CN107056911B

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

Info

Publication number: CN107056911B
Application number: CN201710524266.XA
Authority: CN
Inventors: 张俊祥; 孙一平; 张志宏; 石伟佳; 王保田
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2017-06-30
Filing date: 2017-06-30
Publication date: 2020-09-29
Anticipated expiration: 2037-06-30
Also published as: CN107056911A

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, and being characterized in that the sequence of the full-length coding region of a strawberry WRKY46 gene for promoting early flowering of plants is shown as a sequence table SEQ ID NO. 1; the amino acid sequence is shown in a sequence table SEQ ID NO. 2. The invention provides a gene FvWRKY46 for promoting early flowering of a strawberry plant and is used for constructing a plant expression vector of the FvWRKY46 gene, the constructed plant expression vector is used for transforming arabidopsis thaliana through agrobacterium to obtain a transgenic arabidopsis thaliana plant and bloom in advance, and the gene is shown to have the function of inducing the arabidopsis thaliana to bloom in advance. The invention provides a technical means and a theoretical basis for regulating and controlling the flowering time of the strawberries by utilizing a genetic engineering technology, and has great application value.

Description

Strawberry transcription factor for promoting early flowering of plants and application thereof

The technical field is as follows:

the invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a strawberry transcription factor for promoting plants to bloom in advance and application thereof.

Background art:

the strawberry is a plant of the genus strawberry of the family Rosaceae, the strawberry fruit is fresh, red and tender, the pulp is juicy, the strawberry is sour, sweet and delicious, and the strawberry has special strong fruit fragrance. The strawberry has high nutritive value and good color, fragrance and taste. Because of its unique flavor and rich nutrient substances, it is one of the most widely cultivated berries and has the reputation of "fruit queen".

Flowering is an important process of plant transformation from vegetative to reproductive growth, an important agronomic trait that determines whether a plant is adapted to a particular cultivation area and growing season. Plant flowering is the key to the change of plant growth from vegetative to reproductive growth. Under the influence of external environment and internal factors, plants can bloom at proper time and then grow in a reproductive mode. By adjusting the flowering period, the plants are enabled to bloom in a delayed or advanced way, the vegetative growth or reproductive growth of the plants can be controlled, and the harm of adverse circumstances to crops is avoided. The flowering in the most suitable period can achieve the maximum benefit, promote the accumulation, distribution and effective utilization of resources and have great significance for improving the crop yield.

In the long-term evolution process, plants form a complete set of mechanisms for regulating the growth and development of the plants to adapt to or resist external biotic and abiotic stresses, and the regulation process is controlled by a complex network formed by multiple genes on a molecular level and is realized by single or synergistic regulation of a plurality of transcription factors.

Transcription factors, also known as trans-acting factors, have a major function of activating or inhibiting the transcriptional effects of genes (Arce et al 2008; Jain M2008). The WRKY transcription factor family is a specific transcription factor discovered in the last decade, is named because the N-terminal of the WRKY transcription factor family contains a highly conserved WRKYGQK amino acid sequence, and plays an important role in regulating and controlling the growth, development, metabolism and stress response of plants (Rushton et al 2010; Su Qi 2007). The most important research on WRKY transcription factors is the mechanism related to disease resistance, mainly involving in biotic and abiotic stress response and plant senescence, and few reports on the research on plant development regulation are reported, and only about the research on the regulation of seed and epidermal hair development (Yudi et al 2006; Yanchong et al 2010). So far, there are few reports on their involvement in plant flowering regulation.

The invention content is as follows:

(1) the invention provides a strawberry WRKY transcription factor FvWRKY46 for regulating plant flowering, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 1. The protein coded by the strawberry FvWRKY46 gene for regulating and controlling early blossoming of the plant has an amino acid sequence shown in SEQ ID NO.2 in a sequence table.

(2) The invention provides a plant expression vector pRI101-GFP containing the strawberry WRKY transcription factor FvWRKY 46. The FvWRKY46 gene was cloned into pRI101-GFP to obtain pRI101-GFP-CaMV35S-FvWRKY 46.

(3) The gene FvWRKY46 is applied to cultivating early flowering plants. Specifically, the FvWRKY46 gene is transferred into a target plant through an expression vector. The plant is preferably the model plant Arabidopsis thaliana.

(4) The invention has the beneficial effects that: the existing plant genetic engineering technology and the gene expression analysis, gene cloning and sequence analysis technology are utilized to separate and identify the related gene sequence information of strawberry flowering, the gene is transferred into arabidopsis thaliana by an agrobacterium-mediated transformation method, and identification proves that the bolting and flowering time of a transgenic plant is obviously earlier than that of a wild type (figure 5), so that the FvWRKY46 participates in the flowering process of regulating the plant.

Description of the drawings:

FIG. 1 result of amplification of the sequence of the coding region of FvWRKY46 gene.

FIG. 2 fluorescent quantitative PCR analysis of FvWRKY46 gene expression in different tissues and organs.

FIG. 3 construction of recombinant vector pRI101-GFP-CaMV35S-FvWRKY 46.

(a) And E.coli WRKY46-pMD18-T PCR detection electrophoresis result.

(b) And (3) detecting an electrophoresis result of the PCR of the Escherichia coli pRI101-GFP-CaMV35S-FvWRKY 46.

(c) Sequencing result of pRI101-GFP-CaMV35S-FvWRKY 46.

FIG. 4 identification of resistance in transgenic Arabidopsis

FIG. 5 comparison of flowering time of transgenic Arabidopsis and wild type control shows that transgenic plants bloom earlier than wild type.

The specific implementation mode is as follows:

example 1

Cloning of strawberry WRKY46 Gene

(1) Diploid forest strawberry 'Ruegen' is used as a test material, and the material is grown in a greenhouse.

(2) RNA extraction: and (3) extracting the total RNA of the test material by using a CTAB method, wherein the whole operation process is carried out according to the RNA extraction process of the CTAB method, and then carrying out reverse transcription by using the total RNA as a template to obtain a first cDNA chain.

(3) Cloning of the genes: and (3) performing PCR amplification by using a reverse transcription fruit cDNA first chain as a template and using primers WRKY46-F and WRKY46-R, and recovering a PCR product to obtain a 1107bp target fragment.

WRKY46-F：GCAGGTACCATGTCAAATGAAAAGAAAAGCCC

WRKY46-R：GCAGAATTCTGGCTCCTCCAGCTTGTGAC

Note: the first nine bases in the primer sequences of WRKY46-F and WRKY46-R, namely GCAGGTACC and GCAGAATTC, are needed for constructing a vector, and enzyme cutting sites and protective bases which are not in the FvWRKY46 gene sequence are artificially added. The WRKY46-R primer eliminates the stop codon for the construction of GFP fusion expression vector.

Example 2

Construction of plant expression vectors

(1) After recovering the target fragment, the gel was ligated to pMD18-T vector (purchased from TaKaRa), transformed into E.coli competent cell Trans5 alpha (purchased from Beijing Quanjin Biotechnology Co., Ltd.), screened for positive single colonies, extracted for plasmids, and sequenced.

(2) Selecting EcoRI and Kpn I to respectively carry out double enzyme digestion on WRKY46-pMD18-T recombinant plasmid and pRI101-GFP plasmid with correct sequencing, recovering a large vector fragment and a target gene fragment, transforming escherichia coli Trans5 alpha competent cells (purchased from Beijing all-purpose gold biotechnology, Inc.) after connecting by using T4 ligase, and identifying recombinants to obtain a plant expression vector with target genes.

Example 3

Transformation of Arabidopsis thaliana for functional verification

(1) The flower dipping method infests Arabidopsis thaliana.

An Agrobacterium GV3101 positive clone containing the gene of interest WRKY46 was selected, inoculated into LB + Kan (kanamycin) 50mg/L + Rif (rifampicin) 25mg/L liquid YEP medium, cultured at 28 ℃ for 24 hours with shaking at 200rpm, and 1ml of the cultured bacterial solution was added to 50ml of liquid YEP medium to activate it so that OD 600 became about 0.8.

Transferring the bacterial liquid into a sterile 50ml centrifuge tube, centrifuging at 5000rpm for 10min to collect strains, then re-suspending the agrobacterium with the same volume of MS re-suspension (1/2MS +0.5g/L MES + 5% Sucrose, pH5.7), and adding a surfactant Silwet to make the final concentration reach 0.03% (300 mu L/L).

The inflorescences of freshly flowering Arabidopsis plants were soaked in this solution for 30 s. And (3) covering a membrane on the arabidopsis inflorescence dipped with the bacterial liquid for moisture preservation, culturing in the dark for about 24 hours, uncovering the membrane, and normally managing and maturing under the illumination to obtain seeds.

(2) Resistance identification of transgenic plants

Collecting the seeds of the current generation transgenic plants (T)₀Passage), seeded on 1/2MS solid medium containing 30mg/L kanamycin for selection. Transplanting the green resistant plants growing for about 2 weeks into a nutrition pot, wherein the ratio of the matrix of grass carbon, vermiculite and perlite is 5: 3: 1 (volume ratio). Collecting T₁And (5) seed generation. Sowing the seeds on 1/2MS culture medium containing 30mg/L kanamycin, selecting positive plants, collecting T₂Generation of seeds, sowing the seeds on 1/2MS medium containing 30mg/L kanamycin, the seeds are all green seedlings on the medium, proving T₂The generation is a homozygous system.

(3) And (5) carrying out phenotype identification on the transgenic plant.

Transgenic Arabidopsis thaliana T₂The generation plant (transgenic Arabidopsis thaliana transferred with pRI101-GFP-CaMV35S-FvWRKY46 expression vector) and the wild Arabidopsis thaliana were cultured under the condition of short day (8h light/16 h dark). Each strain was tested for 15 individuals and the results are shown in table 1.

TABLE 1

Phenotype	Flowering time	Lotus leaf
			Wild type Arabidopsis thaliana	33	13.3±1.3
Line 1	19	6.7±0.9
			Line 2	19	6.9±1.1

Compared with wild arabidopsis, the transgenic arabidopsis bolting and flowering have only 7 leaves of rosette leaves, different transgenic lines flower earlier than the wild arabidopsis, under the same short-day culture condition, the wild arabidopsis needs 33 days to flower, and the transgenic arabidopsis transformed with the FvWRKY46 gene only needs 19 days to flower. According to results, over-expression of FvWRKY46 can promote early flowering of Arabidopsis. These results indicate that strawberry FvWRKY46 is a functional gene and has the effects of promoting reproductive growth and shortening flowering time.

Sequence listing

SEQ ID NO:1

ATGTCAAATGAAAAGAAAAGCCCTTACCTACAGTATGACCCTTTCGATTACAACCCCCACGAAATCGACAGGTCAAGCTTTCCATTCTTCAATTACAGCTCTAGTTCCATATACAATATTCCCCAAACACCAGCAGACCCCCAAACTCAAAGCCTACATGGATTTGAATCTGATCCTTCGTATTTGATGAGCTTCGCCGACTGCTTAAATGGGTCAATGGACTACACCACCCTCTCAAAAGCCTTTGATATTTCTTGTTCATCATCTGAAGTCATTTCTCCGCCGCTGTTAGATCAGGACGATCATTCGAAAAACAAGGCTGCAGCTGCTGTTGTAGGAGATCAAAATCCATCCACACCGAACTCATCGATATCTTGTTCATCTAATGAAGCTGGTGCTGCTCGTCATCATGAGCATGAAAACAATCAAGATTCAGATAAGATCAGCAAGAAAGATAAGCATCAGAAGGTAGTACAGTCAGCTGAAGAAGCTCCTGGAGATCATGAAGACGAGTCAAAGAAAGTGAACAAGGCAAAAAAGAAAGAGAAACGGCAGAGGGAACCGCGGTTCGCGTTCTTGACCAAGAGTGAAATTGATCATCTTGAAGATGGCTACAGATGGAGAAAGTACGGACAGAAGGCAGTCAAGAATAGCCCTTATCCTAGAAGTTATTACAGATGCACTACTCAGAAGTGCAATGTAAAGAAACGTGTGGAGAGATCGTTCCACGATCCGGCGACTGTGATCACAACATATGAAGGGCAGCACAACCATCAATGTCCAGCGACACTTCGAGGCAGTATGAATGCTGTTGGCATGTTGACACCTTCCTTTTTGGGAGGATTCGGGACCAGTAACAACGGATCCCCAAGATTTCCACACCAATTATTGTTAACTCAACTGCTTACTCCAGTCGATAACAACCAACTTCAACTGCAAGCTCAACATCATTATGATCCAGCAGGTTCTATTTTCTACTCAAATCTCATGAGTACTCTTCATCAGAATCAACCGCAGCAGCAGCAGCAGCATGTACATGTTCCTCATGACTACAATTTGTTGCAAGATTTAGCTCCCTCATTTAGTCACAAGCTGGAGGAGCCATGA

SEQ ID NO:2

MSNEKKSPYLQYDPFDYNPHEIDRSSFPFFNYSSSSIYNIPQTPADPQTQSLHGFESDPSYLMSFADCLNGSMDYTTLSKAFDISCSSSEVISPPLLDQDDHSKNKAAAAVVGDQNPSTPNSSISCSSNEAGAARHHEHENNQDSDKISKKDKHQKVVQSAEEAPGDHEDESKKVNKAKKKEKRQREPRFAFLTKSEIDHLEDGYRWRKYGQKAVKNSPYPRSYYRCTTQKCNVKKRVERSFHDPATVITTYEGQHNHQCPATLRGSMNAVGMLTPSFLGGFGTSNNGSPRFPHQLLLTQLLTPVDNNQLQLQAQHHYDPAGSIFYSNLMSTLHQNQPQQQQQHVHVPHDYNLLQDLAPSFSHKLEEP

SEQUENCE LISTING

<110> Shenyang agriculture university

<120> strawberry transcription factor for promoting early flowering of plants and application thereof

<130>2017-6-30

<160>2

<170>PatentIn version 3.3

<210>1

<211>1107

<212>DNA

<213> Artificial Synthesis

<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

aaagagaaacggcagaggga 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> Artificial Synthesis

<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 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

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.