CN111961675B

CN111961675B - Clonotus sinensis-free Clinopodium polycephalum closed flower gene CsCly and application thereof

Info

Publication number: CN111961675B
Application number: CN202010900883.7A
Authority: CN
Inventors: 张吉宇; 吴凡; 闫启
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2020-09-01
Filing date: 2020-09-01
Publication date: 2022-03-18
Anticipated expiration: 2040-09-01
Also published as: CN111961675A

Abstract

The invention relates to the technical field of biology, in particular to a cryptophyte dichotoma closed-flower gene CsCly without miscanthus and application thereof, the cryptophyte dichotoma closed-flower gene CsCly without miscanthus can regulate and control the floral organs of plants, induce the closed-flower pollination of the plants, avoid the leakage of anthers and the spread of pollen, and can be used in the field of plant genetic engineering to prevent gene drift and quickly obtain the inbred line of cross-pollinated plants; meanwhile, the expression vector of the closed cell gene CsCly constructed by the invention and a host containing the vector can transfer the closed cell gene into a plant to induce the closed cell pollination of the plant; the application is verified by rice plants, and the result shows that after the CsCly gene without the cryptospermum formosanum is transferred, rice flowers are mutated, anthers are not exposed, and the gene can effectively induce plant closed flower pollination.

Description

Clonotus sinensis-free Clinopodium polycephalum closed flower gene CsCly and application thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of biology, in particular to a cryptophyte dichotoma gene CsCly and application thereof.

[ background of the invention ]

The cryptospermum gracile (Cleistogenes songorica) is a perennial and super-drought grassy plant of cryptospermum gracile of Gramineae, grows well in an area with annual rainfall of 100mm, is a population establishing species and a dominant species of desert grasslands, has high feeding value, strong drought resistance and cold resistance, is suitable for ecological restoration, slope greening, urban greening and the like in an area with annual rainfall of 120mm, and is widely applied in northwest regions.

The cryptospermum aridum belongs to a facultative pollination plant, namely, flowering pollination and closed-flower pollination exist in the same single plant, spikelets are hidden in leaf sheaths for complete closed-flower pollination, and a spike-top flower device is used for flowering pollination, so that the chance of cross-flower pollination is increased. For the same plant, the pollination of both flowers and closed flowers can not only increase the fertilization mode, but also improve the success rate of sexual reproduction. This is achieved by closed pollination, in which the plant is subjected to environmental conditions unsuitable for pollination and fertilization, with the aim of obtaining the maximum number of seeds at the minimum cost to propagate progeny. The characteristic of closed-flower pollination is a developmental strategy for plants to effectively resist adverse environments during long-term evolution. The closed pollinator appears earlier than the flowering pollinator, and the flowering pollinator appears due to specific photoperiod and temperature induction; closed pollination has been determined early in development to be characterized by reduced stamens and reduced stamens size and number as compared to flowering pollinators. In addition, the glumes of closed-flower pollination are not opened, anthers cannot be exposed, pollen cannot be spread outwards, and the plants can be kept pure by avoiding the interference of external pollen, so that the cultivation of a new transgenic variety with the closed-flower pollination characteristic is an ideal strategy for inhibiting gene drift and reducing environmental risk on the premise of not influencing other agronomic traits. However, the gene regulating closed flowers of the apocynum manypearum as the grass with flowering and closed flower pollination is not reported.

[ summary of the invention ]

In view of the above, there is a need to provide cryptogamus formosanus closed flower gene CsCly and application thereof, wherein the gene can regulate and control the development of plant floral organs, induce closed flower pollination of transgenic plants, and avoid pollen leakage and transmission.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the closterium gracile gene CsCly has a nucleic acid sequence shown as SEQ ID NO.1, and a coding sequence of the closterium gracile gene is shown as SEQ ID NO. 2.

The invention also comprises a primer pair for amplifying the closterium gracile gene CsCly coding sequence, wherein the primer pair is a primer pair 1, the upstream sequence of the primer pair is shown as SEQ ID NO.4, and the downstream sequence of the primer pair is shown as SEQ ID NO. 5.

The invention also comprises a protein coded by the cryptophyte formosana closed flower gene CsCly, and the amino acid sequence of the protein is shown as SEQ ID NO. 3.

The invention also comprises an overexpression vector, wherein the overexpression vector contains the nucleic acid sequence of the CsCly gene.

The invention also includes a host cell containing the expression vector.

The invention also comprises application of the cryptophyte formosana closed-flower gene CsCly in inducing plant closed-flower pollination.

Further, the plant is a gramineous plant; the Gramineae is Cryptocarya formosana and/or rice.

The invention also comprises a construction method of the overexpression vector, which comprises the following steps: constructing a primer pair 2 used by an overexpression vector, wherein the upstream sequence is shown as SEQ ID NO.6, and the downstream sequence is shown as SEQ ID NO. 7; and then carrying out PCR amplification by taking young leaf cDNA of the cryptocarya formosana as a template to obtain a target gene fragment, and connecting the target gene fragment with a vector pART-CAM to obtain an overexpression vector, wherein the molar ratio of the target gene fragment to the vector pART-CAM is 5: 1.

The invention also comprises a construction method of the host cell, wherein the method is obtained by transferring the overexpression vector into the agrobacterium tumefaciens GV 3101.

The invention also comprises a method for introducing the cryptophyte formosana closed flower gene CsCly into plants, which comprises the following steps: culturing the embryonic callus of the plant, and then infecting the embryonic callus with the host cell.

Further, the plant is rice.

The invention has the following beneficial effects:

the cryptophyte scandens closed-flower gene CsCly can regulate and control the development of floral organs of plants, induce the closed-flower pollination of the plants, avoid the leakage of pollen and the spread of the pollen, and can be used in the field of plant genetic engineering to prevent gene drift and quickly obtain the inbred line of cross-pollinated plants; meanwhile, the expression vector of the closed cell gene CsCly constructed by the invention and a host containing the vector can transfer the closed cell gene into a plant to induce the closed cell pollination of the plant; the application is verified by rice plants, and the result shows that after the CsCly gene without the cryptospermum formosanum is transferred, the variant anther of the rice flower is not exposed, and the gene can effectively induce the closed-flower pollination of plants.

[ description of the drawings ]

FIG. 1 is an electrophoretogram for examining the coding sequence (CDS) of the CsCly gene of the present invention; in the figure, M is 2000 marker, which is sequentially from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp; 1 and 2 are both CsCly;

FIG. 2 is a PCR electrophoretogram of a colony of example 3 of the present invention; in the figure, M is 2000 marker, which is sequentially from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, 1-6 are all positive colonies;

FIG. 3 is a floral phenotype trait result of the over-expressed plants of example 5 of the present invention; the Wild Type (WT) rice florets are shown on the left, and rice florets (OE _ CsCly) transformed with the CsCly gene are shown on the right.

[ detailed description ] embodiments

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Example 1:

obtaining the closed flower gene CsCly of the cryptospermum formosanum of the embodiment:

(1) taking the young leaves of the cryptospermum formosanum which contain the cryptospermum formosanum closed flower gene CsCly, grinding the young leaves in liquid nitrogen, and extracting the total RNA through a kit. In this embodiment, young leaves of Tenggeli without Cryptospermum formosanum are taken, fully ground in liquid nitrogen, and then total RNA is extracted by using a plant total RNA extraction kit TRIzol (Invitrigen);

(2) total RNA was extracted and reverse transcribed to cDNA. In this example, reverse transcription of RNA into cDNA was carried out using TaKaRa Prime Script TM II 1st strand cDNA Synthesis Kit, the specific operations were performed as described;

(3) taking cDNA as a template, and carrying out PCR amplification on the cDNA through a primer pair 1; wherein, the forward primer sequence of the primer pair 1 is as follows: 5'-ATGGCGGCCACCAAGAGAGCCTTC-3' (SEQ ID NO.4), the reverse primer sequence of primer pair 1 is: 5'-TCAGCTGCTGGGCCTGGGAACTGTG-3' (SEQ ID NO. 5);

the total volume of the PCR reaction system was 20. mu.L, including 1. mu.L of cDNA (50 ng. mu.L-1), 0.5. mu.L of each of the forward and reverse primers (10. mu.M), 10.0. mu.L of Prime STAR Max Premix (2X), sterilized ddH₂O8 mu L, and the PCR amplification program is pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min, and performing 30 cycles; most preferablyThen, the resulting mixture was extended at 72 ℃ for 8min and stored at 4 ℃.

(4) After the PCR reaction in step (3) is finished, agarose Gel electrophoresis is firstly carried out on the amplification product, and then Gel cutting recovery and sequencing are carried out through a Gel recovery Kit (AxyPrep TM DNA Gel Extraction Kit, Axygen can be adopted). Sequencing results show that the sequence of the coding sequence (CDS) of the Clinopodium polycephalum hance Clinopodium gene CsCly is shown in SEQ ID NO.2, and the electrophoretogram of the coding sequence is shown in FIG. 1.

Example 2:

the coding sequence (CDS) of the CsCly gene according to example 1 the amino acid sequence of the protein encoded by SEQ ID NO.2 is shown in SEQ ID NO. 3.

Example 3:

an overexpression vector is constructed according to the CsCly gene in the embodiment 1, the expression vector can insert the cryptophyte formosanus closed flower gene CsCly in the embodiment 1 into a plant expression vector, and a promoter for promoting the expression of a corresponding target gene is an enhanced promoter 35S, and the construction method of the overexpression vector comprises the following steps:

(1) taking the young leaves without the cryptospermum polycarpum, extracting RNA and carrying out reverse transcription to obtain cDNA. Wherein the variety of the miscanthus free miscanthus is Tenggeli miscanthus free.

(2) Primer set 2 designed for amplification of the CsCly gene inserted into the vector pART-CAM, wherein primer set 2

The forward primer sequence is: 5'-GGAGAGGACACGCTCGAGATGGCGGCCACCAAGAGAGCCTTC-3' (SEQ ID NO. 6);

the reverse primer sequence is 5'-TTAAAGCAGGACTCTAGATCAGCTGCTGGGCCTGGGAACTGTG-3' (SEQ ID NO. 7);

and carrying out PCR amplification by using the full-length cDNA as a template to obtain a PCR product. In this example, high fidelity enzyme PrimeSTAR HS DNA Polymerase (TaKaRa Code: DR010A) was used, and the total volume of the reaction system was 20. mu.L: 1 mu L of template cDNA; forward and reverse primers (10. mu.M) 0.5. mu.L each, PrimeSTAR Max Premix (2X) 10.0. mu.L, sterilized ddH₂O8. mu.L. The PCR amplification program is pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min, and performing 30 cycles; finally, the cells were extended at 72 ℃ for 8min and stored at 4 ℃.

(3) And (3) performing gel electrophoresis on the PCR product obtained in the step (2) and recovering. Preparing 1% agarose gel, adding the PCR product into the gel hole, placing the gel hole in an electrophoresis tank for electrophoresis, wherein the voltage is 60-100V, and the sample moves from the negative electrode (black) to the positive electrode (red). When bromophenol blue moved about 1cm from the lower edge of the gel plate, the electrophoresis was stopped. Rapidly cutting the gel containing the target strip under an ultraviolet lamp, transferring the gel into a 2.0mL centrifuge tube, recovering the target segment according to the instruction of a gel recovery kit (Whitecker gel recovery kit), and storing at-20 ℃ for later use.

(4) Linearization of vector pART-CAM: plasmid pART-CAM was double digested with XhoI and XbaI, and the total volume of the double digestion reaction system was 20. mu.l: 2x Tango buffer 4.0. mu.l, XhoI 1. mu.l, 2XbaI 2. mu.l, plasmid 1. mu.g, dH₂O12. mu.l. Carrying out enzyme digestion at 37 ℃ for 2.5 h; inactivating at 65 deg.C for 20 min. After enzyme digestion, carrying out result detection by agarose gel electrophoresis, cutting gel of a corresponding strip of the large fragment of the vector, and recovering an enzyme digestion product by a Baitach gel recovery kit;

(5) and (3) connecting the target fragment with the vector: and (3) mixing the target DNA fragment in the step (3) and the linearized vector in the step (4) in a ratio of 5:1 is added into an EP tube to carry out recombination reaction; mixing, standing at 37 deg.C for 20 min; the transformation is carried out immediately and the remaining ligation solution can be stored at 4 ℃ or-20 ℃ until use.

(6) And (3) transforming escherichia coli: a tube of 100. mu.l of DH5a (E.coli) competent cells was thawed on ice and the tube wall was flicked to resuspend the cells. Adding 10 μ l of the connecting liquid obtained in the step (5) into the competent cells, and incubating on ice for 45min under flicking; quickly putting the mixture on ice for 2min after heat shock is carried out for 90s in a water bath at 42 ℃; adding 500 μ l LB liquid culture medium, incubating at 37 deg.C for 60 min; centrifuging at 5000g for 1min to collect thallus, uniformly coating thallus on a plate containing kanamycin antibiotics according to needs, coating with sterilized glass beads, and inverting at 37 ℃ overnight after the bacteria liquid is absorbed by agar.

(7) And (4) plasmid extraction. Inoculating an escherichia coli single colony containing a target gene into an LB liquid culture medium containing 100mg/LKan, performing shake culture at 200rpm in the dark at 37 ℃ for 14h, and extracting plasmid DNA of bacterial liquid by adopting an instruction in a plasmid miniextraction kit (production).

(8) Agrobacterium tumefaciens GV3101 was transformed by electric shock. Soaking a 0.1cm electric shock cup in alcohol for 1h in a superclean workbench, washing with sterilized water for several times, and air drying with filter paper. Taking out the agrobacterium tumefaciens competent cells from a refrigerator at the temperature of-80 ℃ and then unfreezing the agrobacterium tumefaciens competent cells on ice; plasmid (50 ng/. mu.L concentration), electric shock cup, LB medium were placed on ice for precooling. Transferring 200 μ L of thawed competent cells into a centrifuge tube with proper size, adding 1 μ L of plasmid DNA, flicking, mixing, standing on ice for 2min, transferring the mixed solution into a precooled electric shock cup, flicking for several times to disperse, and standing on ice for 2 min. The electric rotating instrument is turned on, the Manual interface is adjusted, and the voltage is adjusted to 2.5 kV. And the electric shock cup is placed in an electric conversion instrument, a pulse key is pressed, 1000 mu L of LB liquid culture medium is quickly added into the electric shock cup after 2 seconds of hearing the buzzer, and the electric shock cup is gently sucked and beaten for two or three times by a 1000 mu L pipette gun and then is moved into a 1.5mL centrifuge tube. Resuscitating at 28 deg.C and 150rpm for 3 h. 30. mu.L of the transformed product was plated on 100mL LB plates and cultured at 28 ℃ for 2 days, and the transformation result was observed.

(9) And (5) identifying positive clones. Colonies of positive clones were selected for PCR identification as shown in fig. 2, where: in the figure, M is 2000 marker, which is sequentially from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, 1-6 are all positive colonies; in FIG. 2, there is a distinct specific band between 1000bp and 2000bp, which is consistent with the position in FIG. 1, indicating that the target gene has been successfully transferred into Agrobacterium tumefaciens GV 3101.

Example 4:

this example is a host cell comprising a vector for overexpression of the Clinopodium polycephalum CsCly gene, which was obtained by transferring the vector for positive expression of example 3 into Agrobacterium tumefaciens GV 3101.

Example 5:

in this example, the host cell of example 4 was used to transfect a rice plant, as follows:

(1) selecting and shelling Japanese fine seeds with plump seeds and no obvious scars or disease spots on the surfaces, and carrying out the following disinfection operations in an ultra-clean workbench: soaking in 75% ethanol for 45s, sterilizing with 2% NaClO solution for 30min (15 min each), and oscillatingddH₂O washing for 5-7 times, each time for about 1min, and then using ddH₂O soaking overnight (4 ℃);

(2) induction of callus and formation of embryogenic callus. The overnight soaked seeds were blotted dry on filter paper, then blow dried for about 10min, and then transferred to mature embryo induction medium MSD_0.5In the above, 3d post-observation of the contamination and transfer of uncontaminated seeds to a new induction medium MSD_0.5Culturing for 4-7 days, transferring the induced callus to a subculture medium MSD₂Subculturing once every 2 weeks, and continuously subculturing for 1-2 times until a faint yellow granular embryonic callus is induced;

(3) preculture of embryogenic callus and infection with agrobacterium. Transferring embryogenic calli formed by subculture to MSD_0.5Dark culture is carried out on a pre-culture medium of AS for 2-3 days; at the same time, a monoclonal of the vector to be infected, which has been transformed into Agrobacterium, is picked up, shaken overnight (about 18 h) at 28 ℃ in LB liquid medium (with corresponding resistance), harvested at 4000rpm for 15min, and MS is used₀Suspending in liquid culture medium, collecting thallus off-line, and performing MS₀(plus 200uMAS) for infection of embryogenic calli that have been pre-cultured; shading light in the infection process, and continuously shaking during the infection process to make the light and the infection fully contact; after infection, sucking dry the agrobacterium liquid on the surface of the thallus by using filter paper, transferring the agrobacterium liquid to a co-culture medium MSD2-AS (adding a piece of filter paper on the culture medium), and culturing for 2-3 days in the dark;

(4) screening and differentiation thereof. The calli after the completion of co-culture were inoculated with ddH containing 1/1000 Cef₂O cleaning for 2-3 times, and transferring to the first screening MS after drying₁In medium, screening for 2 weeks, and switching to MS₂Screening for 2-3 weeks on a screening culture medium, transferring the embryogenic callus to an empty dish containing filter paper, drying for 1-2 days, and transferring to a differentiation culture medium RE₁Culturing on-CH in dark for 4-5 days, transferring to the place under light for 7-9 days, and transferring to differentiation RE₂on-CH, continuously differentiating until resistant seedlings grow out; transferring the resistant seedlings of 2-3 cm into a rooting 1/2MS-MET culture medium.

The transgenic plants (OE _ CsCly) and Wild Type (WT) plants obtained in example 5 were grown simultaneously as controls, and the same cultivation measures were taken and the floral organs of the transgenic plants (OE _ CsCly) and Wild Type (WT) were observed for flowering time.

The results show that, as shown in FIG. 3, the plot is a plot of rice florets after planting comparison, Wild Type (WT) control rice florets on the left, and CsCly gene-transferred rice florets on the right. The figures show that: right-handed CsCly gene rice floret variation, anthers are not exposed; while the Wild Type (WT) plants on the left grew normally and the florets were exposed, indicating that: the CsCly gene of cryptospermum formosanum can change the development of floral organs to cause closed flower pollination.

In summary, the closed cell gene CsCly separated from the cryptospermum gracile can regulate and control the development of the floral organs of plants and induce the closed cell pollination of the plants, and the closed cell gene CsCly is transferred into the plants through the constructed expression vector and host cells of the closed cell gene CsCly to induce the closed cell pollination of the plants; the rice verification shows that the gene has the function of inducing the closed-flower pollination of the plant.

The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Sequence listing

<110> Lanzhou university

<120> cryptophyte dichotoma gene CsCly without miscanthus and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2338

<212> DNA

<213> Elaphanita indica (Cleistogenes songorica Roshev. Ohwi)

<400> 1

tcagctgctg ggcctgggaa ctgtggcgca gcggcggccc agctgctgcc acagtgggaa 60

tcctgatgat gctgcagcca gggagctgct ggcgtcgtag tcgggctgcc ggggccgggc 120

ggcgtacggt ggcacccaat tattgctgcc gtccggctct ggccagcgca tgtgtggctc 180

tgcctcggcg cctccactgc tgccgatgcc gatctggccc tgcgcgtcgg tccgatggcg 240

cccgggtccg tcagtgcatc acgaaagagg aaaacaagga acgaaaaaga ttgcagtttg 300

agagtcctta gcatgcattg gtgcgtcttt catgcagctc agttggaggt ttctgtgtga 360

gttgtgtgtg atgagtgatc gtacgagggc cgtaccatct ggagcatgtg cagcgtgctc 420

ctgctgcccg ggaggcgaga gtggtcgtca tcagctgggt ggcgcccgaa cgcgccgcgg 480

atcggcggcg tggccggaga cggcgatggc ctggtgcgga tgctcctgtt agggtagccg 540

gcgatcgcct catcctcctc ctccggcagc tccaactggt tttccggcat ggaacagatt 600

cagtgaccga gctggttcag tcaggcaatg gaaactatgc acggtaactg taccgttagg 660

ctcgtcctct gcttgcccgg cgcggagttg aatgcctcat cggcgacggt ggtaggatcg 720

acgcccctct cgccgccggc gcagcccagg ctgagctcga ggtcaagctc accgtcccaa 780

gctgcactca caaatacaat tctatttcag aacagcgcaa gaatgtctga atgtgtgatc 840

aatcaagaaa agaaaaacag aacatgtaag gcagactcac attgcagctg gagctcgttg 900

tcgtacgtct ggggatcgaa gttggtcact gcctccttgc caaagcactt gatcgcagcc 960

ttgtcataga ccctgatcaa catggggtta aacacaaaca cgcataatga cacatcacat 1020

aaatgcagag aagttcagtg cagacatact gggcagcctc catctcagtg tcatacaggc 1080

caaggtacac atacctgcaa tgcaaggaaa aatttactcc aaggaaaatt gcgggcttgg 1140

tgtcttggtt ttgctatgaa tgaagcctag aaatgtataa ggatttatta ctttttgccc 1200

atgagctggc cgatcctggc ctcccacttg ccgcacttgt gctgggtgac tccccggaac 1260

ctggagctcc ccctgacgaa gccggcgccc tgtcgccgga ggacctgaac gaactcctct 1320

ttgctgaagt tgttcatctg gcaacgcaat gccgtggcaa gaacaatgtc tgaatcaaac 1380

agtgaaacaa aaaatacaga tgaacagagc agctatctgc tcactcctgt tcatgatgtc 1440

agaaagatgt tctgtgatcg gatcagtgat caccttcttg atgtcatcct tgtagtcgtc 1500

cagggtgaag ttgatgtctg cactcacacc ccggaacttg attgcagcct ggtcgtaggc 1560

tctgcaaagg atcatagaac aaggggattt cagataaaac ttcagacttc agcttctccg 1620

taaaaacaaa gcaagcgtgc agaattgtgc taaccttgca gcagcctgcg ctgtgtcaaa 1680

tccacctaga caaaaaagga aaaatataaa agaataggga tcatccgtta acaaatcatg 1740

gcacatgatt caatgtcaag aaaccacagc aagtgtaggt gttccaactt ccaagtggac 1800

aggaactcac ccaggtacac ctgctttcca cagtccctgc agatcagagc acaggaaaga 1860

cagagaaatg aacacgaaat ggaataggag atgacgaaaa caacttgcaa agcttttttt 1920

tgcaacggaa caattcgcaa agctaagtgc gaagggaagc taggtgactg accagatgtg 1980

cgactcccac cgaccggtgc gccggtagaa ggtgacgccg cggtactgcg agctccggag 2040

accgcggacc gcgccggctc ttcctcaacg ccggtcgggc ccacgggtca gcggcgaccg 2100

cgtggcactg ctccattccc tgttgctgat gctgccgcgc cggcgccggc gccggcactg 2160

ccaccgccac cgtcgccggg aagaactgct gggtgacagc ggccggcctg cctggcggcc 2220

caggcaccgg gaagccaaag atcatcctgg agccttcctc cggctggcca ccctgcagct 2280

cctcctcgcg tccaatgtcg tcgtcgacgc cgtggaaggc tctcttggtg gccgccat 2338

<210> 2

<211> 1320

<212> DNA

<213> Elaphanita indica (Cleistogenes songorica Roshev. Ohwi)

<400> 2

atggcggcca ccaagagagc cttccacggc gtcgacgacg acattggacg cgaggaggag 60

ctgcagggtg gccagccgga ggaaggctcc agaatgatct ttggcttccc ggtgcctggg 120

ccgccaggca ggccggccgc tgtcacccag cagttcttcc cggcgacggt ggcggtggca 180

gtgccggcgc cggcgccggc gcggcagcat cagcaacagg gaatggagca gtgccacgcg 240

gtcgccgctg acccgtgggc ccgaccggcg ttgaggaaga gccggcgcgg tccgcggtcc 300

cgcagctcgc agtaccgcgg cgtcaccttc taccggcgca ccggtcggtg ggagtcgcac 360

atctgggact gtggaaagca ggtgtacctg ggtggatttg acacagcgca ggctgctgca 420

agagcctacg accaggctgc aatcaagttc cggggtgtga gtgcagacat caacttcacc 480

ctggacgact acaaggatga catcaagaag atgaacaact tcagcaaaga ggagttcgtt 540

caggtcctcc ggcgacaggg cgccggcttc gtcaggggga gctccaggtt ccggggagtc 600

acccagcaca agtgcggcaa gtgggaggcc aggatcggcc agctcatggg caaaaagtat 660

gtgtaccttg gcctgtatga cactgagatg gaggctgccc aggtctatga caaggctgcg 720

atcaagtgct ttggcaagga ggcagtgacc aacttcgatc cccagacgta cgacaacgag 780

ctccagctgc aatcttggga cggtgagctt gacctcgagc tcagcctggg ctgcgccggc 840

ggcgagaggg gcgtcgatcc taccaccgtc gccgatgagg cattcaactc cgcgccgggc 900

aagcagagga cgagcctaac gttggagctg ccggaggagg aggatgaggc gatcgccggc 960

taccctaaca ggagcatccg caccaggcca tcgccgtctc cggccacgcc gccgatccgc 1020

ggcgcgttcg ggcgccaccc agctgatgac gaccactctc gcctcccggg cagcaggagc 1080

acgctgcaca tgctccagat gggccagatc ggcatcggca gcagtggagg cgccgaggca 1140

gagccacaca tgcgctggcc agagccggac ggcagcaata attgggtgcc accgtacgcc 1200

gcccggcccc ggcagcccga ctacgacgcc agcagctccc tggctgcagc atcatcagga 1260

ttcccactgt ggcagcagct gggccgccgc tgcgccacag ttcccaggcc cagcagctga 1320

<210> 3

<211> 439

<212> PRT

<213> Elaphanita indica (Cleistogenes songorica Roshev. Ohwi)

<400> 3

Met Ala Ala Thr Lys Arg Ala Phe His Gly Val Asp Asp Asp Ile Gly

1 5 10 15

Arg Glu Glu Glu Leu Gln Gly Gly Gln Pro Glu Glu Gly Ser Arg Met

20 25 30

Ile Phe Gly Phe Pro Val Pro Gly Pro Pro Gly Arg Pro Ala Ala Val

35 40 45

Thr Gln Gln Phe Phe Pro Ala Thr Val Ala Val Ala Val Pro Ala Pro

50 55 60

Ala Pro Ala Arg Gln His Gln Gln Gln Gly Met Glu Gln Cys His Ala

65 70 75 80

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

85 90 95

Gly Pro Arg Ser Arg Ser Ser Gln Tyr Arg Gly Val Thr Phe Tyr Arg

100 105 110

Arg Thr Gly Arg Trp Glu Ser His Ile Trp Asp Cys Gly Lys Gln Val

115 120 125

Tyr Leu Gly Gly Phe Asp Thr Ala Gln Ala Ala Ala Arg Ala Tyr Asp

130 135 140

Gln Ala Ala Ile Lys Phe Arg Gly Val Ser Ala Asp Ile Asn Phe Thr

145 150 155 160

Leu Asp Asp Tyr Lys Asp Asp Ile Lys Lys Met Asn Asn Phe Ser Lys

165 170 175

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

180 185 190

Gly Ser Ser Arg Phe Arg Gly Val Thr Gln His Lys Cys Gly Lys Trp

195 200 205

Glu Ala Arg Ile Gly Gln Leu Met Gly Lys Lys Tyr Val Tyr Leu Gly

210 215 220

Leu Tyr Asp Thr Glu Met Glu Ala Ala Gln Val Tyr Asp Lys Ala Ala

225 230 235 240

Ile Lys Cys Phe Gly Lys Glu Ala Val Thr Asn Phe Asp Pro Gln Thr

245 250 255

Tyr Asp Asn Glu Leu Gln Leu Gln Ser Trp Asp Gly Glu Leu Asp Leu

260 265 270

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

275 280 285

Thr Val Ala Asp Glu Ala Phe Asn Ser Ala Pro Gly Lys Gln Arg Thr

290 295 300

Ser Leu Thr Leu Glu Leu Pro Glu Glu Glu Asp Glu Ala Ile Ala Gly

305 310 315 320

Tyr Pro Asn Arg Ser Ile Arg Thr Arg Pro Ser Pro Ser Pro Ala Thr

325 330 335

Pro Pro Ile Arg Gly Ala Phe Gly Arg His Pro Ala Asp Asp Asp His

340 345 350

Ser Arg Leu Pro Gly Ser Arg Ser Thr Leu His Met Leu Gln Met Gly

355 360 365

Gln Ile Gly Ile Gly Ser Ser Gly Gly Ala Glu Ala Glu Pro His Met

370 375 380

Arg Trp Pro Glu Pro Asp Gly Ser Asn Asn Trp Val Pro Pro Tyr Ala

385 390 395 400

Ala Arg Pro Arg Gln Pro Asp Tyr Asp Ala Ser Ser Ser Leu Ala Ala

405 410 415

Ala Ser Ser Gly Phe Pro Leu Trp Gln Gln Leu Gly Arg Arg Cys Ala

420 425 430

Thr Val Pro Arg Pro Ser Ser

435

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggcggcca ccaagagagc cttc 24

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcagctgctg ggcctgggaa ctgtg 25

<210> 6

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggagaggaca cgctcgagat ggcggccacc aagagagcct tc 42

<210> 7

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttaaagcagg actctagatc agctgctggg cctgggaact gtg 43

Claims

1. Clinopodium polycephalum closed-flower geneCsClyCharacterized in thatCsClyThe nucleic acid sequence of the gene is shown as SEQ ID NO.1, and the coding sequence is shown as SEQ ID NO. 2.

2. Amplifying the gene of the closed flower of cryptomeria formosana of claim 1CsClyThe primer pair of the coding sequence is characterized in that the primer pair is a primer pair 1, the upstream sequence of the primer pair is shown as SEQ ID NO.4, and the downstream sequence of the primer pair is shown as SEQ ID NO. 5.

3. The closed flower gene of cryptomeria formosana as claimed in claim 1CsClyThe coded protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 3.

4. An overexpression vector comprising the vector of claim 1CsClyThe coding sequence of the gene is SEQ ID NO. 2.

5. The closed flower gene of cryptomeria formosana according to claim 1CsClyThe application in inducing closed-flower pollination of plants.

6. The method for constructing the overexpression vector according to claim 4, wherein the method comprises the following steps: constructing a primer pair 2 used by an overexpression vector, wherein the upstream sequence is shown as SEQ ID NO.6, and the downstream sequence is shown as SEQ ID NO. 7; and then carrying out PCR amplification by taking young leaf cDNA of the cryptocarya formosana as a template to obtain a target gene fragment, and connecting the target gene fragment with a vector pART-CAM to obtain an overexpression vector, wherein the molar ratio of the target gene fragment to the vector pART-CAM is 5: 1.

7. A gene encoding the plant of claim 1CsClyA method for introducing a plant, said method comprising: culturing embryonic callus of the plant; constructing an overexpression vector containing a sequence of SEQ ID NO.2, transferring the overexpression vector into agrobacterium tumefaciens GV3101, and infecting the embryogenic callus with the agrobacterium tumefaciens GV 3101.

8. The method of claim 7, wherein the plant is rice.