Application of transcription factor SlWRKY41 in improvement of drought resistance of tomatoes

Abstract

The invention discloses application of a transcription factor SlWRKY41 in improvement of drought resistance of tomatoes, functional verification is carried out on the SlWRKY41 through a transgenic technology, an application method of a drought-resistant tomato material is created, and research finds that compared with a wild tomato plant, the drought resistance of a transgenic tomato strain overexpressing the SlWRKY41 is improved, a large number of organic infiltration substances are accumulated, and the drought resistance of the tomato plant is improved. The SlWRKY41 has the advantages that the activity of antioxidant enzymes is improved, the accumulation of active oxygen under stress is effectively relieved, the SlWRKY41 is a positive response factor for regulating the drought tolerance of the tomatoes, the regulation and control mechanism for the drought tolerance of the plants is further known, and the SlWRKY41 has important significance on the drought tolerance improvement of the tomatoes.

Description

Technical Field

[0001] The invention relates to the technical field of tomato genetic engineering, and in particular to application of a transcription factor SlWRKY41 in improving drought resistance of tomatoes. Background Art

[0002] Tomato (Solanum lycopersicum L.) is an important vegetable and cash crop, and is very popular around the world. my country is the largest producer of fresh tomatoes and the third largest producer of processed tomatoes, and plays a pivotal role in the world tomato market. Tomatoes belong to the Solanaceae vegetable crops, and are most sensitive to water deficit during the seedling stage. In recent years, global population growth, frequent extreme weather, and water scarcity have exacerbated the impact of drought stress on tomato production. Therefore, analyzing the molecular mechanism of tomato drought resistance and cultivating drought-resistant tomato varieties have become urgent issues to be solved in the process of improving quality and efficiency.

[0003] The growth and development of plants are inevitably affected by many abiotic stresses. In order to adapt to the environment, plants themselves need to cope with stress through various physiological and biochemical adjustments. In the long-term evolutionary process, a series of regulatory mechanisms that are beneficial to themselves and suitable for their own growth and development will gradually form, and transcription factors play an important role in these regulatory processes. Transcription factors, also known as trans-acting factors, can specifically bind to cis-acting elements in the promoter region of eukaryotic genes and activate or inhibit gene transcription. Transcription factors (TFs) are a class of important regulatory proteins that regulate gene expression. TF binds to its related cis-regulatory sequences, acts as a molecular switch in gene expression, and regulates plant signal transduction pathways.

[0004] The WRKY transcription factor family is named after its unique heptapeptide sequence WRKYGQK and is one of the largest transcription factor families in plants. According to the number of domains and zinc finger structure characteristics, WRKY is mainly divided into three categories: Class I contains two WRKY domains, which can be divided into two subclasses (Ia and Ib, with zinc finger structures of C2H2 type and C2HC type, respectively); Class II and Class III contain one WRKY domain, of which Class II zinc finger structure is C2H2 type (IIa-IIe, 5 subclasses), and Class III zinc finger structure is C2HC type. A large number of studies have shown that members of the WRKY transcription factor family are widely involved in responding to various biotic and abiotic stresses. As the main component of responding to adverse stress, WRKY transcription factors bind to the cis-acting element W-box in the promoter region, induce the expression of stress-related target genes, and improve plant stress resistance. When GmWRKY16 was overexpressed in Arabidopsis, the drought tolerance of Arabidopsis was significantly enhanced (Ma et al., 2018); overexpression of GmWRKY54 enhanced the drought tolerance of soybean, while its interference made soybean more sensitive to drought (Wei et al., 2019); compared with the wild type, transgenic rice overexpressing AtWRKY57 showed better physiological state under drought conditions, such as lower leaf water loss rate, cell mortality rate and MDA content (Jiang et al., 2016); SlWRKY81 negatively regulates drought stress response by regulating H2O2-mediated stomatal closure in guard cells (Ahammed et al., 2020); These research results have, to a certain extent, clarified that WRKY transcription factors play an important role in the regulatory network of plant response to drought stress, and can positively regulate downstream gene expression by recognizing and binding to W-box (TTGACC / T) in the promoter, which confers drought stress tolerance to plants through various physiological processes.

[0005] Tomato is one of the most commonly grown solanaceous vegetables in my country, and it has a high demand for water during its growth. As one of the most widely studied transcription factor families in recent years, WRKY transcription factors play an important role in plant resistance to a variety of biotic and abiotic stresses. However, the specific functional mechanisms of most tomato WRKY transcription factors have yet to be fully explored. Therefore, in-depth analysis of the mechanism of action of WRKY transcription factors in regulating tomato drought tolerance is of great significance for the targeted improvement of tomato tolerance to drought stress and the enrichment of tomato germplasm resources. Summary of the invention

[0006] The object of the present invention is to provide an application of a transcription factor SlWRKY41 in improving drought resistance of tomatoes, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: an application of a transcription factor SlWRKY41 in improving drought resistance of tomatoes, wherein the cDNA sequence of the transcription factor SlWRKY41 is shown in SEQ ID NO: 1.

[0008] A method for constructing an overexpression vector of the transcription factor SlWRKY41 for regulating drought tolerance of tomatoes comprises the following steps:

[0009] Step 1: Using Ailsa Craig tomato leaf cDNA as a template, PCR (PCR amplification of SlWRKY41 primer sequence, wherein the forward primer is shown in SEQ ID NO: 4; the reverse primer is shown in SEQ ID NO: 5) polymerase is used to perform PCR amplification to obtain the target fragment, the PCR product is detected by 1% agarose gel electrophoresis, the target fragment is recovered by a recovery kit, PBI121 empty vector is double-digested with SacI and XbaI, the linearized backbone vector is purified and recovered, the gel-recovered target fragment is homologously recombined with the purified backbone vector to construct an overexpression vector;

[0010] Step 2: Use the heat shock method to transform the recombinant product into E. coli DH5α competent cells, and spread it on kanamycin-resistant LB solid medium, pick a single clone bacterial spot for colony PCR identification of positive clones;

[0011] Step 3: The selective marker gene of the PBI121 vector is kanamycin, and the PCR positive clone plasmid is extracted for sequencing verification to obtain the recombinant expression vector of the SlWRKY41 gene, PBI121-SlWRKY41.

[0012] A method for cultivating transgenic plants, the method is applied to the above-mentioned transcription factor SlWRKY41 overexpression vector for regulating drought tolerance of tomatoes, and the coding gene of SlWRKY41 is introduced into a recipient plant to obtain a transgenic plant, comprising the following steps:

[0013] Step 1: The correctly sequenced PBI121-SlWRKY41 was transferred into Agrobacterium tumefaciens GV3101 by freeze-thaw method, and the monoclonal positive strain was screened and verified and stored at -80°C;

[0014] Step 2: Using the plant tissue culture method, inducing callus tissue of wild-type tomato plants, using a bacterial solution containing the target gene and an OD value of 0.4 to prepare a suspension for infiltration, and obtaining transgenic positive plants after cultivation;

[0015] Step 3: Extract DNA from the transgenic T0 generation positive plants, design primers based on the tag sequence on the vector, perform PCR amplification, purify and sequence the amplified products, transplant the identified positive plants to the field for seed preservation, and use the T3 generation plants to observe drought phenotypes and measure physiological indicators.

[0016] Furthermore, the primer sequences used in the PCR amplification for identifying the positive plants in step 3, the primers for identifying the overexpressing plants are: JC-WRKY41-F, as shown in SEQ ID NO: 6, and JC-WRKY41-R, as shown in SEQ ID NO: 7.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention found through transcriptome analysis that the expression of a large number of WRKY family genes under drought stress changed significantly, especially the expression of SlWRKY41 gene increased significantly. The cDNA sequence of the gene is shown in the list SEQ ID NO: 1, which contains 1511 base pairs; the CDS sequence is shown in the list SEQ ID NO: 2, which contains 1012 base pairs; the amino acid sequence of its amino acid coding region is shown in SEQ ID NO: 3, which contains 336 amino acids. Based on the coding region sequence of the SlWRKY41 gene, the present invention obtained overexpression transgenic tomato plants using transgenic technology, and observed their phenotypes and measured their physiological and biochemical indicators after drought stress, and finally preliminarily determined that the gene can improve the drought resistance of tomatoes.

[0019] (2) The present invention uses transcriptome sequencing to analyze the differentially expressed genes of the cultivated tomato variety 'Zhongza No. 9' under normal growth and drought stress, and identifies a gene SlWRKY41 that positively responds to drought, whose expression level is significantly increased under drought stress. Further studies have found that compared with wild-type tomato plants, transgenic tomato lines overexpressing SlWRKY41 have increased drought tolerance and accumulated a large amount of organic osmotic substances, increased antioxidant enzyme activity, and effectively alleviated the accumulation of reactive oxygen under stress, indicating that SlWRKY41 is a positive response factor regulating tomato drought tolerance. Further understanding of the regulatory mechanism of tomato drought tolerance is of great significance for improving tomato drought tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention provides a genetic transformation flow chart of transgenic tomatoes, wherein A is: preparing explants; B is: callus differentiation; C is: generating resistant buds; D is: rooting culture;

[0021] Figure 2This is the PCR verification of the T0 generation transgenic tomatoes provided in the embodiment of the present invention; among them, 8 plants were positive after WRKY41 was transformed to AC (except for 5 and 8, all others were positive);

[0022] Figure 3 The present invention provides an embodiment of the present invention to observe the phenotype of transgenic tomato plants overexpressing WRKY41 under drought conditions, wherein A is: the phenotype of wild-type and overexpression strain tomato seedlings on the 6th day after drought stress treatment (overall front view); B is: the phenotype of wild-type and overexpression strain tomato seedlings on the 6th day after drought stress treatment (overall top view); C is: the phenotype of wild-type and overexpression strain tomato seedlings on the 6th day after drought stress treatment (single plant overall view);

[0023] Figure 4 The present invention provides an example of an analysis of the differences in osmotic regulation substances, oxidase system and ROS accumulation between wild type and overexpression strains under drought stress conditions, wherein A refers to: proline content (mg / g fresh weight); B refers to soluble sugar content (μg / g fresh weight); C refers to: soluble protein content (μg / g fresh weight); D refers to: MDA content (μmol / g fresh weight); E refers to: O2 - content (μg / g fresh weight); F refers to H2 O2 content (μmol / g fresh weight); G refers to: SOD activity (U / g fresh weight); H refers to: POD activity (U / g fresh weight); I refers to: CAT activity (U / g fresh weight);

[0024] Figure 5 It is a flow chart of cultivating tomatoes of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If not otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the reagents used are all conventional commercial products.

[0026] Example 1: A transcription factor SlWRKY41 that regulates drought tolerance in tomato, the cDNA sequence of which is shown in SEQ ID NO: 1.

[0027] Example 2: A transcription factor SlWRKY41 that regulates drought tolerance of tomato, whose CDS sequence is shown in SEQ ID NO: 2.

[0028] Example 3: A protein of the transcription factor SlWRKY41 that regulates drought tolerance of tomato, the amino acid sequence of which is shown in SEQ ID NO:3.

[0029] Example 4: A method for constructing an overexpression vector of the transcription factor SlWRKY41 for regulating drought tolerance of tomatoes, the construction method comprising the following steps:

[0030] Step 1: Using 'Ailsa Craig' tomato leaf cDNA as a template, PCR polymerase (Takara PrimeSTARGXL DNA Polymerase) was used to amplify the target fragment, and the PCR product was detected by 1% agarose gel electrophoresis. The target fragment was recovered using a recovery kit, and the PBI121 empty vector was double-digested with SacI and XbaI, and the linearized backbone vector was purified and recovered. The gel-recovered target fragment was homologously recombined with the purified backbone vector to construct an overexpression vector.

[0031] Step 2: Use the heat shock method to transform the recombinant product into Escherichia coli DH5α competent cells, and spread it on kanamycin-resistant LB solid culture medium, pick single clone bacterial spots for colony PCR identification of positive clones.

[0032] Step 3: The selective marker gene of the PBI121 vector is kanamycin, and the PCR positive clone plasmid is extracted for sequencing verification to obtain the recombinant expression vector of the SlWRKY41 gene, PBI121-SlWRKY41.

[0033] In the above step 1, the primer sequences for PCR amplification of SlWRKY41 are as shown in SEQ ID NO: 4: 5'ATGGAGAAAGTTAAAAGTATGGAGAAAAAG 3'; and the reverse primer is as shown in SEQ ID NO: 5: 5'TTAAATGAAGAATTCTTCAATGTCAAATGTG 3'.

[0034] Example 5: A method for cultivating transgenic plants, comprising introducing the coding gene of SlWRKY41 into a recipient plant to obtain a transgenic plant.

[0035] Step 1: The correctly sequenced overexpression recombinant plasmid was transferred into Agrobacterium tumefaciens GV3101 by freeze-thaw method, and the monoclonal positive strain was screened for verification and stored at -80°C for subsequent Agrobacterium infection.

[0036] Step 2: Use the plant tissue culture method to induce callus tissue of wild-type tomato plants. Use a bacterial solution containing the target gene and an OD value of 0.4 to prepare a suspension for infiltration, and obtain transgenic positive plants through co-cultivation, bud induction differentiation, and rooting.

[0037] Step 3: Extract DNA from the transgenic T0 generation positive plants, design primers based on the tag sequence on the vector, perform PCR amplification, purify and sequence the amplified products, and finally transplant the identified positive plants to the field for seed preservation, and use the T3 generation plants for drought phenotype observation and determination of physiological indicators.

[0038] The primer sequences used in the PCR amplification for identifying positive plants in the above step 3 and the primers for identifying overexpressing plants are: JC-WRKY41-F, as shown in SEQ ID NO: 6: 5'-ATGACGCACAATCCCACTATC-3';

[0039] JC-WRKY41-R, as shown in SEQ ID NO: 7: 5'-ATGGACCTTCAAGCCCATTTA-3'.

[0040] Example 6: Application of a transcription factor SlWRKY41 for regulating drought resistance of tomatoes. The SlWRKY41 gene increases drought resistance of tomatoes by affecting osmotic regulating substances, ROS system and oxidase.

[0041] Combination Figure 1-4 The application principle of the present invention is described in detail.

[0042] The application method of the SlWRKY41 gene for regulating drought tolerance of tomatoes provided in the embodiment of the present invention is: selecting leaves of the cultivated tomato variety 'Zhongza No. 9' under normal growth and drought stress for transcriptome sequencing, and analyzing the differentially expressed genes, from which a gene SlWRKY41 that positively responds to drought stress is identified, and its expression level is significantly increased under drought stress, and its function is annotated as a WRKY type transcription factor. Subsequently, it is found that it is located on chromosome 1 by comparison on the tomato website https: / / solgenomics.net. According to the CDS sequence of the SlWRKY41 gene, an overexpression vector was constructed, and a genetic transformation experiment was carried out to obtain positive plants to determine the function of the gene. The cDNA sequence of the SlWRKY41 gene provided by the present invention is shown in the sequence list SEQ ID NO: 1, which contains 1511 base pairs; the CDS sequence is shown in the sequence list SEQ ID NO: 2, which contains 1012 base pairs; the amino acid sequence of its coding region is shown in the sequence list SEQ ID NO: 3, which contains 336 amino acids.

[0043] Primers were designed according to the full-length CDS sequence shown in SEQ ID NO: 2 in the sequence listing, and PCR amplification was performed using cDNA of Ailsa Craig roots as a template and Takara PrimeSTAR GXL DNA Polymerase enzyme.

[0044] PCR reaction system: cDNA template 1uL, Takara PrimeSTAR GXL DNA Polymerase 25uL, Primer F 1uL, Primer F 1uL, ddH2O 22uL. Purify the PCR product and connect it to the vector backbone before testing.

[0045] The overexpression recombinant plasmid with correct sequencing was transformed into Agrobacterium GV3101, and then transformed into Ailsa Craig using the recombinant Agrobacterium-mediated transformation. Figure 1 .

[0046] The identified PBI121-SlWRKY41 recombinant Agrobacterium was activated by overnight culture in liquid LB medium until the bacterial liquid OD600 = 0.4. The bacteria were collected by centrifugation at 5000 rpm for 10 min, and the bacteria were resuspended in an equal volume of liquid MS medium. The explants were immersed in the resuspension for 10 min and then placed on the co-culture medium and cultured in the dark at 28°C for 2 days, and then cultured under normal light.

[0047] Through tissue culture technology, after co-cultivation, bud induction differentiation, rooting and resistant plant screening, two overexpression transgenic positive plants were obtained, namely OE-WRKY41-1 and OE-WRKY41-5.

[0048] When the complete plants were formed, some leaves were cut to extract DNA for the detection of SlWRKY41 gene in transgenic tomatoes. The overexpression transgenic plants were detected using primers JC-WRKY41-F and JC-WRKY41-R, with sequences of 5'-ATGACGCACAATCCCACTATC-3' and

[0049] 5'-ATGGACCTTCAAGCCCATTTA-3'.

[0050] The DNA of the obtained overexpressed transgenic plants and wild-type plants was extracted respectively. The above DNA was used as a template for PCR amplification reaction, and the PCR product was detected by 1.5% agarose gel electrophoresis. Figure 2 .

[0051] The positive plants identified were transplanted to the field for management, and T0 generation seeds were harvested. After germination in 55-degree warm water, the seeds were sown in plug trays, and when they had three leaves and one heart, they were transferred to the greenhouse for cultivation to obtain T1 generation transgenic plants. According to this step, subculture was continued to T2 generation for seed retention for subsequent physiological and biochemical index determination.

[0052] Verification of the correlation between the SlWRKY41 gene and the antioxidant system, osmotic regulation system, ROS accumulation and phosphorus metabolism indicators under drought stress.

[0053] The transgenic tomato seeds were cleaned, soaked, germinated, and sown; when the seeds had three leaves and one heart, the seedlings with good growth and uniform size were selected, the surface of the root system was cleaned, and the seedlings were planted in 1 / 2 times Japanese Yamazaki formula tomato nutrient solution for 5 days. After the seedlings were grown, the solution was replaced with 1 times Japanese Yamazaki formula nutrient solution, wherein the normal treatment was 1 times Japanese Yamazaki formula nutrient solution, and the drought treatment was to add 10% (g / L) polyethylene glycol 6000 (PEG 6000) to the 1 times nutrient solution. Six days after the drought stress treatment, 2-3 functional leaves and roots below the growth point were taken and quickly frozen by liquid nitrogen and stored in a -80°C refrigerator for subsequent determination of physiological indicators.

[0054] After the plants were treated with drought for 6 days, the phenotypes of the plants were observed, and it was found that the transgenic tomatoes overexpressing SlWRKY41 had a good growth trend and a well-developed root system, indicating that the transgenic tomatoes overexpressing SlWRKY41 enhanced the drought tolerance of the plants. Figure 3 .

[0055] The activities of antioxidant enzyme systems (SOD, POD and CAT), ROS accumulation (MDA, H2O2, O2 - ), the content of osmotic regulating substances (proline, soluble protein, soluble sugar). Figure 4 As shown in the results, the osmotic regulation ability, oxidase activity and ROS scavenging ability of transgenic plants overexpressing SlWRKY41 under drought stress were significantly improved, indicating that SlWRKY41 can improve the drought tolerance of tomato plants.

[0056] Sequence Listing

[0057] SEQ ID NO: 1. cDNA sequence of SlWRKY41 gene:

[0058]

[0059] SEQ ID NO: 2. CDS sequence of SlWRKY41 gene:

[0060]

[0061] SEQ ID NO: 3. Protein encoded by SlWRKY41 gene

[0062] MEKVKSMEKKKLINELTQGKEFVNQLKNQIIGPLASPEECDLLIGKILSTLEKSLSILNLKALLLEGGINNINNSTSSCSSISFLGSSNNNISPKSEVFDSSREKLDKNMVSKKRKKTQQWINQTSISVNGLEGPCEDGFSWRKYGQKDILGASHPRAYYRCTHRNTQ GCLATKQVQKSDANSTIFEVTYKGRHSCKVAQSEIFSLNNQKRQKCNKKQQEEMVIFNSATPNHNAENFNIITAKDEEVFTAFPFPPAPLHIENIIEENKFFPDSSELPLYLASPNDEFGMEHMILQSSDSDLTELISTPTSISNSSFGGEGVDYEPEITFDIEEFFI

[0063] SEQ ID NO: 4, DNA artificial sequence

[0064] ATGGAGAAAGTTAAAAGTATTGGAGAAAAAG

[0065] SEQ ID NO: 5, DNA artificial sequence

[0066] TTAAATGAAGAATTCTTCAATGTCAAATGTG

[0067] SEQ ID NO: 6, DNA artificial sequence

[0068] ATGACGCACAATCCCACTATC

[0069] SEQ ID NO: 7, DNA artificial sequence

[0070] ATGGACCTTCAAGCCCATTTA.

[0071] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application of a transcription factor SlWRKY41 in improving drought resistance of tomatoes, wherein the cDNA sequence of the transcription factor SlWRKY41 is shown in SEQ ID NO:

1.

2. A method for constructing an overexpression vector of the transcription factor SlWRKY41 for regulating drought tolerance of tomato, comprising the following steps: Step 1: Using Ailsa Craig tomato leaf cDNA as a template, PCR polymerase was used to amplify the target fragment, the PCR product was detected by 1% agarose gel electrophoresis, the target fragment was recovered by a recovery kit, PBI121 empty vector was double-digested with SacI and XbaI, the linearized backbone vector was purified and recovered, the gel-recovered target fragment and the purified backbone vector were homologously recombined to construct an overexpression vector; Step 2: Use the heat shock method to transform the recombinant product into E. coli DH5α competent cells, and spread it on kanamycin-resistant LB solid medium, pick a single clone bacterial spot for colony PCR identification of positive clones; Step 3: The selective marker gene of the PBI121 vector is kanamycin, and the PCR positive clone plasmid is extracted for sequencing verification to obtain the recombinant expression vector of the SlWRKY41 gene, PBI121-SlWRKY41.

3. The method for constructing a transcription factor SlWRKY41 overexpression vector for regulating drought tolerance of tomatoes according to claim 2, characterized in that: In step 1, PCR was performed to amplify the primer sequence of SlWRKY41, wherein the forward primer was shown as SEQ ID NO: 4; and the reverse primer was shown as SEQ ID NO:

5.

4. A method for cultivating transgenic plants, the method being applied to the transcription factor SlWRKY41 overexpression vector for regulating drought tolerance of tomatoes as claimed in claim 2, introducing the coding gene of SlWRKY41 into a recipient plant to obtain a transgenic plant.

5. A method for cultivating transgenic plants according to claim 4, characterized in that: The steps include: Step 1: The correctly sequenced PBI121-SlWRKY41 was transferred into Agrobacterium tumefaciens GV3101 by freeze-thaw method, and the monoclonal positive strain was screened and verified and stored at -80°C; Step 2: Using the plant tissue culture method, inducing callus tissue of wild-type tomato plants, using a bacterial solution containing the target gene and an OD value of 0.4 to prepare a suspension for infiltration, and obtaining transgenic positive plants after cultivation; Step 3: Extract DNA from the transgenic T0 generation positive plants, design primers based on the tag sequence on the vector, perform PCR amplification, purify and sequence the amplified products, transplant the identified positive plants to the field for seed preservation, and use the T3 generation plants to observe drought phenotypes and measure physiological indicators.

6. The method for cultivating transgenic plants according to claim 4, characterized in that: The primer sequences used in the PCR amplification for identifying the positive plants in step 3, the primers for identifying the overexpressing plants are: JC-WRKY41-F, as shown in SEQ ID NO: 6, and JC-WRKY41-R, as shown in SEQ ID NO: 7.

Images