Cold-resistant gene trwrky10a540, expression vector and breeding method thereof

Abstract

The application provides a cold-resistant gene TrWRKY10a540, an expression vector, application and breeding method thereof, and belongs to the field of plant molecular genetics and genetic improvement. Specifically disclosed is a cold-resistant gene TrWRKY10a540 and an expression vector derived from Trifolium repens L. and application thereof in improving the cold resistance of plants, and the problem of insufficient research on low-temperature stress related genes of Trifolium repens in the prior art is solved. The application further discloses a breeding method for regulating the cold stress resistance of plants by the cold-resistant gene TrWRKY10a540, and research results show that the gene can significantly improve the cold resistance and antioxidant level of plants under low-temperature stress conditions, and has good functional effects. The application can be widely applied to plant molecular breeding, is particularly suitable for improving the low-temperature resistance of economic crops such as forage grasses, provides a new genetic resource for the breeding of cold-resistant plants, and has important significance for revealing the regulation mechanism of the cold resistance of plants.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular genetics and genetic improvement, and specifically relates to a WRKY transcription factor gene TrWRKY10a540 derived from white clover and its application in improving plant cold resistance. Background Technology

[0002] White clover ( Trifolium repens White clover (L.) is a perennial leguminous plant widely distributed in temperate and cold temperate regions, renowned for its high yield, high quality, and strong adaptability. However, white clover is highly susceptible to abiotic stresses during its growth, especially during the harsh winters in high-latitude regions, which often leads to abnormal mortality, thus severely restricting its production and promotion.

[0003] Plant cold resistance mechanisms are regulated by a variety of transcription factors, among which the WRKY family members have attracted much attention due to their functions in regulating stress response pathways and enhancing antioxidant systems. However, the WRKY family members from the forage grass white clover have not been fully studied, making further investigation into the cold resistance mechanisms of WRKY family members particularly important. Summary of the Invention

[0004] This invention addresses the technological gap in existing research on genes related to low-temperature stress in white clover (Trifolium repens L.) by providing a cold-resistance gene TrWRKY10a540 derived from white clover and its application in improving plant cold resistance.

[0005] A gene for improving plant cold resistance, characterized in that the gene is the white clover TrWRKY10a540 gene.

[0006] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0007] Furthermore, the amino acid sequence encoded by the gene is shown in SEQ ID NO.4.

[0008] Application of the TrWRKY10a540 gene in plant cold stress resistance.

[0009] An expression vector to improve plant cold resistance, wherein the expression vector is a pCAMBIA1300-TrWRKY10a540 expression vector containing the TrWRKY10a540 gene.

[0010] Application of pCAMBIA1300-TrWRKY10a540 expression vector in plant cold stress resistance.

[0011] A breeding method for regulating the cold stress resistance of plants, the steps of which are as follows:

[0012] S1: The coding sequence of white clover TrWRKY10a540 was cloned using primers to obtain the gene clone sequence;

[0013] S2: The gene clone sequence is ligated into the pCAMBIA1300 vector to obtain the expression vector;

[0014] S3: The expression vector obtained in S2 is introduced into Agrobacterium to obtain recombinant Agrobacterium;

[0015] S4: Obtain transgenic plants by infecting plants with the recombinant Agrobacterium obtained in S3.

[0016] Furthermore, the Agrobacterium mentioned in S3 is GV3101.

[0017] Furthermore, the plant described in S4 is Arabidopsis thaliana.

[0018] Furthermore, the infection described in S4 is an inflorescence infection method.

[0019] Beneficial effects

[0020] The above experiments verified that the TrWRKY10a540 gene enhances the cold resistance and antioxidant capacity of plants under low-temperature stress. Arabidopsis thaliana overexpressing this gene showed better growth after low-temperature treatment, with significantly increased activities of superoxide dismutase (SOD) and catalase (CAT). These results indicate that the TrWRKY10a540 gene has important application value in plant cold-resistance breeding and can serve as an excellent low-temperature stress response gene resource, widely used in research on improving the cold resistance of forage crops and other economic crops. Attached Figure Description

[0021] Figure 1 Image of PCR identification of TrWRKY10a540 gene-positive colonies;

[0022] Figure 2 Predicted domain structure of TrWRKY10a540 protein;

[0023] Figure 3 Image showing PCR positive screening of T3 generation Arabidopsis thaliana;

[0024] Figure 4 Phenotypic diagrams of transgenic and wild-type Arabidopsis thaliana after low-temperature stress;

[0025] Figure 5 Figure showing the expression level of the TrWRKY10a540 gene in transgenic plants;

[0026] Figure 6The image shows the results of physiological and biochemical index detection in transgenic Arabidopsis thaliana under low temperature stress. A represents chlorophyll (CHL) content, B represents malondialdehyde (MDA) content, C represents proline (Pro) content, D represents catalase (CAT) content, E represents peroxidase (POD) content, and F represents superoxide dismutase (SOD) content. Detailed Implementation

[0027] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0029] The primers used in the following examples were synthesized by Shanghai Sangon Biotech Co., Ltd.; sequencing was performed by Shanghai Sangon Biotech Co., Ltd.; restriction endonucleases, ligases, pMD™18-TVector Cloning Kit, DNA Marker, PrimeSTAR Max Premix (2X), and reverse transcription kits used in the experiments were all purchased from Takara Bio Inc.; DH5α competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.; plasmid extraction kits, gel extraction kits, and genome extraction kits were all purchased from TransGen Biotech Co., Ltd.; and RNA extraction kits were purchased from Tiangen Biotech Co., Ltd.

[0030] The naming of TrWRKY10a540: Tr represents white clover ( Trifolium repens L.), WRKY indicates that this gene belongs to the WRKY transcription factor family. This gene is located on chromosome 10 (Chr10), at the gene locus chr10.jg540. TrWRKY10a540 belongs to subfamily IIa (Group IIa) of the WRKY transcription factor family. Its gene structure contains 3 introns and encodes a protein of 288 amino acids in length.

[0031] The nucleotide sequence of TrWRKY10a540 is shown in SEQ ID NO.3; the amino acid sequence of TrWRKY10a540 is shown in SEQ ID NO.4.

[0032] Example 1. Cloning and sequencing of the TrWRKY10a540 gene.

[0033] 1. Total RNA extraction and cDNA synthesis.

[0034] First, total RNA was extracted from fresh white clover (Trifolium repens L.) leaves using an RNA extraction kit, following standard operating procedures. The extracted RNA was analyzed using a NanoDrop 2000 spectrophotometer to ensure its purity met the requirements for subsequent experiments. Subsequently, the total RNA was reverse transcribed using a PrimeScript™ RT kit to synthesize cDNA. This process ensured the absence of genomic DNA contamination.

[0035] 2. PCR amplification of the TrWRKY10a540 gene.

[0036] The above cDNA was diluted 5 times and used as a template. Specific primers TrWRKY10a540-F and TrWRKY10a540-R for the TrWRKY10a540 gene were designed using Primer5 software. PCR amplification of the open reading frame (ORF) region of the white clover TrWRKY10a540 gene was performed using primers TrWRKY10a540-F and TrWRKY10a540-R.

[0037] The sequences of primers TrWRKY10a540-F and TrWRKY10a540-R are as follows:

[0038] TrWRKY10a540-F: AATCAACCAACCCTATAT (SEQ ID NO.1);

[0039] TrWRKY10a540-R: ATGAATATGATTGTGTTAT (SEQ ID NO. 2).

[0040] The PCR reaction system (50 μL) is as follows: 1 μL cDNA template, 1 μL each of primers TrWRKY10a540-F and TrWRKY10a540-R, 25 μL PrimeSTAR Max Premix (2X), and 22 μL dd H2O; all sample loading steps are performed on ice.

[0041] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min, 98℃ denaturation for 10 s, 52℃ annealing for 15 s, 72℃ extension for 15 s, for 35 cycles, and finally 72℃ extension for 10 min.

[0042] The reaction system and procedure ensured the efficient amplification of the TrWRKY10a540 gene, and the obtained PCR amplification products were detected by agarose gel electrophoresis to confirm the size and purity of the target band.

[0043] 3. Agarose gel electrophoresis and product purification.

[0044] PCR amplification products were separated by agarose gel electrophoresis. After electrophoresis, the target band was cut and purified using a gel extraction kit. The purified PCR product was ligated into the pMD18-T vector. The ligation reaction mixture consisted of 2 μL of the gel-extracted product and 1 μL of pMD18-T, followed by the addition of 5 μL Solution I and 1 μL ddH2O. The mixture was then incubated in a PCR instrument at 16°C for 30 min to obtain the ligation product, pMD18T-TrWRKY10a540 plasmid. The ligation product was transformed into DH5α competent E. coli for subsequent cloning screening.

[0045] 4. Clone screening and sequencing.

[0046] Transformed DH5α *Escherichia coli* single clones were screened on LB agar plates containing ampicillin (Amp). Single colony clones were selected for PCR. Figure 1 The sequencing was performed, and the sequencing results were compared using the bioinformatics software DNAMAN to confirm the correctness of the target gene sequence, ultimately obtaining the complete sequence of the TrWRKY10a540 gene. Simultaneously, 300 μL of the validated cloned bacterial culture was mixed with 300 μL of 50% glycerol and stored at -80℃.

[0047] Example 2: Physicochemical property analysis and tertiary structure prediction of the white clover TrWRKY10a540 gene.

[0048] 1. In this embodiment, the protein characterization of the white clover transcription factor TrWRKY10a540 was analyzed. Using Proparam prediction, the molecular formula of the protein encoded by TrWRKY10a540 is C0. 1405 H 2250 N 408 O 453 S 14 With a molecular weight of 32554.62 kDa and a theoretical isoelectric point of 6.90, and an instability index of 44.24, it is inferred that TrWRKY10a540 protein is an unstable acidic protein.

[0049] 2. Subcellular localization prediction using WoLF PSORT showed that the TrWRKY10a540 protein accounted for 61.0%, 34.1%, and 4.9% of the cell nucleus, cell membrane, and cytoskeleton, respectively.

[0050] 3. The spatial structure of the trimer assembly of the protein encoded by the TrWRKY10a540 gene was predicted using AlphaFold 2, with pTM=0.62 and ipTM=0.61. The results were visualized using ChimeraX (version 1.9) software. The results are shown below. Figure 2 As shown.

[0051] Example 3: Constructing an expression vector for the TrWRKY10a540 gene in plants.

[0052] 1. Using the pMD18T-TrWRKY10a540 plasmid obtained in Example 1 as a template, double digestion was performed using BamHI and PstI enzymes. Simultaneously, the pCAMBIA1300 plasmid was also double digested using BamHI and PstI enzymes. The double digestion system was as follows: 3 μL of pMD18T-TrWRKY10a540 plasmid or pCAMBIA1300 plasmid, 0.5 μL each of HindIII and BamHI, 1 μL of 1×K buffer, and 5 μL of ddH2O. The mixture was reacted in a PCR instrument at 37℃ for 30 min.

[0053] 2. After enzymatic digestion of the target gene fragment and vector plasmid, the target fragment was recovered by agarose gel electrophoresis. The target gene fragment was then ligated into the vector using T4 ligase. The ligation system was as follows: 2 μL pCAMBIA1300, 6 μL LtrWRKY10a540 gene fragment, 1 μL T4 ligase, and 1 μL 10× buffer. Ligation was carried out at 16℃ for 4-5 h.

[0054] 3. The ligation product obtained above was transformed into Escherichia coli DH5α competent cells by heat shock. After being cultured overnight in LB solid medium containing kanamycin (Kan), colony PCR identification was performed. Single colonies that were successfully identified were picked and cultured in LB liquid medium containing Kan at 37°C and 200 rpm overnight for 12 h with shaking. The plasmid was extracted and sent for sequencing. The correct plasmid was named pCAMBIA1300-TrWRKY10a540.

[0055] Example 4: Construction and identification of Arabidopsis thaliana overexpression lines infected with the TrWRKY10a540 gene.

[0056] 1. The pCAMBIA1300-TrWRKY10a540 plasmid obtained in Example 3 was transformed into Agrobacterium GV3101 by freeze-thaw method.

[0057] Add 1 μg of recombinant plasmid to 100 μL of GV3101 Agrobacterium competent cells, mix gently, and then sequentially incubate on ice for 10 min, flash freeze in liquid nitrogen for 5 min, incubate in water at 37°C for 5 min, and incubate on ice for 5 min. Add 800 μL of antibiotic-free YEB liquid medium and incubate in centrifuge tubes at 28°C and 200 rpm for 2–3 h with shaking. Spread 500 μL of the bacterial culture onto YEB solid medium containing the corresponding antibiotics (Rif 50 mg / L, Kan 50 mg / L). Incubate in an inverted incubator at 28°C for 48–72 h. Pick single colonies and incubate in liquid YEB medium (containing antibiotics Rif 50 mg / L, Kan 50 mg / L) with shaking at 28°C and 200 rpm. After the bacterial culture becomes turbid, perform bacterial PCR detection. Add the positive bacterial cultures that pass the test to glycerol and store at -80°C.

[0058] 2. Agrobacterium-mediated genetic transformation of Arabidopsis thaliana.

[0059] Agrobacterium GV3101 glycerol bacteria containing the pCAMBIA1300-TrWRKY10a540 overexpression vector, preserved under ultra-low temperature conditions, were activated twice in YEB liquid medium and cultured with shaking at 28°C and 200 rpm. When the OD value of the bacterial culture reached 0.6-0.8, it was centrifuged at 5000 r / min at room temperature, and the supernatant was discarded. The above bacterial culture was precipitated and resuspended in a 5% sucrose solution containing 200 μL / Lsilwet-77 (OD=0.4-0.5).

[0060] 3. Infection of Arabidopsis thaliana inflorescences.

[0061] The Arabidopsis inflorescences were infected using the resuspended solution obtained above. Robust Arabidopsis plants that had grown for approximately 30 days, with pods already formed in the main inflorescence and lateral inflorescences 2-10 cm in length were selected. The flowering inflorescences and pods were removed, and the unflowered but budding inflorescences were immersed sequentially in the resuspended solution obtained above for approximately 30 seconds. After watering, the plants were cultured in darkness for 24 hours, then transferred to normal light conditions. The treated plants were then restored to normal light and water / fertilizer management and cultured until the seeds matured, at which point T0 generation seeds were harvested.

[0062] 4. Positive screening of transgenic seedlings.

[0063] Positive screening was performed on the obtained T0 generation seeds. After surface disinfection, the T0 generation seeds were evenly sown on 1 / 2 solid MS selective medium containing Kan resistance and cultured at 22°C under 16 hours of light for about 7 days. Plants showing good growth and well-developed root systems were selected and transplanted into sterilized nutrient soil for continued growth. Plump T1 generation seeds were selected, rinsed once with sterile water, then soaked in 75% alcohol for 30 seconds for disinfection, thoroughly rinsed with sterile water, then disinfected with 1% sodium hypochlorite for 5 minutes, and rinsed 5-6 times with sterile water. After disinfection, they were evenly placed in 1 / 2 solid MS medium containing Kan resistance and vernalized at 4°C for 2 days, followed by normal cultivation conditions (24 / 22°C (day / night), 60% humidity, 16 h light / 8 h dark). After two weeks of cultivation, Arabidopsis thaliana with good growth and normal development were transplanted into plastic pots filled with nutrient soil. PCR verification was performed using Arabidopsis thaliana leaf DNA as a template. The results are as follows: Figure 3 As shown, products with the same target band were sent to BGI for sequencing and comparison; three lines with high expression levels were selected from the first generation of positive lines and self-crossed and screened to the T3 generation (named S2, S4 and S7) to obtain seeds of overexpressing transgenic Arabidopsis thaliana lines with stable expression, which were used for subsequent functional analysis and stress resistance-related experiments.

[0064] Example 5: Application of the TrWRKY10a540 gene in improving plant cold resistance.

[0065] 1. Transgenic Arabidopsis cultivation.

[0066] Wild-type and T3 generation transgenic Arabidopsis seeds were disinfected and cleaned, then sown in 1 / 2 solid MS medium. Vernalization was carried out at 4°C in the dark for 3 days, followed by two weeks of growth in a light incubator. Uniformly growing Arabidopsis seedlings were then planted in small square pots containing a 1:1 ratio of nutrient soil and vermiculite (culture conditions: 24 / 22°C (day / night), 60% humidity, 16 h light / 8 h dark) and cultured for another 2 weeks. T3 generation transgenic Arabidopsis and wild-type Arabidopsis were cultured under 4°C low-temperature stress and normal culture conditions, respectively, and their growth was observed. Results are as follows: Figure 4 Compared with the wild type, S2, S4 and S7 have significantly improved low-temperature resistance.

[0067] Plants were divided into two groups: a control group cultured under normal conditions and an experimental group subjected to 24 h of low-temperature stress (16 h light / 8 h darkness). Samples were collected from both groups, flash-frozen in liquid nitrogen, and stored at -80°C. The relative expression level of TrWRKY10a540 in transgenic Arabidopsis was analyzed. The results are as follows: Figure 5 The gene TrWRKY10a540 was detected in S2, S4 and S7.

[0068] The effects of TrWRKY10a540 overexpression on physiological and biochemical parameters of Arabidopsis thaliana under low temperature were investigated. These included the following six parameters: chlorophyll (CHL) content, malondialdehyde (MDA) content, proline (Pro) content, catalase (CAT) content, peroxidase (POD) content, and superoxide dismutase (SOD) content. Results are shown below. Figure 6 .

[0069] The results showed that under normal temperature conditions, there were no significant differences in various physiological indicators between transgenic and wild-type plants. However, after low-temperature stress treatment, the transgenic lines showed significant advantages in several indicators. First, the chlorophyll content of the transgenic plants was significantly higher than that of the wild-type plants (…). Figure 6 The presence of MDA (acid content) indicates that it can effectively slow down chlorophyll degradation under low-temperature conditions, helping to maintain photosynthetic efficiency. Secondly, the MDA content is significantly lower than that of the wild type (…). Figure 6 The B result indicates a milder degree of membrane lipid peroxidation and less damage to cell membrane integrity. Furthermore, the transgenic plants showed a significant increase in proline accumulation (B). Figure 6 The C content suggests that it has a stronger ability to adapt to osmotic regulation and low-temperature stress relief. Regarding antioxidant enzyme activity, the transgenic plants showed significantly higher CAT, POD, and SOD activities than the wild type. Figure 6 D- Figure 6 The results (F) indicate that it is more efficient in scavenging reactive oxygen species and alleviating oxidative stress. This series of physiological improvements comprehensively demonstrates the crucial role of the TrWRKY10a540 gene in enhancing plant tolerance to low-temperature stress.

Claims

1. The application of the white clover TrWRKY10a540 gene in improving plant cold resistance, characterized in that, The nucleotide sequence of the TrWRKY10a540 gene is shown in SEQ ID NO.3; the plant is Arabidopsis thaliana.

2. The application of a pCAMBIA1300-TrWRKY10a540 expression vector containing the TrWRKY10a540 gene in improving plant cold resistance, characterized in that, The plant is Arabidopsis thaliana; the nucleotide sequence of TrWRKY10a540 is shown in SEQ ID NO.

3.

3. A breeding method for improving the cold resistance of Arabidopsis thaliana, characterized in that, The breeding method involves the following steps: S1: The nucleotide sequence of the white clover TrWRKY10a540 gene, as shown in SEQ ID NO.3, was cloned using primers to obtain the gene clone sequence; S2: The gene clone sequence is ligated into the pCAMBIA1300 vector to obtain the expression vector; S3: The expression vector obtained in S2 is introduced into Agrobacterium to obtain recombinant Agrobacterium; S4: Transgenic Arabidopsis thaliana is obtained by infecting plants with the recombinant Agrobacterium obtained in S3.

4. The breeding method according to claim 3, characterized in that, The Agrobacterium in S3 is GV3101.

5. The breeding method according to claim 3, characterized in that, The infection method in S4 is the inflorescence infection method.

Images