

(In Table 1, “denaturation” refers to a reaction in which double-stranded DNA is melt into single strand and secondary structure is eliminated, “primer extension” refers to an synthesizing of the new complementary strand, and “30 cycles” means that three basic steps of denaturation-annealing-primer extension are repeated with 30 cycles.

As a result, DNA fragments (having expected length of approximately 1500 bp) of (sucrose:fructan 6-fructosyltransferase and invertase) cDNAs were amplified by the above PCR with the pair of (sucrose:fructan 6-fructosyltransferase and invertase) cDNA-specific degenerated primers having nucleotide sequence of SEQ.ID No.3 and nucleotide sequence of SEQ.ID No.4. Theses amplified DNA fragments were then isolated and subsequently sequenced using a DNA sequencer (Model 373S by ABI Co.) according to a conventional method. By comparing the sequences with known sucrose:fructan 6-fructosyltransferase cDNA and know invertase cDNA, it were confirmed that novel sucrose:fructan 6-fructosyltransferase cDNA fragments and novel invertase cDNA fragments were isolated.

3) Isolation and Nucleotide Sequencing of Full Length of cDNAs Encoding Sucrose:fructan 6-fructosyltransferase and Invertase of the Present Invention

About 1×10 5 recombinant plagues from the cDNA library obtained in the manner described in the above were subjected to a hybridization assay by using filters lifted with 1×10 5 recombinant plaques, and using probes prepared by labeling (with 32 P) each novel (sucrose:fructan 6-fructosyltransferase and invertase) cDNA fragment obtained in the above.

The hybridization reaction was carried out for 16 hours at 42° C., in a solution containing 50% formamide, 5×SSPE, 5×Denhardt's solution, 0.5% SDS and 0.2 mg/ml salmon sperm DNA with 32 P-labeled each probe.

The filters were then washed twice in a solution containing 2×SSC and 0.1% SDS at 65° C. for 10 min. Afterwards, the filters were washed twice with another washing solution containing 0.1×SSC and 0.1% SDS, at 65° C. for 15 min. Detection of each positive plaque binding to 32 P-labeled probe was performed by exposing above washed filters to X-ray films.

About 35 positive recombinant plaques obtained in the above were subjected to nucleotide sequencing with DNA Sequencer by ABI Co. Analysis of the nucleotide sequences of these recombinant plaques revealed that novel sucrose:fructan 6-fructosyltransferase cDNA having a nucleotide sequence corresponding to the amino acid sequence listed as SEQ.ID No. 1 in FIG. 1 and novel invertase cDNA having a nucleotide sequence corresponding to the amino acid sequence listed as SEQ.ID No. 2 in FIG. 2 had been isolated from the winter wheat.

In fact, what were isolated were i) a novel wheat-derived sucrose:fructan 6-fructosyltransferase cDNA having a nucleotide sequence corresponding to the amino acid sequence listed as SEQ.ID. No.1 in FIG. 1, comprising 1848 nucleotides/616 amino acids and having 93% identity (on amino acid sequence level) with barley-derived sucrose:fructan 6-fructosyltransferase cDNA, ii) a novel wheat-derived invertase cDNA having a nucleotide sequence corresponding to the amino acid sequence listed as SEQ.ID. No.2 in FIG. 2, comprising 1986 nucleotides/662 amino acids and having 55% identity (on amino acid sequence level) with sugarcane-derived invertase cDNA.

Investigation of Enzymatic Activity

In order to investigate enzymatic activities of the novel wheat-derived sucrose:fructan 6-fructosyltransferase cDNA and the novel wheat-derived invertase cDNA of the present invention, enzymatic reactions were conducted under the following conditions with the use of culture solutions containing the novel proteins secreted by recombinant yeast (into which novel sucrose:fructan 6-fructosyltransferase cDNA and the novel invertase cDNA of the present invention have been introduced).

[Enzymatic Reaction Condition]

Buffer solution (20 mM citric acid/phosphoric acid), pH 5.5

Final substrate concentration: 50 mM sucrose

Reaction temperature: 4° C., reaction time: 17 hours.

As a result, it was confirmed that a culture solution containing novel protein secreted by recombinant yeast (into which novel sucrose:fructan 6-fructosyltransferase cDNA of the present invention has been introduced), had a function of synthesizing a kestotriose (a trisaccharide) and a kestotetraose (a tetrasaccharide) using sucrose (disaccharide) as a substrate. Also, it was confirmed that a culture solution containing novel protein secreted by recombinant yeast (into which novel invertase cDNA of the present invention has been introduced), had a function of synthesizing a kestotriose (a trisaccharide) using sucrose (disaccharide) as a substrate.

The nucleotide sequences of the novel cDNAs obtained in the present invention are listed in the following.

6

1

616

Triticum aestivum

1

Met Gly Ser His Gly Lys Pro Pro Leu Pro Tyr Ala Tyr Lys Pro Leu

1 5 10 15

Pro Ser Asp Ala Asp Gly Glu Arg Ala Gly Cys Thr Arg Trp Arg Val

20 25 30

Cys Ala Val Ala Leu Thr Ala Ser Ala Met Val Val Val Val Val Gly

35 40 45

Ala Thr Leu Leu Ala Gly Phe Arg Val Asp Gln Ala Val Asp Glu Glu

50 55 60

Ala Ala Gly Gly Phe Pro Trp Ser Asn Glu Met Leu Gln Trp Gln Arg

65 70 75 80

Ser Gly Tyr His Phe Gln Thr Ala Lys Asn Tyr Met Ser Asp Pro Asn

85 90 95

Gly Leu Met Tyr Tyr Arg Gly Trp Tyr His Met Phe Phe Gln Tyr Asn

100 105 110

Pro Val Gly Thr Asp Trp Asp Asp Gly Met Glu Trp Gly His Ala Val

115 120 125

Ser Arg Asn Leu Val Gln Trp Arg Thr Leu Pro Ile Ala Met Val Ala

130 135 140

Asp Gln Trp Tyr Asp Ile Leu Gly Val Leu Ser Gly Ser Met Thr Val

145 150 155 160

Leu Pro Asn Gly Thr Val Ile Met Ile Tyr Thr Gly Ala Thr Asn Ala

165 170 175

Ser Ala Val Glu Val Gln Cys Ile Ala Thr Pro Ala Asp Pro Thr Asp

180 185 190

Pro Leu Leu Arg Arg Trp Thr Lys His Pro Ala Asn Pro Val Ile Trp

195 200 205

Ser Pro Pro Gly Val Gly Thr Lys Asp Phe Arg Asp Pro Met Thr Ala

210 215 220

Trp Tyr Asp Glu Ser Asp Asp Thr Trp Arg Thr Leu Leu Gly Ser Lys

225 230 235 240

Asp Asp Asn Asn Gly His His Asp Gly Ile Ala Met Met Tyr Lys Thr

245 250 255

Lys Asp Phe Leu Asn Tyr Glu Leu Ile Pro Gly Ile Leu His Arg Val

260 265 270

Glu Arg Thr Gly Glu Trp Glu Cys Ile Asp Phe Tyr Pro Val Gly Arg

275 280 285

Arg Thr Ser Asp Asn Ser Ser Glu Met Leu His Val Leu Lys Ala Ser

290 295 300

Met Asp Asp Glu Arg His Asp Tyr Tyr Ser Leu Gly Thr Tyr Asp Ser

305 310 315 320

Ala Ala Asn Arg Trp Thr Pro Ile Asp Pro Glu Leu Asp Leu Gly Ile

325 330 335

Gly Leu Arg Tyr Asp Trp Gly Lys Phe Tyr Ala Ser Thr Ser Phe Tyr

340 345 350

Asp Pro Ala Lys Lys Arg Arg Val Leu Met Gly Tyr Val Gly Glu Val

355 360 365

Asp Ser Lys Arg Ala Asp Val Val Lys Gly Trp Ala Ser Ile Gln Ser

370 375 380

Val Pro Arg Thr Ile Ala Leu Asp Glu Lys Thr Arg Thr Asn Leu Leu

385 390 395 400

Leu Trp Pro Val Glu Glu Ile Glu Thr Leu Arg Leu Asn Ala Thr Glu

405 410 415

Leu Ser Asp Val Thr Leu Asn Thr Gly Ser Val Ile His Ile Pro Leu

420 425 430

Arg Gln Gly Thr Gln Leu Asp Ile Glu Ala Thr Phe His Leu Asp Ala

435 440 445

Ser Ala Val Ala Ala Leu Asn Glu Ala Asp Val Gly Tyr Asn Cys Ser

450 455 460

Ser Ser Gly Gly Ala Val Asn Arg Gly Ala Leu Gly Pro Phe Gly Leu

465 470 475 480

Leu Val Leu Ala Ala Gly Asp Arg Arg Gly Glu Gln Thr Ala Val Tyr

485 490 495

Phe Tyr Val Ser Arg Gly Leu Asp Gly Gly Leu His Thr Ser Phe Cys

500 505 510

Gln Asp Glu Leu Arg Ser Ser Arg Ala Lys Asp Val Thr Lys Arg Val

515 520 525

Ile Gly Ser Thr Val Pro Val Leu Asp Gly Glu Ala Phe Ser Met Arg

530 535 540

Val Leu Val Asp His Ser Ile Val Gln Gly Phe Ala Met Gly Gly Arg

545 550 555 560

Thr Thr Met Thr Ser Arg Val Tyr Pro Met Glu Ala Tyr Gln Glu Ala

565 570 575

Lys Val Tyr Leu Phe Asn Asn Ala Thr Gly Ala Ser Val Thr Ala Glu

580 585 590

Arg Leu Val Val His Glu Met Asp Ser Ala His Asn Gln Leu Ser Asn

595 600 605

Met Asp Asp His Ser Tyr Val Gln

610 615

2

662

Triticum aestivum

2

Met Asp Ser Ser Arg Val Ile Leu Ile Pro Gly Thr Pro Pro Leu Pro

1 5 10 15

Tyr Ala Tyr Glu Gln Leu Pro Ser Ser Ser Ala Asp Ala Lys Gly Ile

20 25 30

Glu Glu Glu Arg Ala Gly Gly Gly Gly Leu Arg Trp Arg Ala Cys Ala

35 40 45

Ala Val Leu Ala Ala Ser Ala Val Val Ala Leu Val Val Ala Ala Ala

50 55 60

Val Phe Gly Ala Ser Gly Ala Gly Trp Asp Ala Val Ala Ala Ser Val

65 70 75 80

Pro Ala Thr Pro Ala Thr Glu Phe Pro Arg Ser Arg Gly Lys Glu His

85 90 95

Gly Val Ser Glu Lys Thr Ser Gly Ala Tyr Ser Ala Asn Ala Phe Pro

100 105 110

Trp Ser Asn Ala Met Leu Gln Trp Gln Arg Thr Gly Tyr His Phe Gln

115 120 125

Pro Asp Lys Tyr Tyr Gln Asn Asp Pro Asn Gly Pro Val Tyr Tyr Gly

130 135 140

Gly Trp Tyr His Phe Phe Tyr Gln Tyr Asn Pro Ser Gly Ser Val Trp

145 150 155 160

Glu Pro Gln Ile Val Trp Gly His Ala Val Ser Lys Asp Leu Ile His

165 170 175

Trp Arg His Leu Pro Pro Ala Leu Val Pro Asp Gln Trp Tyr Asp Ile

180 185 190

Lys Gly Val Leu Thr Gly Ser Ile Thr Val Leu Pro Asp Gly Lys Val

195 200 205

Ile Leu Leu Tyr Thr Gly Asn Thr Glu Thr Phe Ala Gln Val Thr Cys

210 215 220

Leu Ala Glu Pro Ala Asp Pro Ser Asp Pro Leu Leu Arg Glu Trp Val

225 230 235 240

Lys His Pro Ala Asn Pro Val Val Phe Pro Pro Pro Gly Ile Gly Met

245 250 255

Lys Asp Phe Arg Asp Pro Thr Thr Ala Trp Phe Asp Glu Ser Asp Gly

260 265 270

Thr Trp Arg Thr Ile Ile Gly Ser Lys Asn Asp Ser Asp His Ser Gly

275 280 285

Ile Val Phe Ser Tyr Lys Thr Lys Asp Phe Leu Ser Tyr Glu Leu Met

290 295 300

Pro Gly Tyr Met Tyr Arg Gly Pro Lys Gly Thr Gly Glu Tyr Glu Cys

305 310 315 320

Ile Asp Leu Tyr Ala Val Gly Gly Gly Arg Lys Ala Ser Asp Met Tyr

325 330 335

Asn Ser Thr Ala Glu Asp Val Leu Tyr Val Leu Lys Glu Ser Ser Asp

340 345 350

Asp Asp Arg His Asp Trp Tyr Ser Leu Gly Arg Phe Asp Ala Ala Ala

355 360 365

Asn Lys Trp Thr Pro Ile Asp Glu Glu Leu Glu Leu Gly Val Gly Leu

370 375 380

Arg Tyr Asp Trp Gly Lys Tyr Tyr Ala Ser Lys Ser Phe Tyr Asp Pro

385 390 395 400

Val Lys Lys Arg Arg Val Val Trp Ala Tyr Val Gly Glu Thr Asp Ser

405 410 415

Glu Arg Ala Asp Ile Thr Lys Gly Trp Ala Asn Leu Gln Ser Ile Pro

420 425 430

Arg Thr Val Glu Leu Asp Glu Lys Thr Arg Thr Asn Leu Val Gln Trp

435 440 445

Pro Val Glu Glu Leu Asp Ala Leu Arg Ile Asn Thr Thr Asp Leu Ser

450 455 460

Gly Ile Thr Val Gly Ala Gly Ser Val Ala Phe Leu Pro Leu His Gln

465 470 475 480

Thr Ala Gln Leu Asp Ile Glu Ala Thr Phe Arg Ile Asp Ala Ser Ala

485 490 495

Ile Glu Ala Leu Asn Glu Ala Asp Val Ser Tyr Asn Cys Thr Thr Ser

500 505 510

Ser Gly Ala Ala Thr Arg Gly Ala Leu Gly Pro Phe Gly Leu Leu Val

515 520 525

Leu Ala Asn Arg Ala Leu Thr Glu Gln Thr Gly Val Tyr Phe Tyr Val

530 535 540

Ser Lys Gly Leu Asp Gly Gly Leu Arg Thr His Phe Cys His Asp Glu

545 550 555 560

Leu Arg Ser Ser His Ala Ser Asp Val Val Lys Arg Val Val Gly Ser

565 570 575

Thr Val Pro Val Leu Asp Gly Glu Asp Phe Ser Val Arg Val Leu Val

580 585 590

Asp His Ser Ile Val Gln Ser Phe Ala Met Gly Gly Arg Leu Thr Ala

595 600 605

Thr Ser Arg Ala Tyr Pro Thr Glu Ala Ile Tyr Ala Ala Ala Gly Val

610 615 620

Tyr Met Phe Asn Asn Ala Thr Gly Thr Ser Val Thr Ala Glu Lys Leu

625 630 635 640

Val Val His Asp Met Asp Ser Ser Tyr Asn His Ile Tyr Thr Asp Asp

645 650 655

Asp Leu Val Val Val Asp

660

3

atgaatcgat cccnaatcgg 20

4

ccngtngcag ttagttagaa 20

5

atggggtcac acggcaagcc accgctaccg tacgcgtaca agccactgcc ctccgacgcc 60

gacggcgagc gggccggctg cacgaggtgg cgcgtgtgcg ccgtcgcgct gacggcctcg 120

gccatggtgg tggtggtggt cggcgccacg ctcctggcag ggttccgggt ggaccaggcc 180

gtcgacgagg aggcggcggg cgggttcccg tggagcaacg agatgctgca gtggcagcgc 240

agtggctacc atttccagac ggccaagaac tacatgagcg atcccaacgg tctgatgtac 300

taccgtggat ggtaccacat gttcttccag tacaacccgg tgggcaccga ctgggacgac 360

ggcatggagt ggggccacgc cgtgtcccgg aacctcgtcc aatggcgcac cctccctatt 420

gccatggtgg ccgaccagtg gtacgacatc ctcggggttc tatcgggctc tatgaccgtg 480

ctacccaatg gcacggtcat catgatctac acgggggcca ccaacgcctc cgccgttgag 540

gtccagtgca tcgccactcc cgccgaccct accgaccccc tcctccgccg ctggaccaag 600

caccccgcca accccgtcat ctggtcgccg ccgggggtcg gcaccaagga tttccgagac 660

ccgatgaccg cttggtacga tgaatctgat gacacatggc gcaccctgct cgggtccaag 720

gacgacaaca acggccacca cgatggcatc gccatgatgt acaagaccaa ggacttcctt 780

aactacgagc tcatcccggg catcttgcat cgggtcgagc gcaccggcga gtgggagtgc 840

atcgacttct accctgtcgg tcgccgcacc agcgacaact catcggagat gttgcacgtg 900

ttgaaggcga gcatggacga cgaacggcat gactactact cgctaggcac gtacgactct 960

gcggcaaaca ggtggacgcc gatcgacccg gagctcgact tggggatcgg gttgagatac 1020

gactggggta agttctacgc gtccacctcg ttctatgatc cggcgaagaa gcgacgcgtg 1080

ctgatggggt acgtcggcga ggtcgactcc aagcgggctg atgtggtgaa gggatgggcc 1140

tcaattcagt cagttccaag gacaattgct ctcgacgaga agacccggac gaacctcctc 1200

ctctggcccg tggaggagat tgagaccctc cgcctcaacg ccaccgaact cagcgacgtc 1260

acccttaaca ccggctccgt catccatatc ccgctccgcc aaggcactca gctcgacatc 1320

gaggccactt tccaccttga tgcttctgcc gtcgctgccc tcaatgaggc cgatgtgggc 1380

tacaactgca gcagcagcgg cggtgctgtt aaccgcggcg cgctaggccc cttcggcctc 1440

ctcgtcctcg ctgccggtga ccgccgtggc gagcaaacgg cggtgtactt ctacgtgtcc 1500

agggggctcg acggaggcct ccataccagc ttctgccaag acgagttacg gtcgtcacgg 1560

gccaaggacg tgacgaagcg agtgattggg agcacggtgc cggtgctcga cggcgaggct 1620

ttctcgatga gggtgctcgt ggaccactcc atcgtgcagg gcttcgcgat gggcgggagg 1680

accacgatga cgtcacgggt gtacccgatg gaggcctatc aggaggcaaa agtgtacttg 1740

ttcaacaatg ccaccggtgc cagcgtcacg gcggaaaggc tcgtcgtgca cgagatggac 1800

tcagcgcaca accagctctc caatatggac gatcactcgt atgttcaatg a 1851

6

atggattcgt ctcgcgtcat actcatcccc ggcacgccgc cgctgccgta cgcctacgag 60

cagctgccgt cctcctccgc ggacgccaag ggcatcgagg aggagcgggc cggcggcggt 120

ggcctgaggt ggcgcgcgtg cgccgccgtg ctggccgcct cggccgtggt ggcgctcgtc 180

gtcgccgccg cggtcttcgg ggccagcggg gcgggctggg acgcggtggc cgcctccgtg 240

ccggcgaccc cggcgacgga gttcccgagg agcaggggca aggagcacgg cgtgtcggag 300

aagacgtcgg gggcctactc cgccaacgcg ttcccgtgga gcaacgccat gctgcagtgg 360

cagcgcaccg gctaccattt ccagccggac aagtactacc agaacgatcc caacggtccg 420

gtttactatg gcggatggta ccacttcttc taccagtaca acccgtcggg ctccgtgtgg 480

gagccccaaa tcgtgtgggg ccacgccgtg tccaaggacc tcattcactg gcgccacctc 540

ccgccggcct tggtgcccga ccagtggtac gacatcaagg gcgtcctcac cggctccatc 600

accgtgctcc ccgacggcaa ggtcatcctc ctctacacgg ggaacaccga gacctttgcg 660

caggtgacct gcctcgcgga gcccgccgac ccgagcgatc ccctcctccg cgagtgggtc 720

aagcaccccg ccaaccccgt cgtgttcccg ccccccggca tcggcatgaa ggacttccgc 780

gaccccacca ccgcgtggtt cgacgagtcc gacggcacgt ggcgcaccat catcggctcc 840

aagaacgact cggaccactc cggcatcgtc ttctcctaca agaccaagga cttcctcagc 900

tacgagctga tgccggggta catgtaccgc ggccccaagg gcaccggcga gtacgagtgc 960

atcgacctct acgccgtcgg cgggggccgc aaggccagcg acatgtacaa ctcgaccgcc 1020

gaggacgtgc tgtacgtgct caaggagagc agcgacgacg accggcacga ctggtactcg 1080

ctgggccggt tcgacgccgc cgccaacaag tggacgccga tcgacgagga gctggagctc 1140

ggcgtcgggc tgcggtacga ctggggcaag tactacgcgt ccaagtcctt ctacgacccc 1200

gtgaagaagc ggcgcgtcgt gtgggcgtac gtcggcgaga ccgactcgga gcgcgccgac 1260

atcaccaagg ggtgggccaa cctccagtcg attccgagga cagtggagct tgacgagaag 1320

acccggacga acctcgtcca atggcctgtg gaggagctcg atgccctccg catcaacacc 1380

accgatctca gcggcatcac cgtcggcgcc ggctccgttg ccttcctccc cctccatcag 1440

accgctcagc tcgacatcga ggcaaccttc cgcatcgatg cctccgccat tgaggccctc 1500

aacgaggccg atgttagcta caactgcacc accagcagcg gggctgccac ccgcggcgcg 1560

cttggcccct tcggcctcct tgtcctcgcc aaccgcgccc tgaccgaaca gacgggagtg 1620

tacttctatg tgtccaaggg cctcgacggt ggtcttcgga ctcacttctg ccacgacgag 1680

ttgcgctcgt cgcatgctag tgacgtggtg aagcgggtgg tgggtagcac ggtgccagtg 1740

ctcgacggcg aagatttttc cgttagggtg ctcgtggacc actccattgt gcagagcttc 1800

gcgatgggcg ggaggttgac agcaacgtcg agggcgtacc cgaccgaggc catctacgcg 1860

gcagcggggg tctacatgtt caacaacgcc accggcacta gcgtcaccgc cgagaagctt 1920

gtcgtgcatg atatggactc gtcgtacaac catatataca cagatgatga cttggtagtc 1980

gtcgattag 1989

The advantages of the present invention may be concluded as follows.

1) With the use of the present invention, it has become possible to provide i) a novel wheat-derived sucrose:fructan 6-fructosyltransferase cDNA encoding different amino acid sequence from that of a conventional sucrose:fructan 6-fructosyltransferase cDNA, and ii) a novel wheat-derived invertase cDNA encoding different amino acid sequence from that of a conventional invertase cDNA.

2) Since the novel wheat-derived sucrose:fructan 6-fructosyltransferase cDNA and the novel wheat-derived invertase cDNA of the present invention are all capable of expressing at a low temperature environment so as to produce (synthesize) a desired fructan in a low temperature environment. Therefore, when either or both of the cDNAs of the present invention are introduced into a plant, it is sure to efficiently increase the cold resistance of the plant, thereby making it possible to produce new plant variety having an increased cold resistance.

Further, since the novel wheat-derived sucrose:fructan 6-fructosyltransferase cDNA and the novel wheat-derived invertase cDNA of the present invention are all capable of synthesizing a desired fructan, these cDNAs are useful for industrially producing a fructo-oligosaccharide which can be used to prevent a carious tooth and for increasing intestinal bifid bacteria.

While the presently preferred embodiments of the this invention have been shown and described above, it is to be understood that these disclosures are for the purpose of illustration and that various changes and modifications may be made without departing from the scope of the invention as set forth in the appended claims.