CN107056911B - Strawberry transcription factor for promoting early flowering of plants and application thereof - Google Patents

Abstract

The invention relates to a strawberry transcription factor for promoting early flowering of plants and application thereof, belonging to the field of genetic engineering in molecular biology. NO: 1; the amino acid sequence is shown in SEQ ID NO: 2 of the sequence listing. The invention provides a strawberry early flowering-promoting gene FvWRKY46 and is used to construct a plant expression vector of the FvWRKY46 gene. The constructed plant expression vector is transformed into Arabidopsis thaliana by Agrobacterium to obtain transgenic Arabidopsis plants and early flowering, indicating that the gene has the ability to induce Arabidopsis thaliana. mustard early flowering function. The invention provides technical means and theoretical basis for regulating the flowering time of strawberries by using genetic engineering technology, and has great application value.

Description

A strawberry transcription factor that promotes early flowering of plants and its application

Technical field:

The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to a strawberry transcription factor for promoting early flowering of plants and its application.

Background technique:

Strawberry is a plant of the Rosaceae Strawberry genus. The strawberry fruit is delicious, red and tender, the pulp is juicy, sweet and sour, and has a special rich fruit aroma. Strawberries have high nutritional value and are excellent in color, aroma and taste. It is one of the most widely cultivated berries and is known as the 'Queen of Fruits' due to its unique flavor and rich nutrient content.

Flowering is an important process in which plants change from vegetative growth to reproductive growth. It is an important agronomic trait that determines whether plants are adapted to specific cultivation areas and growing seasons. Plant flowering is the key to the change of plants from vegetative to reproductive growth. Under the influence of external environment and internal factors, plants will flower and reproduce at the right time. By adjusting the flowering period to delay or advance flowering of plants, the vegetative or reproductive growth of plants can be controlled and the damage to crops by adversity can be avoided. Flowering at the most suitable period can achieve the greatest benefit, promote the accumulation, distribution and effective utilization of resources, which is of great significance to improving crop yield.

In the long-term evolution process, plants have formed a complete set of mechanisms for regulating their own growth and development to adapt to or resist external biotic and abiotic stresses. This regulatory process is at the molecular level by the complex network formed by multiple genes. Regulation is achieved by many transcription factors alone or in concert.

Transcription factors are also called trans-acting factors, and their main function is to activate or repress the transcriptional effects of genes (Arce et al. 2008; Jain M 2008). The WRKY transcription factor family is a class of plant-specific transcription factors discovered in the past ten years. It is named because its N-terminal contains the highly conserved WRKYGQK amino acid sequence. It plays a role in regulating plant growth, development, metabolism and stress response. important role (Rushton et al. 2010; Su Qi 2007). The most studied WRKY transcription factor is its mechanism related to disease resistance, which is mainly involved in biotic and abiotic stress responses and plant senescence. There are few reports on the regulation of plant development, only the regulation of the development of seeds and epidermal hairs ( Yu Diqiu et al. 2006; Yu Yanchong et al. 2010). To date, little has been reported on their involvement in plant flowering regulation.

Invention content:

(1) The present invention provides a strawberry WRKY transcription factor FvWRKY46 that regulates plant flowering, and the nucleotide sequence of the gene is shown in SEQ ID NO.1. The protein encoded by the strawberry FvWRKY46 gene that regulates early flowering of plants has the amino acid sequence described in SEQ ID NO. 2 in the sequence listing.

(2) The present invention provides a plant expression vector pRI101-GFP containing the above strawberry WRKY transcription factor FvWRKY46. The FvWRKY46 gene was cloned into pRI101-GFP to obtain pRI101-GFP-CaMV35S-FvWRKY46.

(3) Application of the gene FvWRKY46 of the present invention in cultivating early flowering plants. Specifically, the FvWRKY46 gene is transferred into the target plant through an expression vector. The plant is preferably the model plant Arabidopsis thaliana.

(4) Beneficial effect of the present invention: utilize existing plant genetic engineering technology, utilize gene expression analysis, gene cloning and sequence analysis technology, isolate and identify the relevant gene sequence information of strawberry flowering, and by the transformation method mediated by Agrobacterium tumefaciens The gene was transformed into Arabidopsis thaliana, and the identification showed that the bolting and flowering time of the transgenic plants were significantly earlier than those of the wild type (Fig. 5), indicating that FvWRKY46 was involved in regulating the flowering process of plants.

Description of drawings:

Figure 1 Amplification results of the coding region sequence of FvWRKY46 gene.

Figure 2 Fluorescence quantitative PCR analysis of the expression of FvWRKY46 gene in different tissues and organs.

Figure 3 Construction of recombinant vector pRI101-GFP-CaMV35S-FvWRKY46.

(a) Electrophoresis results of E. coli WRKY46-pMD18-T PCR detection.

(b) PCR electrophoresis results of E. coli pRI101-GFP-CaMV35S-FvWRKY46.

(c) Sequencing results of pRI101-GFP-CaMV35S-FvWRKY46.

Figure 4. Resistance identification of transgenic Arabidopsis

Fig. 5 Comparison of flowering time between transgenic Arabidopsis and wild-type control, transgenic plants flowered earlier than wild-type.

Detailed ways:

Example 1

Cloning of Strawberry WRKY46 Gene

(1) The diploid forest strawberry ‘Ruegen’ was used as the test material, and the material was grown in a greenhouse.

(2) RNA extraction: the total RNA of the test material was extracted by the CTAB method. The whole operation process followed the RNA extraction process of the CTAB method, and then reverse transcribed the total RNA as a template to obtain the first strand of cDNA.

(3) Gene cloning: The first strand of the reverse transcribed fruit cDNA was used as the template, and the primers WRKY46-F and WRKY46-R were used for PCR amplification, and the PCR product was recovered to obtain the target fragment of 1107 bp.

WRKY46-F: GCAGGTACCATGTCAAATGAAAAGAAAAGCCC

WRKY46-R: GCAGAATTCTGGCTCCTCCAGCTTGTGAC

Note: The first nine bases in the WRKY46-F and WRKY46-R primer sequences, namely GCAGGTACC and GCAGAATTC, are required for the construction of the vector, and are artificially added restriction sites and protective bases that do not belong to the FvWRKY46 gene sequence. The stop codon of the WRKY46-R primer was removed due to the need for constructing a GFP fusion expression vector.

Example 2

Construction of plant expression vector

(1) after the target fragment was recovered by the gel, it was connected to the pMD18-T vector (purchased from TaKaRa company), then transformed into Escherichia coli competent cell Trans5α (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the positive single colony was screened, Plasmids were extracted and sequenced.

(2) Double-enzyme digestion of the correctly sequenced WRKY46-pMD18-T recombinant plasmid and pRI101-GFP plasmid was performed with EcoRI and KpnⅠ, and the large vector fragment and the target gene fragment were recovered, ligated with T4 ligase, and transformed into E. coli Trans5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), after identifying the recombinant, a plant expression vector with the target gene was obtained.

Example 3

Transforming Arabidopsis for functional validation

(1) Infecting Arabidopsis thaliana by dipping flower method.

Pick an Agrobacterium GV3101 positive clone containing the target gene WRKY46, inoculate it in the liquid YEP medium of LB+Kan (Kanamycin) 50mg/L+Rif (rifampicin) 25mg/L, and shake it at 28°C and 200rpm. 24h, take 1ml of cultured bacterial liquid, add 50ml liquid YEP medium to activate, make OD 600=0.8.

The bacterial solution was transferred to a sterile 50ml centrifuge tube, centrifuged at 5000rpm for 10min to collect the bacteria, and then resuspended with the same volume of MS resuspended solution (1/2MS+0.5g/L MES+5%Sucrose, pH5.7). Agrobacterium was suspended, and the surfactant Silwet was added to make the final concentration 0.03% (300 μl/L).

Immerse freshly bloomed Arabidopsis plant inflorescences in this solution for 30 s. The Arabidopsis thaliana inflorescence immersed in the bacterial solution was covered with a moisturizing film, cultivated in the dark for about 24 hours, and then the film was removed and placed in the light for normal management to mature and harvest the seeds.

(2) Resistance identification of transgenic plants

The seeds of contemporary transgenic plants (T ₀ generation) were collected and sown on 1/2 MS solid medium containing 30 mg/L kanamycin for selection. The green resistant plants that had grown for about 2 weeks were transferred into a nutrient pot, and the substrate was peat, vermiculite and perlite in a ratio of 5:3:1 (volume ratio). T ₁ generation seeds were collected. The seeds were sown on 1/2MS medium containing 30mg/L kanamycin, positive plants were selected, the T ₂ generation seeds were collected, and the seeds were sown in 1/2MS medium containing 30mg/L kanamycin Above, the seeds on the medium were all green shoots, which proved that the T ₂ generation was a homozygous line.

(3) Phenotypic identification of transgenic plants.

Transgenic Arabidopsis T ₂ generation plants (transgenic Arabidopsis transformed into pRI101-GFP-CaMV35S-FvWRKY46 expression vector) and wild-type Arabidopsis were cultured under short-day (8h light/16h dark) conditions. 15 individual plants were tested for each line, and the test results are shown in Table 1.

Table 1

Phenotype flowering time rosette leaves wild type Arabidopsis 33 13.3±1.3 Strain 1 19 6.7±0.9 Strain 2 19 6.9±1.1

Compared with wild-type Arabidopsis, transgenic Arabidopsis had only 7 leaves for bolting and rosettes at flowering, and different transgenic lines bloomed earlier than wild-type Arabidopsis. Under the same short-day culture conditions, wild-type Arabidopsis thaliana had a The mustard flower takes 33 days, while the transgenic Arabidopsis thaliana transformed with the FvWRKY46 gene takes only 19 days to flower. According to the results, overexpression of FvWRKY46 can promote early flowering in Arabidopsis. These results indicate that strawberry FvWRKY46 is a functional gene, which can promote reproductive growth and shorten flowering time.

sequence listing

SEQ ID NO: 1

ATGTCAAATGAAAAGAAAAGCCCTTACCTACAGTATGACCCTTTCGATTACAACCCCCACGAAATCGACAGGTCAAGCTTTCCATTCTTCAATTACAGCTCTAGTTCCATATACAATATTCCCCAAACACCAGCAGACCCCCAAACTCAAAGCCTACATGGATTTGAATCTGATCCTTCGTATTTGATGAGCTTCGCCGACTGCTTAAATGGGTCAATGGACTACACCACCCTCTCAAAAGCCTTTGATATTTCTTGTTCATCATCTGAAGTCATTTCTCCGCCGCTGTTAGATCAGGACGATCATTCGAAAAACAAGGCTGCAGCTGCTGTTGTAGGAGATCAAAATCCATCCACACCGAACTCATCGATATCTTGTTCATCTAATGAAGCTGGTGCTGCTCGTCATCATGAGCATGAAAACAATCAAGATTCAGATAAGATCAGCAAGAAAGATAAGCATCAGAAGGTAGTACAGTCAGCTGAAGAAGCTCCTGGAGATCATGAAGACGAGTCAAAGAAAGTGAACAAGGCAAAAAAGAAAGAGAAACGGCAGAGGGAACCGCGGTTCGCGTTCTTGACCAAGAGTGAAATTGATCATCTTGAAGATGGCTACAGATGGAGAAAGTACGGACAGAAGGCAGTCAAGAATAGCCCTTATCCTAGAAGTTATTACAGATGCACTACTCAGAAGTGCAATGTAAAGAAACGTGTGGAGAGATCGTTCCACGATCCGGCGACTGTGATCACAACATATGAAGGGCAGCACAACCATCAATGTCCAGCGACACTTCGAGGCAGTATGAATGCTGTTGGCATGTTGACACCTTCCTTTTTGGGAGGATTCGGGACCAGTAACAACGGATCCCCAAGATTTCCACACCAATTATTGTTAACTCAACTGCTTACTCCAGTCGATAACAACCAACTTCAACTGCAAGCTCAACATCATTATGATCCAGCAGGTTCTATTTTCTACTCAAATCTCATGAGTACTCTTC ATCAGAATCAACCGCAGCAGCAGCAGCAGCATGTACATGTTCCTCATGACTACAATTTGTTGCAAGATTTAGCTCCCTCATTTAGTCACAAGCTGGAGGAGCCATGA

SEQ ID NO: 2

MSNEKKSPYLQYDPFDYNPHEIDRSSFPFFNYSSSSIYNIPQTPADPQTQSLHGFESDPSYLMSFADCLNGSMDYTTLSKAFDISCSSSEVISPPLLDQDDHSKNKAAAAVVGDQNPSTPNSSISCSSNEAGAARHHEHENNQDSDKISKKDKHQKVVQSAEEAPGDHEDESKKVNKAKKKEKRQREPRFAFLTKSEIDHLEDGYRWRKYGQKAVKNSPYPRSYYRCTTQKCNVKKRVERSFHDPATVITTYEGQHNHQCPATLRGSMNAVGMLTPSFLGGFGTSNNGSPRFPHQLLLTQLLTPVDNNQLQLQAQHHYDPAGSIFYSNLMSTLHQNQPQQQQQHVHVPHDYNLLQDLAPSFSHKLEEP

SEQUENCE LISTING

<110> Shenyang Agricultural University

<120> A strawberry transcription factor that promotes early flowering of plants and its application

<130> 2017-6-30

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1107

<212> DNA

<213> Synthetic

<400> 1

atgtcaaatg aaaagaaaag cccttaccta cagtatgacc ctttcgatta caacccccac 60

gaaatcgaca ggtcaagctt tccattcttc aattacagct ctagttccat atacaatatt 120

ccccaaacac cagcagaccc ccaaactcaa agcctacatg gatttgaatc tgatccttcg 180

tatttgatga gcttcgccga ctgcttaaat gggtcaatgg actacaccac cctctcaaaa 240

gcctttgata tttcttgttc atcatctgaa gtcatttctc cgccgctgtt agatcaggac 300

gatcattcga aaaacaaggc tgcagctgct gttgtaggag atcaaaatcc atccacaccg 360

aactcatcga tatcttgttc atctaatgaa gctggtgctg ctcgtcatca tgagcatgaa 420

aacaatcaag attcagataa gatcagcaag aaagataagc atcagaaggt agtacagtca 480

gctgaagaag ctcctggaga tcatgaagac gagtcaaaga aagtgaacaa ggcaaaaaag 540

aaagagaaac ggcagaggga accgcggttc gcgttcttga ccaagagtga aattgatcat 600

cttgaagatg gctacagatg gagaaagtac ggacagaagg cagtcaagaa tagcccttat 660

cctagaagtt attacagatg cactactcag aagtgcaatg taaagaaacg tgtggagaga 720

tcgttccacg atccggcgac tgtgatcaca acatatgaag ggcagcacaa ccatcaatgt 780

ccagcgacac ttcgaggcag tatgaatgct gttggcatgt tgacaccttc ctttttggga 840

ggattcggga ccagtaacaa cggatcccca agatttccac accaattatt gttaactcaa 900

ctgcttactc cagtcgataa caaccaactt caactgcaag ctcaacatca ttatgatcca 960

gcaggttcta ttttctactc aaatctcatg agtactcttc atcagaatca accgcagcag 1020

cagcagcagc atgtacatgt tcctcatgac tacaatttgt tgcaagattt agctccctca 1080

tttagtcaca agctggagga gccatga 1107

<210> 2

<211> 368

<212> PRT

<213> Synthetic

<400> 2

Met Ser Asn Glu Lys Lys Ser Pro Tyr Leu Gln Tyr Asp Pro Phe Asp

1 5 10 15

Tyr Asn Pro His Glu Ile Asp Arg Ser Ser Phe Pro Phe Phe Asn Tyr

20 25 30

Ser Ser Ser Ser Ile Tyr Asn Ile Pro Gln Thr Pro Ala Asp Pro Gln

35 40 45

Thr Gln Ser Leu His Gly Phe Glu Ser Asp Pro Ser Tyr Leu Met Ser

50 55 60

Phe Ala Asp Cys Leu Asn Gly Ser Met Asp Tyr Thr Thr Leu Ser Lys

65 70 75 80

Ala Phe Asp Ile Ser Cys Ser Ser Ser Glu Val Ile Ser Pro Pro Leu

85 90 95

Leu Asp Gln Asp Asp His Ser Lys Asn Lys Ala Ala Ala Ala Val Val

100 105 110

Gly Asp Gln Asn Pro Ser Thr Pro Asn Ser Ser Ile Ser Cys Ser Ser

115 120 125

Asn Glu Ala Gly Ala Ala Arg His His Glu His Glu Asn Asn Gln Asp

130 135 140

Ser Asp Lys Ile Ser Lys Lys Asp Lys His Gln Lys Val Val Gln Ser

145 150 155 160

Ala Glu Glu Ala Pro Gly Asp His Glu Asp Glu Ser Lys Lys Val Asn

165 170 175

Lys Ala Lys Lys Lys Lys Glu Lys Arg Gln Arg Glu Pro Arg Phe Ala Phe

180 185 190

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

195 200 205

Lys Tyr Gly Gln Lys Ala Val Lys Asn Ser Pro Tyr Pro Arg Ser Tyr

210 215 220

Tyr Arg Cys Thr Thr Gln Lys Cys Asn Val Lys Lys Arg Val Glu Arg

225 230 235 240

Ser Phe His Asp Pro Ala Thr Val Ile Thr Thr Tyr Glu Gly Gln His

245 250 255

Asn His Gln Cys Pro Ala Thr Leu Arg Gly Ser Met Asn Ala Val Gly

260 265 270

Met Leu Thr Pro Ser Phe Leu Gly Gly Phe Gly Thr Ser Asn Asn Gly

275 280 285

Ser Pro Arg Phe Pro His Gln Leu Leu Leu Thr Gln Leu Leu Thr Pro

290 295 300

Val Asp Asn Asn Gln Leu Gln Leu Gln Ala Gln His His Tyr Asp Pro

305 310 315 320

Ala Gly Ser Ile Phe Tyr Ser Asn Leu Met Ser Thr Leu His Gln Asn

325 330 335

Gln Pro Gln Gln Gln Gln Gln His Val His Val Pro His Asp Tyr Asn

340 345 350

Leu Leu Gln Asp Leu Ala Pro Ser Phe Ser His Lys Leu Glu Glu Pro

355 360 365

Claims (2)

1. An application of a strawberry transcription factor in promoting early flowering of arabidopsis thaliana is characterized in that a coding region sequence of the strawberry transcription factor is shown as SEQ ID No. 1.

2. The use according to claim 1, wherein the amino acid sequence is as shown in SEQ ID No. 2.

Images