CN1164748C - Wheat cyclin gene and its separating method and use - Google Patents

Abstract

The present invention relates to the clone, the recombination and the functional analysis of a gene WcycD2 of the wheat cell cycle protein CycD2, which belongs to the field of molecular biology and biotechnology. Total RNA is extracted from a wheat root tip, and then the total RNA of 2 micrograms is inversely transcribed into cDNA. A primer is designed according to amino acid sequences conserved into the cell cycle protein CycD2 of other plants for carrying out the conventional polymerase chain reaction (PCR). Then the primer is connected with a pGEM-T vector to be converted into a DH5 alpha cell for carrying out sequence measurement. Then 3' end fast amplification and 5' end fast amplification are carried out for obtaining the cDNA of whole length. A right expression vector is further constructed to be converted into arabidopis thaliana. Compared with the wild plants, the transgenic plants have the advantages that the growth rate is obviously increased, the life cycle is shortened, and the maturation is advanced. The excess expression of the gene in the arabidopsis thaliana can greatly accelerate the plant growing progress. Thus, the gene can be used for being transferred into crops of wheat, paddy rice, corn, etc. to obtain the purpose of accelerating the plant growing process. The gene has an important application prospect.

Description

Wheat cyclin gene and separation method thereof and application

(1) technical field:

The present invention relates to clone, reorganization and functional analysis and the application of wheat cyclin PROTEIN C ycD2 gene WcycD2, belong to molecular biology and biological technical field.

(2) background technology:

Higher plant ontogeny relate to the increase of cell number and the g and D of cell, particularly reproductive development also are based upon on the fissional basis.Fissional main mode is mitotic division, is called the cell cycle at continuous splitted cell from once dividing all processes that finishes to division next time finishes to be experienced.Complete cell cycle comprise duplicate early stage (G1), replicative phase (S), duplicate the later stage (G2) and division stage (M) four-stage.Thought in the past that the M phase was mitotic main period, found afterwards G1-S-G2 in whole cell cycle elapsed-time standards than long many of M phase, and complicated biological chemistry incident takes place, as duplicating with proteinic synthetic of DNA.Discover that the protein kinase that many cyclin bletillas depend on cyclin plays an important role in the cell cycle process, these cyclins are divided into 5 types of A, B, C, D, E, and the cyclin of D type mainly works in the initial reference mark G1 phase of cell cycle and controls the reaction to extracellular signal of cell fission and cell.Wherein CycD3 enters S at cell and works during the phase, and the CycD2 expression of gene is being activated in early days of G1, and it is considered to an important regulatory factor of fissional upstream.Cockcroft is with CycD2 gene overexpression in tobacco of Arabidopis thaliana, the transfer-gen plant growth velocity increases, the growth of beginning to reach from seedling to sophisticated all stages of opening of leaf development is all quickened, show that cell fission is the main factor of decision original hase activity and growth velocity, can reach the purpose of coordinate plant growth speed by the adjusting G1 phase, be illustrated in quark if people such as Ford in the growth velocity of the cyclin D of 2000 " nature " controlling plant, 405:575-579 (Cockcroft CE et al., 2000, Nature, Cyclin D control of growth rate in plants, 405:575-579).

The inventor is according to the aminoacid sequence of the cyclin CycD2 that delivers in other plant, and the design primer utilizes reverse transcription one polymerase chain reaction, isolates Codocyte cyclin CycD2 gene WcycD2 from wheat.Further construction of expression vector, transformation mode plant Arabidopis thaliana is compared with wild-type plant, and the transfer-gen plant growth velocity obviously increases, and life cycle shortens, ripen in advance.

(3) summary of the invention:

The present invention isolates the cDNA of Codocyte cyclin CycD2 total length first from wheat, be connected on the expression vector, utilizes Agrobacterium to infect arabidopsis thaliana transformation, and the life cycle of transfer-gen plant is compared about 7 days in advance than wild.

From the wheat tip of a root, extract total RNA, then the total RNA reverse transcription of 2 micrograms is become cDNA.Aminoacid sequence according to conservative among the cyclin CycD2 in other plant, design a pair of primer:

Forward primer: 5 '-ATCGATTGGATTTGGAAGGT-3 '

Reverse primer: 5 '-(GC) AGCTG (AC) GTCATCCA-3 of TGC (CT) A (AG),

Carry out conventional polymerase chain reaction (PCR), get 2 μ l PCR products and be connected on the pGEM-T carrier, transform DH5 α cell, carry out sequencing.Carry out 3 then ' and 5 ' terminal rapid amplifying obtain the cDNA of total length.Concrete PCR reagent and condition are:

10 * reaction buffer, 5 μ l

Deoxynucleoside acid mixture (dNTP) 4 μ l

Forward primer (5 μ M) 4 μ l

Reverse primer (5 μ M) 4 μ l

Template cDNA 4 μ l

Taq archaeal dna polymerase 0.5 μ l

Cumulative volume 50 μ l

The PCR reaction conditions is: 94 ℃ 3 minutes, enter following circulation then: 94 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 1 minute, totally 35 circulations, last 72 ℃ were extended 10 minutes.

Result's dna fragmentation that increases.Get 2 μ l PCR products and be connected to pGEM-T carrier (Promega company product), transform DH5 α cell (common carrier host cell), dull and stereotyped incubated overnight.The picking bacterial plaque is extracted plasmid DNA, is used for sequencing.

Through 3 ' and the cDNA that 5 ' terminal rapid amplifying obtains total length be 182 1bp, comprise upstream sequence and poly A tract crust before the initiator codon.Open reading frame partly is 1059bp, push away thus 353 amino acid whose one section sequences of tool, this aminoacid sequence is retrieved in international gene pool, show with cyclin D2 in the corn of having delivered and compare, amino acid identity shows the gene that has obtained Codocyte cyclin CycD2 through above-mentioned clone's step up to 69%.

Sequence table

(1) information of SEQ ID NO 1

(a) sequence signature

* length: 1815 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: cDNA

(c) suppose: not

(d) antisense: not

(e) initial source: wheat

(f) sequence description: SEQ IN NO.1

1 TAATTGGCCA?CGCGTCAAAA?TAGTAAGGAG?GAGGGGGGGG?GATATAGTTT?ATCCTTTTCG 60

61 TTTATCTCTT?TGACGGATCA?AGAACAGGGG?AGGGAGATTG?AAGGGGGGGA?GGAAGCTATG 120

121 CATCCAACGC?GTTGGGAGCT?CTCCCATATG?GTCGACCTGC?AGGCGGCCGC?GAATTCACTA 180

181 GTGATTTCTC?TAGATAAATC?AGTTCTCGCA?TCGGTTTATT?GATTGCTCAT?TAACCCAGCA 240

241 TTCCCGCCGC?TGCCATTCTT?GGGAGGAGTC?AGCATGGTTC?CGTCCGGGTA?CGACTGCGCC 300

301 GCCTCCGTCC?TGCTCTGCGC?CGAGGACAAC?GCCGCCATCC?TCGGCCTCGA?CGACGACGAG 360

361 GAAGACTGCT?CCTGGGCGGC?GGCGGCCGCC?ACCCCGCCGC?GCATCGCCGC?CGATGCCGCC 420

421 GCGGCGGCGG?AGGGGTTCCT?GGTGGACCAC?CCCGTGCAGT?CCGATGAGTG?CGTGGCGGCG 480

481 CTCGTCGAGA?CGGAGAAGGA?GCACATGCCC?GCCGACGGGT?ACCCCCAGAT?GCTGCTGCGC 540

541 CGGCCCGGGG?CCCTGGATTT?GGCCGCCGTC?AGGAGGGACG?CCATAGATTG?GATTTGGGAG 600

601 GTCATTGAGC?ATTTCAATTT?CGCGCCGTTG?ACTGCGGTCC?TGTCTGTCAA?CTACCTTGAT 660

661 AGGTTCCTCT?CCGTCTATCC?CCTTCCTGAA?GGCAAAGCTT?GGGTGACACA?GCTCTTGGCA 720

721 GTGGCTTGCT?TGTCCCTCGC?TTCAAAGATG?GAGGAGACCT?ATGTGCCGCT?CCCCGTCGAC 780

781 CTGCAGGTGG?TTGAGGCAAA?TTCTGCGTTC?GAGGGGAGGA?CCATAAAAAG?GATGGAGCTT 840

841 TTGGTGCTCA?GCACCTTAAA?ATGGAGGATG?CAAGCTGTTA?CTGCTTGCTC?ATTTATTGAC 900

901 TACTTCCTGC?GCAAATTCAA?TGATCATGAC?GCGCCCTCCA?TGCTCGCATT?CTCCCGCTCG 960

961 ACCGACCTCA?TCCTGAGCAC?AGCTAAAGGA?GCTGATTTTT?TGGTGTTCAG?ACCTTCAGAG 1020

1021?ATTGCTGCAA?GTGTCGCACT?TGCCGCATTT?GGGGAGCGCA?ATACTTCAGT?AGTCGAGCGG 1080

1081?GCTACAACTA?CTTGCAAGTT?CATAAACAAG?GAGCGAGTGT?TAAGATGCTA?CGAACTGATT 1140

1141?CAAGACAAGG?TAGCAATGGG?AACCATTGTC?CTAAAGTCAG?CTGGATCATC?AATGTTCTCT 1200

1201?GTGCCGCAAA?GCCCGATAGG?CGTGTCGGAT?GCTGCTGCAT?GTCTCAGCCA?ACAGAGTGAT 1260

1261?GATACTGCTG?TTGGATCTCC?AGCAACATGC?TACCAAGCCT?CTTCGGCAAG?CAAAAGGAGG 1320

1321?AGAATTGGCA?GATGACCGAT?CTCATGTCGT?GCTGTGCTTC?AGGTGTGTTG?CTCACCGTTT 1380

1381?TGGCTGTTTT?GTTGTTTCAA?ACATCTCAAT?TCAGTTTGGT?CACTCGAGAA?CTATAGTGTG 1440

1441?GTCAAGTAGT?TGCTGGAAGG?AACAAAAACA?CCATAGTCAA?TACTTGACCC?ATAAGAACAA 1500

1501?ACAGTTCGCC?GCGAGCCTGT?TCATGCAATC?GACATATGTA?AGGGGCATGC?TAAGCTGTTA 1560

1561?GTGTCCTGAG?ATGCATGCTG?TGTAGGCAGA?GATGGAGCAA?GGAAGTTGCA?TGTTGTAGAG 1620

1621?ATGAGAGCTC?TATTGCCCTG?TTTTGTATTT?TGGTTCCATA?TTCAGTTTCT?AATGAACTTT 1680

1681?AGGCAAGGGC?TGCTCTGATC?ATTGAGGCGG?TGAATGAAAA?TGATCCTAAA?ACGGATCGGC 1740

1741?TGAGATACAG?AACAGTTTAT?ATTTGTTCTG?ATGAGTTATA?AATTTTGTCG?ACAAAAAAAA 1800

1801?AAAAAAAAAA?AAAAA?1815

(2) information of SEQ IN NO.2

(a) sequence signature

* length: 353 amino acid

* type: amino acid

* chain: strand

* topological framework: linearity

(b) molecule type: protein

(c) sequence description

1 MVPSGYDCAA?SVLLCAEDNA?AILGLDDDEE?DCSWAAAAAT?PPRIAADAAA?AAEGFLVDHP 60

61 VQSDECVAAL?VETEKEHMPA?DGYPQMLLRR?PGALDLAAVR?RDAIDWIWEV?IEHFNFAPLT 120

121?AVLSVNYLDR?FLSVYPLPEG?KAWVTQLLAV?ACLSLASKME?ETYVPLPVDL?QVVEANSAFE 180

181?GRTIKRMELL?VLSTLKWRMQ?AVTACSFIDY?FLRKFNDHDA?PSMLAFSRST?DLILSTAKGA 240

241?DFLVFRPSEI?AASVALAAFG?ERNTSVVERA?TTTCKFINKE?RVLRCYELIQ?DKVAMGTIVL 300

301?KSAGSSMFSV?PQSPIGVSDA?AACLSQQSDD?TAVGSPATCY?QASSASKRRR?IGR 353

According to above-mentioned sequence, the primer of design construction expression vector:

Forward primer: 5 '-TCTCTAGATAAATCAGTTCTCGCATC-3 '

Reverse primer: 8 '-TAGAGCTCCACAGCACGACATGAGATCG-3 '

CDNA with total RNA reverse transcription of the tip of a root is a template, carries out the sequence that pcr amplification goes out to contain the total length encoder block, is connected on the pGEM-T carrier evaluation of checking order earlier.Downcut this fragment with XbaI and SacI then, be inserted into the downstream of the 35S promoter of expression vector PBI121.Change the expression vector that builds over to Agrobacterium EHA105.

Utilize the osmose process arabidopsis thaliana transformation, the seed of results screens containing on the LB solid medium of kantlex.T3 is observed for the growth and development of plants that isozygotys, compare with wild-type, the transfer-gen plant growth velocity obviously increases, and can shift to an earlier date about 7 days ripe.

According to above-mentioned technology, from wheat, isolate the gene of Codocyte cyclin CycD2, this gene overexpression can cause the growth velocity of transgenic arabidopsis to increase, and life cycle shortens.The increase that shows this gene expression dose is enough to promote the acceleration of cell fission and plant-growth.Therefore, allow this gene overexpression in farm crop such as wheat, paddy rice and corn and some other ornamental plants, can quicken the growth and development of plant process, shorten life cycle, will have very important economic benefit and social benefit.

(4) concrete invention embodiment:

The cloning process of cyclin CycD2 gene in embodiment 1. wheats

1. the extraction of total RNA: adopt single stage method or RNA kit to extract total RNA

2.cDNA article one chain is synthetic: get the total RNA of 2 micrograms, add 5 * reaction buffer, 4 μ l, 10mM thymus nucleic acid (dNTP) 2 μ l, ribonuclease inhibitor (40-200u/ μ l) 0.5 μ l, primer oligodT (1 μ g/ μ l) 1 μ l, ThermoScript II (10u/ μ l) 2 μ l, 42 ℃ were reacted 60 minutes, 85 ℃ of 10 minutes termination reactions are diluted to 200 μ l.

3.PCR reaction: polymerase chain reaction (PCR) reagent and condition are

At first following reagent is mixed:

10 * reaction buffer, 5 μ l

Deoxynucleoside acid mixture (dNTP) 4 μ l

Forward primer (5 μ M) 4 μ l

Reverse primer (5 μ M) 4 μ l

Template cDNA 4 μ l

Taq archaeal dna polymerase 0.5 μ l

Cumulative volume 50 μ l

The PCR reaction conditions is: 94 ℃ 3 minutes, enter following circulation then: 94 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 1 minute, totally 35 circulations, last 72 ℃ were extended 10 minutes.

4. gene clone: get 2 μ l PCR and be connected with the pGEM-T carrier, operation steps is undertaken by Promega company product pGEM-T and pGEM-Teasy Vector system specification sheets.Connect product transformed into escherichia coli DH5 α bacterial strain then, be coated with grow overnight on the LB flat board that contains penbritin (100 mcg/ml) of 5-bromo-4-chloro-3-indoles-β-D-galactoside and X-gal on the surface.The picking white colony, overnight incubation in the LB liquid nutrient medium.

5. the extraction of plasmid DNA: alkaline process extracts plasmid DNA.

6. sequencing: originally be operated in Dalian Bao Bio-Engineering Company and carry out.

7.3 ' with the separating of 5 ' sequence: the SMART RACE cDNA Amplification Kit specification sheets by Clontech company carries out

8. homology retrieval: utilize BLAST software that the sequence in isolated sequence and the gene library is compared.

Embodiment 2: wheat cyclin PROTEIN C ycD2 gene WcycD2 has following sequence:

(1) information of SEQ ID NO 1

(a) sequence signature

* length: 1815 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: cDNA

(c) suppose: not

(d) antisense: not

(e) initial source: wheat

(f) sequence description: SEQ IN NO.1

1 TAATTGGCCA?CGCGTCAAAA?TAGTAAGGAG?GAGGGGGGGG?GATATAGTTT?ATCCTTTTCG 60

61 TTTATCTCTT?TGACGGATCA?AGAACAGGGG?AGGGAGATTG?AAGGGGGGGA?GGAAGCTATG 120

121 CATCCAACGC?GTTGGGAGCT?CTCCCATATG?GTCGACCTGC?AGGCGGCCGC?GAATTCACTA 180

181 GTGATTTCTC?TAGATAAATC?AGTTCTCGCA?TCGGTTTATT?GATTGCTCAT?TAACCCAGCA 240

241 TTCCCGCCGC?TGCCATTCTT?GGGAGGAGTC?AGCATGGTTC?CGTCCGGGTA?CGACTGCGCC 300

301 GCCTCCGTCC?TGCTCTGCGC?CGAGGACAAC?GCCGCCATCC?TCGGCCTCGA?CGACGACGAG 360

361 GAAGACTGCT?CCTGGGCGGC?GGCGGCCGCC?ACCCCGCCGC?GCATCGCCGC?CGATGCCGCC 420

421 GCGGCGGCGG?AGGGGTTCCT?GGTGGACCAC?CCCGTGCAGT?CCGATGAGTG?CGTGGCGGCG 480

481 CTCGTCGAGA?CGGAGAAGGA?GCACATGCCC?GCCGACGGGT?ACCCCCAGAT?GCTGCTGCGC 540

541 CGGCCCGGGG?CCCTGGATTT?GGCCGCCGTC?AGGAGGGACG?CCATAGATTG?GATTTGGGAG 600

601 GTCATTGAGC?ATTTCAATTT?CGCGCCGTTG?ACTGCGGTCC?TGTCTGTCAA?CTACCTTGAT 660

661 AGGTTCCTCT?CCGTCTATCC?CCTTCCTGAA?GGCAAAGCTT?GGGTGACACA?GCTCTTGGCA 720

721 GTGGCTTGCT?TGTCCCTCGC?TTCAAAGATG?GAGGAGACCT?ATGTGCCGCT?CCCCGTCGAC 780

781 CTGCAGGTGG?TTGAGGCAAA?TTCTGCGTTC?GAGGGGAGGA?CCATAAAAAG?GATGGAGCTT 840

841 TTGGTGCTCA?GCACCTTAAA?ATGGAGGATG?CAAGCTGTTA?CTGCTTGCTC?ATTTATTGAC 900

901 TACTTCCTGC?GCAAATTCAA?TGATCATGAC?GCGCCCTCCA?TGCTCGCATT?CTCCCGCTCG 960

961 ACCGACCTCA?TCCTGAGCAC?AGCTAAAGGA?GCTGATTTTT?TGGTGTTCAG?ACCTTCAGAG 1020

1021?ATTGCTGCAA?GTGTCGCACT?TGCCGCATTT?GGGGAGCGCA?ATACTTCAGT?AGTCGAGCGG 1080

1081?GCTACAACTA?CTTGCAAGTT?CATAAACAAG?GAGCGAGTGT?TAAGATGCTA?CGAACTGATT 1140

1141?CAAGACAAGG?TAGCAATGGG?AACCATTGTC?CTAAAGTCAG?CTGGATCATC?AATGTTCTCT 1200

1201?GTGCCGCAAA?GCCCGATAGG?CGTGTCGGAT?GCTGCTGCAT?GTCTCAGCCA?ACAGAGTGAT 1260

1261?GATACTGCTG?TTGGATCTCC?AGCAACATGC?TACCAAGCCT?CTTCGGCAAG?CAAAAGGAGG 1320

1321?AGAATTGGCA?GATGACCGAT?CTCATGTCGT?GCTGTGCTTC?AGGTGTGTTG?CTCACCGTTT 1380

1381?TGGCTGTTTT?GTTGTTTCAA?ACATCTCAAT?TCAGTTTGGT?CACTCGAGAA?CTATAGTGTG 1440

1441?GTCAAGTAGT?TGCTGGAAGG?AACAAAAACA?CCATAGTCAA?TACTTGACCC?ATAAGAACAA 1500

1501?ACAGTTCGCC?GCGAGCCTGT?TCATGCAATC?GACATATGTA?AGGGGCATGC?TAAGCTGTTA 1560

1561?GTGTCCTGAG?ATGCATGCTG?TGTAGGCAGA?GATGGAGCAA?GGAAGTTGCA?TGTTGTAGAG 1620

1621?ATGAGAGCTC?TATTGCCCTG?TTTTGTATTT?TGGTTCCATA?TTCAGTTTCT?AATGAACTTT 1680

1681?AGGCAAGGGC?TGCTCTGATC?ATTGAGGCGG?TGAATGAAAA?TGATCCTAAA?ACGGATCGGC 1740

1741?TGAGATACAG?AACAGTTTAT?ATTTGTTCTG?ATGAGTTATA?AATTTTGTCG?ACAAAAAAAA 1800

1801?AAAAAAAAAA?AAAAA?1815

(2) information of SEQ IN NO.2

(a) sequence signature

Length: 355 amino acid

Type: amino acid

Chain: strand

Topological framework: linearity

(b) molecule type: protein

(d) sequence description

1 MVPSGYDCAA?SVLLCAEDNA?AILGLDDDEE?DCSWAAAAAT?PPRIAADAAA?AAEGFLVDHP 60

61 VQSDECVAAL?VETEKEHMPA?DGYPQMLLRR?PGALDLAAVR?RDAIDWIWEV?IEHFNFAPLT 120

121?AVLSVNYLDR?FLSVYPLPEG?KAWVTQLLAV?ACLSLASKME?ETYVPLPVDL?QVVEANSAFE 180

181?GRTIKRMELL?VLSTLKWRMQ?AVTACSFIDY?FLRKFNDHDA?PSMLAFSRST?DLILSTAKGA 240

241?DFLVFRPSEI?AASVALAAFG?ERNTSVVERA?TTTCKFINKE?RVLRCYELIQ?DKVAMGTIVL 300

301?KSAGSSMFSV?PQSPIGVSDA?AACLSQQSDD?TAVGSPATCY?QASSASKRRR?IGR 353

Embodiment 3: the structure of expression vector

1. according to the nucleotide sequence of isolated cyclin CycD2 gene, design primer:

Forward primer: 5 '-TCTCTAGATAAATCAGTTCTCGCATC-3 '

Reverse primer: 5 '-TAGAGCTCCACAGCACGACATGAGATCG-3 '

CDNA with total RNA reverse transcription of the tip of a root is a template, carries out the polymerase chain reaction.

2. get 2 μ l PCR and be connected with the pGEM-T carrier, operation steps is undertaken by Promega company product pGEM-T and pGEM-T easyVector system specification sheets.Transformed into escherichia coli DH5 α bacterial strain then is coated with grow overnight on the LB flat board that contains penbritin (100 mcg/ml) of 5-bromo-4-chloro-3-indoles-β-D-galactoside and X-gal on the surface.The picking white colony, overnight incubation in the LB liquid nutrient medium.Alkaline process extracts plasmid DNA, carries out sequencing.

3. with XabI and two restriction enzymes of SacI this gene is downcut from the pGEM-T carrier, the PBI121 that cuts with the same enzyme enzyme is connected.Connect product and transform DH5 α cell, cultivate containing on the LB solid plate of penbritin then, bacterium colony is carried out PCR identifies and the restriction analysis of plasmid DNA.

4. the expression vector that builds is transformed Agrobacterium EHA105.

Embodiment 4: gene function analysis

1. plant and plant Arabidopis thaliana.

2. the Agrobacterium mono-clonal identified of picking is in the LB liquid nutrient medium that contains 50 mg/litre kantlex, 28 ℃ of shaking culture.

3. centrifugal, the Agrobacterium precipitation is with permeating substratum (5% sucrose, 0.1M MgCl ₂, 0.5%Silwet L-77) and suspend bacterium liquid OD ₆₀₀About 0.8.

4. the Arabidopis thaliana inflorescence is immersed in the penetrating fluid, soaked 5 minutes.

5. Shou Huo seed obtains resistant plant in screening culture medium (1 * MS salt, 1% sucrose, pH5.7,0.8% agar, kantlex 30 mg/litre) screening.

With T3 for the homozygous lines seed be placed on wild type seeds under identical condition, measure the height of plant, observe its growth and development state.

Sequence table

＜110〉Shandong Agricultural University

＜120〉wheat cyclin gene and separation method thereof and application

<160>1

<170>patent?In?3.1

<210>1

<211>1815

<212>cDNA

＜213〉wheat (Triticum aestivum)

<221>1-1815

<400>1

taattggcca?cgcgtcaaaa?tagtaaggag?gagggggggg?gatatagttt?atccttttcg 60

tttatctctt?tgacggatca?agaacagggg?agggagattg?aaggggggga?ggaagctatg 120

catccaacgc?gttgggagct?ctcccatatg?gtcgacctgc?aggcggccgc?gaattcacta 180

gtgatttctc?tagataaatc?agttctcgca?tcggtttatt?gattgctcat?taacccagca 240

ttcccgccgc?tgccattctt?gggaggagtc?agcatggttc?cgtccgggta?cgactgcgcc 300

gcctccgtcc?tgctctgcgc?cgaggacaac?gccgccatcc?tcggcctcga?cgacgacgag 360

gaagactgct?cctgggcggc?ggcggccgcc?accccgccgc?gcatcgccgc?cgatgccgcc 420

gcggcggcgg?aggggttcct?ggtggaccac?cccgtgcagt?ccgatgagtg?cgtggcggcg 480

ctcgtcgaga?cggagaagga?gcacatgccc?gccgacgggt?acccccagat?gctgctgcgc 540

cggcccgggg?ccctggattt?ggccgccgtc?aggagggacg?ccatagattg?gatttgggag 600

gtcattgagc?atttcaattt?cgcgccgttg?actgcggtcc?tgtctgtcaa?ctaccttgat 660

aggttcctct?ccgtctatcc?ccttcctgaa?ggcaaagctt?gggtgacaca?gctcttggca 720

gtggcttgct?tgtccctcgc?ttcaaagatg?gaggagacct?atgtgccgct?ccccgtcgac 780

ctgcaggtgg?ttgaggcaaa?ttctgcgttc?gaggggagga?ccataaaaag?gatggagctt 840

ttggtgctca?gcaccttaaa?atggaggatg?caagctgtta?ctgcttgctc?atttattgac 900

tacttcctgc?gcaaattcaa?tgatcatgac?gcgccctcca?tgctcgcatt?ctcccgctcg 960

accgacctca?tcctgagcac?agctaaagga?gctgattttt?tggtgttcag?accttcagag 1020

attgctgcaa?gtgtcgcact?tgccgcattt?ggggagcgca?atacttcagt?agtcgagcgg 1080

gctacaacta?cttgcaagtt?cataaacaag?gagcgagtgt?taagatgcta?cgaactgatt 1140

caagacaagg?tagcaatggg?aaccattgtc?ctaaagtcag?ctggatcatc?aatgttctct 1200

gtgccgcaaa?gcccgatagg?cgtgtcggat?gctgctgcat?gtctcagcca?acagagtgat 1260

gatactgctg?ttggatctcc?agcaacatgc?taccaagcct?cttcggcaag?caaaaggagg 1320

agaattggca?gatgaccgat?ctcatgtcgt?gctgtgcttc?aggtgtgttg?ctcaccgttt 1380

tggctgtttt?gttgtttcaa?acatctcaat?tcagtttggt?cactcgagaa?ctatagtgtg 1440

gtcaagtagt?tgctggaagg?aacaaaaaca?ccatagtcaa?tacttgaccc?ataagaacaa 1500

acagttcgcc?gcgagcctgt?tcatgcaatc?gacatatgta?aggggcatgc?taagctgtta 1560

gtgtcctgag?atgcatgctg?tgtaggcaga?gatggagcaa?ggaagttgca?tgttgtagag 1620

atgagagctc?tattgccctg?ttttgtattt?tggttccata?ttcagtttct?aatgaacttt 1680

aggcaagggc?tgctctgatc?attgaggcgg?tgaatgaaaa?tgatcctaaa?acggatcggc 1740

tgagatacag?aacagtttat?atttgttctg?atgagttata?aattttgtcg?acaaaaaaaa 1800

aaaaaaaaaa?aaaaa 1815

Claims (2)

1. a clone wheat cyclin gene WcycD2 is characterized in that it has the sequence shown in following:

1 TAATTGGCCA?CGCGTCAAAA?TAGTAAGGAG?GAGGGGGGGG?GATATAGTTT?ATCCTTTTCG 60

61 TTTATCTCTT?TGACGGATCA?AGAACAGGGG?AGGGAGATTG?AAGGGGGGGA?GGAAGCTATG 120

121 CATCCAACGC?GTTGGGAGCT?CTCCCATATG?GTCGACCTGC?AGGCGGCCGC?GAATTCACTA 180

181 GTGATTTCTC?TAGATAAATC?AGTTCTCGCA?TCGGTTTATT?GATTGCTCAT?TAACCCAGCA 240

241 TTCCCGCCGC?TGCCATTCTT?GGGAGGAGTC?AGCATGGTTC?CGTCCGGGTA?CGACTGCGCC 300

301 GCCTCCGTCC?TGCTCTGCGC?CGAGGACAAC?GCCGCCATCC?TCGGCCTCGA?CGACGACGAG 360

361 GAAGACTGCT?CCTGGGCGGC?GGCGGCCGCC?ACCCCGCCGC?GCATCGCCGC?CGATGCCGCC 420

421 GCGGCGGCGG?AGGGGTTCCT?GGTGGACCAC?CCCGTGCAGT?CCGATGAGTG?CGTGGCGGCG 480

481 CTCGTCGAGA?CGGAGAAGGA?GCACATGCCC?GCCGACGGGT?ACCCCCAGAT?GCTGCTGCGC 540

541 CGGCCCGGGG?CCCTGGATTT?GGCCGCCGTC?AGGAGGGACG?CCATAGATTG?GATTTGGGAG 600

601 GTCATTGAGC?ATTTCAATTT?CGCGCCGTTG?ACTGCGGTCC?TGTCTGTCAA?CTACCTTGAT 660

661 AGGTTCCTCT?CCGTCTATCC?CCTTCCTGAA?GGCAAAGCTT?GGGTGACACA?GCTCTTGGCA 720

721 GTGGCTTGCT?TGTCCCTCGC?TTCAAAGATG?GAGGAGACCT?ATGTGCCGCT?CCCCGTCGAC 780

781 CTGCAGGTGG?TTGAGGCAAA?TTCTGCGTTC?GAGGGGAGGA?CCATAAAAAG?GATGGAGCTT 840

841 TTGGTGCTCA?GCACCTTAAA?ATGGAGGATG?CAAGCTGTTA?CTGCTTGCTC?ATTTATTGAC 900

901 TACTTCCTGC?GCAAATTCAA?TGATCATGAC?GCGCCCTCCA?TGCTCGCATT?CTCCCGCTCG 960

961 ACCGACCTCA?TCCTGAGCAC?AGCTAAAGGA?GCTGATTTTT?TGGTGTTCAG?ACCTTCAGAG 1020

1021?ATTGCTGCAA?GTGTCGCACT?TGCCGCATTT?GGGGAGCGCA?ATACTTCAGT?AGTCGAGCGG 1080

1081?GCTACAACTA?CTTGCAAGTT?CATAAACAAG?GAGCGAGTGT?TAAGATGCTA?CGAACTGATT 1140

1141?CAAGACAAGG?TAGCAATGGG?AACCATTGTC?CTAAAGTCAG?CTGGATCATC?AATGTTCTCT 1200

1201?GTGCCGCAAA?GCCCGATAGG?CGTGTCGGAT?GCTGCTGCAT?GTCTCAGCCA?ACAGAGTGAT 1260

1261?GATACTGCTG?TTGGATCTCC?AGCAACATGC?TACCAAGCCT?CTTCGGCAAG?CAAAAGGAGG 1320

1321?AGAATTGGCA?GATGACCGAT?CTCATGTCGT?GCTGTGCTTC?AGGTGTGTTG?CTCACCGTTT 1380

1381?TGGCTGTTTT?GTTGTTTCAA?ACATCTCAAT?TCAGTTTGGT?CACTCGAGAA?CTATAGTGTG 1440

1441?GTCAAGTAGT?TGCTGGAAGG?AACAAAAACA?CCATAGTCAA?TACTTGACCC?ATAAGAACAA 1500

1501?ACAGTTCGCC?GCGAGCCTGT?TCATGCAATC?GACATATGTA?AGGGGCATGC?TAAGCTGTTA 1560

1561?GTGTCCTGAG?ATGCATGCTG?TGTAGGCAGA?GATGGAGCAA?GGAAGTTGCA?TGTTGTAGAG 1620

1621?ATGAGAGCTC?TATTGCCCTG?TTTTGTATTT?TGGTTCCATA?TTCAGTTTCT?AATGAACTTT 1680

1681?AGGCAAGGGC?TGCTCTGATC?ATTGAGGCGG?TGAATGAAAA?TGATCCTAAA?ACGGATCGGC 1740

1741?TGAGATACAG?AACAGTTTAT?ATTTGTTCTG?ATGAGTTATA?AATTTTGTCG?ACAAAAAAAA 1800

1801?AAAAAAAAAA?AAAAA?1815

2. the function of wheat cyclin gene according to claim 1 is characterized in that this gene overexpression in Arabidopis thaliana, can quicken the growth course of plant.