CN109456981B - FvAGI gene, expression vector and application - Google Patents

Abstract

The invention provides an FvAGI gene, an expression vector and application, belonging to the technical field of plant genetic engineering, wherein the FvAGI gene has a nucleotide sequence shown in SEQ ID No. 1. The FvAGI gene is transferred into the strawberry, so that a strawberry male sterile plant can be obtained.

Description

FvAGI gene, expression vector and application

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to an FvAGI gene, an expression vector and application.

Background

The fertility is reduced due to poor development of embryo sacs and pollen in strawberry production; in conventional cross breeding, the workload is too large due to frequent emasculation, so that the research on the development of strawberry flowers, particularly gametophytes is important. The key gene for controlling gametogenesis in flower development is AGAMOUS (AG) gene, and has potential application prospect for strawberry breeding.

Genetic transformation is a technique of introducing exogenous genes into recipient cells by means of biology, physics or chemistry to obtain transgenic animals, plants and microorganisms, and transgenes are expressed in the recipient cells, so that the transgenic organisms retain original excellent traits and simultaneously increase a trait controlled by a target gene. Since the first transgenic plant appeared in 1983, the research on plant transgenes has been greatly advanced. Some transgenic crops are planted in a commercial mode, the area is increased continuously, and in 2008, 25 countries in the world have commercial transgenic plants, and the planting area is 1.25 hundred million ha. The key to the development of transgenic material is to clone important and functionally relevant genes.

The model systems of the flower development process are an ABC model, an ABCDE model and a tetramer model. The AGAMOUS (AG) gene is the only C-type homeogene found in Arabidopsis thaliana, which regulates the third and fourth floral organs development of Arabidopsis thaliana and is involved in the differentiation of stamens and carpels of plants. In the aspect of stamen morphogenesis, early AG induces microspore generation through the expression of SPOROCYTELESS (SPL/NZZ) to form pollen; in the later period of flower development, AG controls the synthesis of jasmonic acid by regulating and controlling catalytic enzyme DAD1 encoding the initial step of jasmonic acid biosynthesis, coordinates the cracking of anthers and the prolongation of pollen tubes, but how to play a role in the formation process of anthers and filaments in the middle period of flower development is not clarified. In terms of carpel morphogenesis, AG directs carpel development by regulating SPATULA (SPT) CRABSCHWAW (CRC) GIANT KELLER (GIK) KNUCKLES (KNU). At the level of transcriptional regulation, the expression of the AG gene is influenced by specific elements within its longest intron (AGI) in addition to the regulation of the promoter sequence upstream of the gene. AGI thus acts as an enhancer to regulate the expression of the AG gene and is used to construct organ-specific expression vectors. The experimental results show that AGI is used as an organ-specific enhancer to cause specific expression of different genes in stamens and carpels, cause abnormal flower organs or anther dehiscence abnormality or pollen shape and size difference, and finally cause transgenic plants to show higher sterility, and the evidence indicates that the AGI comprises important regulatory elements and can drive the expression of any gene in male and female organs. Introns have become one of the important elements for increasing the expression of exogenous genes in transgenic plants. However, little is known about the function and regulatory mechanisms of introns in plants. Published studies indicate that arabidopsis thaliana AtAGI and the enhancer sequence of the strong constitutive promoter CaMV 35S directly activate the expression of the reporter gene without a promoter, and that placing AtAGI between the strong constitutive promoter CaMV 35S and the reporter gene results in two different transcription initiation regions, one type of transcription starting from the middle of AtAGI, containing most of the reporter coding sequence, which cannot be translated into a functional reporter protein, meaning that there is an intergenic noncoding RNAs; another type fuses AtAGI from the smallest 35S, producing a functional reporter protein. The above are all functions of AGI as a regulation element.

There are no reports of AGI gene cloning and function verification in strawberry, and no reports that a strong constitutive promoter CaMV 35S is directly connected with FvAGI and applied to a transformed plant, and a transgenic plant generates a plant with the same phenotype as the FvAG mutant and can generate a male sterile plant.

Disclosure of Invention

The invention aims to provide an FvAGI gene which is transferred into strawberries to obtain strawberry male sterile plants.

The invention provides an FvAGI gene, which has a nucleotide sequence shown in SEQ ID No. 1.

The invention also provides an expression vector which comprises the FvAGI gene in the technical scheme.

Preferably, the expression vector is constructed by inserting the FvAGI gene described in the above-mentioned embodiment into the vector pK7WG 2D.

Preferably, the method for constructing the expression vector comprises the following steps:

1) using strawberry genome DNA as a template, and carrying out PCR amplification by using an FvAGI primer pair to obtain an FvAGI gene;

2) connecting the FvAGI gene obtained in the step 1) with a vector pENTR to obtain pENTR-FvAGI;

3) the pENTR-FvAGI obtained in the step 2) is reacted with a vector pK7WG2D to obtain pK7WG 2D-FvAGI.

Preferably, the FvAGI primer pair comprises an FvAGI upstream primer and an FvAGI downstream primer;

the FvAGI upstream primer has a nucleotide sequence shown in SEQ ID No. 2;

the FvAGI downstream primer has a nucleotide sequence shown in SEQ ID No. 3.

The invention also provides application of the FvAGI gene in the technical scheme in cultivating strawberry male sterile plants.

The invention also provides application of the expression vector in the technical scheme in cultivating male sterile plants of strawberries.

Preferably, the method for cultivating strawberry male sterile plants comprises the following steps:

transferring the expression vector into agrobacterium by adopting an electric shock method;

and (3) transfecting the obtained agrobacterium to strawberry, and then culturing to obtain strawberry male sterile plants.

The invention provides an FvAGI gene, which is transferred into strawberries to obtain male sterile plants of the strawberries.

Drawings

FIG. 1 is a diagram of a transgenic FvAGI plant of example 1 of the present invention, wherein A is a vector used for transgene, B is a phenotype of the transgenic plant, C is PCR identification of the transgenic plant, M is 1kb marker, -: negative control, +: positive control; d is a flower surface row of the transgenic plant;

FIG. 2 shows the androecium phenotype of transgenic fertile and sterile plants provided in example 1 of the present invention,

wherein FIG. 2-1 shows anther expression of FvAGI-transferred plants, the left image shows transgenic fertile plants, the middle image shows transgenic sterile plants 1, and the right image shows transgenic sterile plants 2;

FIG. 2-2 shows the detection of pollen viability of the FvAGI-transformed strawberry sterile plant and the wild-type plant, wherein A is the wild-type plant with strong pollen viability, and B is the sterile plant with weak pollen viability;

FIG. 2-3 shows the observation of the forms of pollen of the sterile plants transformed with FvAGI and wild plants, wherein A is the super-long spherical shape of wild pollen, B is the reticulate pattern rule of wild pollen, and C is the long spherical shape of pollen of the sterile plants transformed with FvAGI; d is the irregular reticulate pattern of the pollen of the sterile plant transformed with FvAGI.

Detailed Description

The invention provides an FvAGI gene, which has a nucleotide sequence shown in SEQ ID No.1, and the specific sequence is as follows:

GTATAATCCTAAATTGGTCCTACTCTTCTTCCTCTTCTCAAATTCCT CAAAGTTTCACAAAGTTTTGTGCTTTCCTAGCTTCAAGTGTTCTTGGCA AAAAAGAAGTGTGTTCTTGGCAAAACTTCAGCAAAAACTTTGGTTTTT AAAAGTAATTGGGGACTGGTGTTGAAGATTCTTGCTTCTGCAACAGAT CTTTGGGTGTGGATTCAATCACAGAAACATAATTAATCATTGTTCTGTAC TCAGCTGAGAGAGCCTGAGATGGAGTATTCTGTCTTTCTCTTTCTTGAA CCAAAGAACCAGTACTTGTCTCTTTTTTTCTTCTATTTTTTTTTTTCCTTC TATGTGCTCTAGTTAGTTTTTTTTCATTCTGTTTATGACCAATTTCTTAGC TCTTGGTTTGGTAGAAGCATAGATCTGGTGTTAAACATAGAAAAAATAG TATATAAGGGATGGAAATAGAAACTAGTAGTGGGTTTTGGAATGTATGC AGGGCTCTGAGATCAGCTCCTTGCCTTCCTCCATTGCTGTTTCCTTTTTC TCTTTTTTAGTTTCTCTTTCTCTCTCGCTCACTCTGTGCAACACTCTCTT ATGTCATCTATGAAGCTAAGATGGTCTACAACACAAGCTGTCACGCTCC TATTAGCCCAGTCCCCGATCTCCTTGACACTTCCTCAATAGCACATCAG TTATGGACACCAAGAAAGTAAAAGTACAATTTTATTTTATTTTCTATAAA AAAAAAAGGATAAGACCATGGCTGGAAAAAAGAAAGGGCACAAAAG ATTAGCTAGGGTTTTTTCCAAAACACCCAAATGGGTTATCCTTGAACTG TGTTGGAGTTTACCTTTGTTCTAACAACCAAATCAGGCAACATCTAGTC ACACCAAGATGAGGTATTCCCCAAATCTGACTTTCCTCACCTGGAAAAT CAAACACAGGAGGTTTTAACCTAATTAGGGTTTATTAGCAACTGCAATT AGTAATCACTTGGCCCAAGATCTATACTGGGTCACCCTTAAATCAAGTA AAAATGAGATATTTTCACCCATCCAATCAAAACCCAGTTCCAAAATGTT TTCTTTTCCCTTAAATTTCTGACCTTTTCTGGGAAATGGGTGTGTTCTGG TTAGGGTTAGTTTTGGTCTTTGGTTCACAGAATCTTTGGTCACGTCACC TCGAGATTTTTCGGTTCCTCATTTCTGACAAAAGAGTTCTATGTCATGAT TCACAGTGGGGTTACTTCTCTTTCCTTCTTGAACCCTAAATTTCAATTTC TTTCTTGGATCTCATAGAAAATATTAGCTCTTTCTTTATAGTTTTCTTTCA GATTTGCTCTTAAAGCTCTCAGTCACCCCAGCCAATGAGCTCACAGCT CTCGACTGGGCCATCAATGAGTCACTGTTGGCATTCTGGAGAGACACT TTTAATTTCCTCAGAATCTTGTATAGATATATTTGTGCAAGTTTTGAAGG GTGTGGAGAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG AGAGAGAGAGAGAGAGAGAGAAAGTGAGGAGTGTTGCAGAAACAAA GATATGAAATCTGCGAGCCAATGGGAAGTTCTGAGCTTTTGGCTTATTA TGACTGCTTCTGAAACAGACGGCTTTGATGTTGTCAATGTCATCGGAGT TTCATATAGAAAAGACTGGTTCTTTATTATATATTTACTGTCAGTAAGTTT TTCAAATCCTTTCGAGGTGGGTAATAGTGTAAGGTTTAGGGTAATATCC CTTGGGTAATTGGTGGGAGAGAACATGGCAGAGTGAATTCCGGCCAAT CAGATTGGGGAGCTGTGCCATCGTGGCAACAGGTAGCCAATCACAAGC TCGACGGTTTTAAGGTATTATCAGAGGTGCATAGAAAAGAGTGTGAGT ACTGTTGGATCAATTTGACCGAGGTAGATGTCTTTCTTTATTAGGCTCA GTTAAACTGAGAGAGTGAGAGAGTGAGTTCATTTGTCTACTAGTCTAAT GGCCTAAGTGTGAGCGAGGTTTATGGTTTATCATAATTTCTACTTAACCC TCAATTTTTGGGGTTTTGTTTAGTTATCTCCTGAGCAGAAAGCTGTAAT CTTGGATGTGTATCACATCTGTCACATTTGAAATTGACCCAGTCTAAAA CACACAACTGGGTTTCTTCCTTGGCACATTGAGATTTTCTTGATTCTTAT ATTAGATACAGTTAAGTAGCTAGCTTAGAGATTTGGTTTTTTTTTTTTGA GCTAATGAAGAGTGCAGGTCTTTGCTTTACCAGATATAGAGCATTCAGA CAACCCGTTTTATTCTTTCAACTTCAAGCATTAGTTTGTAGGAACATTTT CAATTTCCCTTTTCACCATAAAAAACTTCGATCAATGACCTTTTCCCTCT ATACATATATCATAATGTTCTAGCTCACCCTTTTGGCTAAATTTTTTAATC TTTCTCCGTTTCCTATTGAGGCGTGGCTCATTGCGTGCCTCATTTAGTTC TATTTTGTGTAAACCAAATTCTTTCATTGTAGATATGTAGTAAACTAAAG TATTAGCGTGCATATTCCTATTCTATTAAAATTCATGATCATAGATTCAAA TTTAGATTATTCTTCATTTATTTGCTTGTTTTAGTAGAGATATATTTTCTTG TTCTATATCAAAGTACTGCTAGTCCTTTATTCTTGGGTTCTGTCTTTTCTC ATATCTCAAAATGGTACATGAGGATATCAGAAATTCTCTTTCACATTCAG TGTTGTGCATGTGTGAATAGATGATGAATGTAAAGCCACACAATAATTC AATAAAACCTGTTCAGTAACTGTAACTTTGATCATGGCCGGGTCAAGAA TTATGCTTTATGTTAATTAGTCACTTCCGGATGTTGTAATATGTACCCTTA TATAGTATCTTATTTCATGCTTTGATTGCTTAGCAG。

in the invention, the FvAGI gene is transferred into strawberry, so that a strawberry male sterile plant can be obtained.

The variety of the strawberry is not particularly limited, and the variety of the forest strawberry can be selected, such as YW5AF7, Rugen, Hawaii and the like.

The invention also provides an expression vector which comprises the FvAGI gene in the technical scheme.

In the present invention, the expression vector is preferably constructed by inserting the FvAGI gene described in the above technical means into the vector pK7WG 2D.

In the present invention, the method for constructing the expression vector preferably comprises the steps of:

1) using strawberry genome DNA as a template, and carrying out PCR amplification by using an FvAGI primer pair to obtain an FvAGI gene;

2) connecting the FvAGI gene obtained in the step 1) with a vector pENTR to obtain pENTR-FvAGI;

3) the pENTR-FvAGI obtained in the step 2) is reacted with a vector pK7WG2D to obtain pK7WG 2D-FvAGI.

The invention takes strawberry genome DNA as a template and uses FvAGI primer pairs to carry out PCR amplification to obtain FvAGI genes.

The method for extracting the strawberry genome DNA is not particularly limited, and a conventional method for extracting the strawberry genome DNA is adopted.

In the present invention, the FvAGI primer pair preferably comprises an FvAGI upstream primer and an FvAGI downstream primer; the FvAGI upstream primer has a nucleotide sequence shown in SEQ ID No.2, and the specific sequence is shown as follows:

5’CACCGTATAATCCTAAATTGGTCCTACTC 3’；

the FvAGI downstream primer has a nucleotide sequence shown in SEQ ID No.3, and is shown as follows:

5’CTGCTAAGCAATCAAAGCATGAAATAAGA3’。

in the invention, the FvAGI primer pair is preferably designed according to gene24852DNA sequence of https:// www.rosaceae.org/search/genes by using Primer5.0, and the 5' end of the upstream primer is added with a recognition sequence CACC of directional cloning to ensure that the sequence is correctly inserted to form an entry clone.

In the present invention, the system used for PCR amplification is preferably 20. mu.LddH₂O is solvent, 10ng-1 μ g of DNA, the final concentration of forward primer is 0.3 μ M, the final concentration of reverse primer is 0.3 μ M, 1 XPfuffer, Pfu2.5units, the final concentration of dNTPS is 0.2 mM.

In the present invention, the procedure of PCR amplification is preferably: 4min at 94 ℃; 30s at 94 ℃, 40s at 58 ℃, 3min at 72 ℃ and 35 cycles; 10min at 72 ℃.

In the present invention, it is preferable to connect the obtained FvAGI gene to a vector pENTR to obtain pENTR-FvAGI.

The method for connecting the FvAGI gene and the vector pENTR is not particularly limited, and a conventional method can be adopted.

In the present invention, the vector pK7WG2D is preferably the vector pK7WG2D II.

In the present invention, the pENTR-FvAGI thus obtained is preferably reacted with a vector pK7WG2D to obtain pK7WG 2D-FvAGI.

The conditions for the reaction between pENTR-FvAGI and the carrier pK7WG2D are not particularly limited, and the conventional reaction conditions are adopted.

The invention also provides application of the FvAGI gene in the technical scheme in cultivating strawberry male sterile plants.

The invention also provides application of the expression vector in the technical scheme in cultivating male sterile plants of strawberries.

In the present invention, the method for cultivating strawberry male sterile plants preferably comprises: transferring the expression vector into agrobacterium by an electric shock method; and (3) transfecting the obtained agrobacterium to strawberry, and then culturing to obtain strawberry male sterile plants.

The method for transfecting the strawberry by the obtained agrobacterium is not particularly limited, and the conventional method for transfecting the strawberry by the agrobacterium is adopted. The conditions for culturing the strawberry after transformation are not particularly limited, and the conventional culture method for culturing the strawberry transfected by the agrobacterium is adopted.

An FvAGI gene, an expression vector and applications of the present invention will be described in further detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.

Example 1

Extracting total DNA of the strawberry:

fresh strawberry leaves are taken and extracted by a method for extracting DNA (described in Wangjing, Zhao Mizhen, in red plum, and the like, development and application of strawberry EST-SSR marker, academic paper of fruit trees, 2011,28(4): 721-.

Amplification of the FvAGI gene:

based on the gene24852DNA sequence of https:// www.rosaceae.org/search/genes, primers were designed using Primer5.0, and the recognition sequence CACC for the targeted clones was added to the 5' end of the upstream primer to ensure that the sequences could be inserted correctly to form entry clones.

Upstream primer AGI F1:5 'CACCGTATAATCCTAAATTGGTCCTACTC 3'

The downstream primer is AGI R1:5 'CTGCTAAGCAATCAAAGCATGAAATAAGA 3'.

And (3) PCR system: final concentration DNA10 ng-1. mu.g, forward primer 0.3. mu.M, reverse primer 0.3. mu.M, 1 XPfu buffer, Pfu2.5units, 0.2mM dNTPS, ddH₂O was metered to a volume of 20. mu.L. The PCR reaction condition is 94 ℃ for 4 min; 30s at 94 ℃, 40s at 58 ℃, 3min at 72 ℃ and 35 cycles; 10min at 72 ℃. Pfu high fidelity enzyme amplification, and recovering specific band and cloning sequencing after electrophoresis of the product by 1% agarose gel.

The construction of the FvAGI gene entry clone, namely, the fragment with correct sequencing is mixed with pENTR vector according to the molar ratio of 1:1 (refer to the instruction of Gateway pENTR directive TOPO Cloning kit of Invitrogen company), the mixture is connected and transferred into DH5 alpha for 5min at room temperature, the connection product is thermally shocked to transform escherichia coli DH5 alpha, then the obtained product is coated on an LB plate containing kanamycin (Kan, 50 mu g/mL) for screening, a single colony is selected for PCR identification, and the extracted plasmid of the positive clone with correct identification is the entry clone with successful construction.

The FvAGI gene expression vector is constructed by referring to the specification of Gateway LR clone II Enzyme Mix of Invitrogen company, wherein 3. mu.L of Entry clone plasmid (50-150 ng), 1. mu.L of pK7WG2D II plasmid (150ng), 4. mu.L of TE buffer (pH 8.0) and 2. mu.L of LR clone II Enzyme Mix are mixed briefly, reacted at 25 ℃ for 1h, and 1. mu.L of protease K is added after the reaction is completed, and the reaction is terminated at 37 ℃ for 10 min. 2 mu L of reaction liquid is taken to thermally excite and transform Escherichia coli D H5 alpha, and then the Escherichia coli D H5 alpha is coated on an LB plate containing spectinomycin resistance (Spec, 100 mu g/mL) for screening, a single colony is selected for PCR identification, the identified correct bacterial liquid is sequenced, and the correct positive clone extraction plasmid is the expression vector which is successfully constructed.

Agrobacterium transformation and transgenic strawberry screening:

the pK7WG2D II-FvAGI expression vector was transferred into Agrobacterium GV3101 by electroporation, the transformed product was spread on plates containing spectinomycin resistance (Spec, 100mg/L), gentamicin resistance (Gent 50. mu.g/mL), rifampicin (RIF 10. mu.g/mL) for screening, and single colonies were picked for PCR identification.

The identified single colony was preserved in Agrobacterium glycerol, and 100. mu.l of the culture broth was added with 5mL of LB (Spec 50. mu.g/mL, Rif 50. mu.g/mL) before transformation, and cultured overnight at 30 ℃ with shaking at 220 rpm. The 5mL of the suspension was poured into 50mL of LB (Spec 50. mu.g/mL, Rif 50. mu.g/mL, Gent 50. mu.g/mL), and cultured at 30 ℃ for 5 hours with shaking at 220 rpm. The cells were collected by centrifugation at 3000rpm for 10 minutes. The supernatant was discarded, and 20ml of a liquid co-culture medium (1 XMS, 2% sucrose, pH5.8 (3.4 mg/l of 6-benzylaminopurine (6-BA), 0.3mg/l of indolebutyric acid (IBA), 200. mu.M Acetosyringone (AS)) was added to suspend the cells for later use.

Forest strawberries (variety YW5AF7) grown in a plant room were taken and young leaves were just spread, and enough leaves were taken for each experiment. Cutting leaves perpendicular to main veins, cutting each leaflet into small leaves according to size, pricking 2-3 small holes on each small leaf, immediately transferring into the previously prepared liquid suspension culture medium, and shaking at 22 ℃ and 40rpm for 1-3 h.

The explants were removed, the bacterial solution was blotted dry with sterile filter paper and transferred to solid co-culture medium (MS +6-BA3.4mg/l, + IBA0.3mg/l + AS 200. mu.M), and cultured in the dark at 22 ℃ for 3 days.

After dark culture, the transformed leaves were washed aseptically with 3 washes at 22 ℃ 200rpm for 5 minutes each, after which the explants were bathed with sterile water supplemented with 500. mu.g/mL carbenicillin (Carb) and 500. mu.g/mL timentin (tim) for 1h, the liquid was blotted with sterile filter paper, transferred to sterile medium (MS + Carb 250. mu.g/mL + timn 250. mu.g/mL, 6-BA3.4mg/L, IBA0.3mg/L), incubated at 22 ℃ for 7 days with low light, and then transferred to 22 ℃ light culture.

Thereafter, the cells were transferred to new selection medium (MS + Carb 250. mu.g/mL + timn 250. mu.g/mL, Kan2.0-5. mu.g/mL (increasing and decreasing), 6-BA3.4mg/L, IBA0.3mg/L) every 3 weeks.

After the adventitious bud (or adventitious bud cluster) is induced, the carrier with GFP fluorescence is used for screening positive plants, and the positive plants are transferred to a rooting culture medium (0.01mg/LIBA, 2% glucose, 1 xMS, pH 5.8) for rooting culture.

And (4) hardening the rooted seedlings, transplanting the rooted seedlings to a soil matrix, and transferring the soil matrix to a plant culture room for culture to obtain the strawberry plant.

Through transgene identification (figure 1), 6 fertile plants and 9 sterile plants are obtained. It is found that from the seedling stage to the bud stage, the transgenic plant has no obvious morphological difference with the wild plant. In the flowering period, the transgenic plant has normal calyx and petal development and no obvious difference with wild plants. However, in the fruiting period, 9 plants were sterile (see FIG. 1 for results).

The androecium phenotype of the transgenic fertile and sterile plants is detected, the development change of the androecium and anther of the transgenic plants is obvious, and the anther is full in the transgenic fertile plants; the transgenic sterile plants show that the anthers are shrunken and not full. The wild type is respectively used as a female parent and a male parent to be hybridized with the sterile plant, and the result shows that the sterile type is male sterility. Transgenic plants had lower pollen numbers and lower vigor than wild type (see figure 2 for results).

The embodiment can show that the FvAGI gene provided by the invention can be transferred into strawberries to obtain strawberry male sterile plants.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> agricultural science and academy of Jiangsu province

<120> FvAGI gene, expression vector and application

<141> 2018-11-21

<160> 3

<170> SIPOSequenceListing 1.0

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gtataatcct aaattggtcc tactcttctt cctcttctca aattcctcaa agtttcacaa 60

agttttgtgc tttcctagct tcaagtgttc ttggcaaaaa agaagtgtgt tcttggcaaa 120

acttcagcaa aaactttggt ttttaaaagt aattggggac tggtgttgaa gattcttgct 180

tctgcaacag atctttgggt gtggattcaa tcacagaaac ataattaatc attgttctgt 240

actcagctga gagagcctga gatggagtat tctgtctttc tctttcttga accaaagaac 300

cagtacttgt ctcttttttt cttctatttt tttttttcct tctatgtgct ctagttagtt 360

ttttttcatt ctgtttatga ccaatttctt agctcttggt ttggtagaag catagatctg 420

gtgttaaaca tagaaaaaat agtatataag ggatggaaat agaaactagt agtgggtttt 480

ggaatgtatg cagggctctg agatcagctc cttgccttcc tccattgctg tttccttttt 540

ctctttttta gtttctcttt ctctctcgct cactctgtgc aacactctct tatgtcatct 600

atgaagctaa gatggtctac aacacaagct gtcacgctcc tattagccca gtccccgatc 660

tccttgacac ttcctcaata gcacatcagt tatggacacc aagaaagtaa aagtacaatt 720

ttattttatt ttctataaaa aaaaaaggat aagaccatgg ctggaaaaaa gaaagggcac 780

aaaagattag ctagggtttt ttccaaaaca cccaaatggg ttatccttga actgtgttgg 840

agtttacctt tgttctaaca accaaatcag gcaacatcta gtcacaccaa gatgaggtat 900

tccccaaatc tgactttcct cacctggaaa atcaaacaca ggaggtttta acctaattag 960

ggtttattag caactgcaat tagtaatcac ttggcccaag atctatactg ggtcaccctt 1020

aaatcaagta aaaatgagat attttcaccc atccaatcaa aacccagttc caaaatgttt 1080

tcttttccct taaatttctg accttttctg ggaaatgggt gtgttctggt tagggttagt 1140

tttggtcttt ggttcacaga atctttggtc acgtcacctc gagatttttc ggttcctcat 1200

ttctgacaaa agagttctat gtcatgattc acagtggggt tacttctctt tccttcttga 1260

accctaaatt tcaatttctt tcttggatct catagaaaat attagctctt tctttatagt 1320

tttctttcag atttgctctt aaagctctca gtcaccccag ccaatgagct cacagctctc 1380

gactgggcca tcaatgagtc actgttggca ttctggagag acacttttaa tttcctcaga 1440

atcttgtata gatatatttg tgcaagtttt gaagggtgtg gagaagagag agagagagag 1500

agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 1560

agagagagag agagagagag agagaaagtg aggagtgttg cagaaacaaa gatatgaaat 1620

ctgcgagcca atgggaagtt ctgagctttt ggcttattat gactgcttct gaaacagacg 1680

gctttgatgt tgtcaatgtc atcggagttt catatagaaa agactggttc tttattatat 1740

atttactgtc agtaagtttt tcaaatcctt tcgaggtggg taatagtgta aggtttaggg 1800

taatatccct tgggtaattg gtgggagaga acatggcaga gtgaattccg gccaatcaga 1860

ttggggagct gtgccatcgt ggcaacaggt agccaatcac aagctcgacg gttttaaggt 1920

attatcagag gtgcatagaa aagagtgtga gtactgttgg atcaatttga ccgaggtaga 1980

tgtctttctt tattaggctc agttaaactg agagagtgag agagtgagtt catttgtcta 2040

ctagtctaat ggcctaagtg tgagcgaggt ttatggttta tcataatttc tacttaaccc 2100

tcaatttttg gggttttgtt tagttatctc ctgagcagaa agctgtaatc ttggatgtgt 2160

atcacatctg tcacatttga aattgaccca gtctaaaaca cacaactggg tttcttcctt 2220

ggcacattga gattttcttg attcttatat tagatacagt taagtagcta gcttagagat 2280

ttggtttttt ttttttgagc taatgaagag tgcaggtctt tgctttacca gatatagagc 2340

attcagacaa cccgttttat tctttcaact tcaagcatta gtttgtagga acattttcaa 2400

tttccctttt caccataaaa aacttcgatc aatgaccttt tccctctata catatatcat 2460

aatgttctag ctcacccttt tggctaaatt ttttaatctt tctccgtttc ctattgaggc 2520

gtggctcatt gcgtgcctca tttagttcta ttttgtgtaa accaaattct ttcattgtag 2580

atatgtagta aactaaagta ttagcgtgca tattcctatt ctattaaaat tcatgatcat 2640

agattcaaat ttagattatt cttcatttat ttgcttgttt tagtagagat atattttctt 2700

gttctatatc aaagtactgc tagtccttta ttcttgggtt ctgtcttttc tcatatctca 2760

aaatggtaca tgaggatatc agaaattctc tttcacattc agtgttgtgc atgtgtgaat 2820

agatgatgaa tgtaaagcca cacaataatt caataaaacc tgttcagtaa ctgtaacttt 2880

gatcatggcc gggtcaagaa ttatgcttta tgttaattag tcacttccgg atgttgtaat 2940

atgtaccctt atatagtatc ttatttcatg ctttgattgc ttagcag 2987

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<213> Artificial Sequence (Artificial Sequence)

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caccgtataa tcctaaattg gtcctactc 29

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctgctaagca atcaaagcat gaaataaga 29

Claims (5)

1.FvAGIApplication of gene in cultivating male sterile plant of strawberry, and the geneFvAGIThe nucleotide sequence of the gene is shown in SEQ ID No. 1.

2. Application of expression vector in cultivating male sterile plants of strawberries, wherein the expression vector is inserted into vector pK7WG2DFvAGIThe gene is constructed, theFvAGIThe nucleotide sequence of the gene is shown in SEQ ID No. 1.

3. The use of claim 2, wherein said method of growing strawberry male sterile plants comprises:

transferring the expression vector into agrobacterium by adopting an electric shock method;

and (3) transfecting the obtained agrobacterium to strawberry, and then culturing to obtain strawberry male sterile plants.

4. The use according to claim 2, wherein the expression vector is constructed by a method comprising the steps of:

1) using strawberry genome DNA as a template, and carrying out PCR amplification by using FvAGI primer pair to obtainFvAGIA gene;

2) subjecting the product obtained in the step 1) toFvAGIConnecting the gene with a vector pENTR to obtain pENTR-FvAGI;

3) performing Gateway reaction on pENTR-FvAGI obtained in the step 2) and a vector pK7WG2D to obtain pK7WG 2D-FvAGI.

5. The use of claim 4, wherein the FvAGI primer pair comprises an FvAGI forward primer and an FvAGI downstream primer;

the nucleotide sequence of the FvAGI upstream primer is shown as SEQ ID No. 2;

the nucleotide sequence of the FvAGI downstream primer is shown in SEQ ID No. 3.

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