Application of tomato SlSPRH2 gene in improvement of tomato heat resistance

Abstract

According to the application of the tomato SlSPRH2 gene in improving the heat resistance of tomatoes, an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 95% homology with the amino acid sequence is included, and the sequence keeps the functional characteristic of regulating and controlling the heat resistance of the tomatoes. According to the present invention, the understanding of the plant heat resistance mechanism is enriched, the new tomato variety capable of resisting the high temperature stress and maintaining the high yield and the high quality is cultivated in the future, the brand new gene resource approach is developed, and the important theoretical guidance significance and the practical application value are provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and relates to application of a tomato gene SlSPRH2 in improving the heat resistance of tomatoes. Background Art

[0002] During the growth and development of plants, temperature is one of the key factors affecting whether plants can grow normally. When plants sense abnormal temperature, changes such as cell dehydration, increased intracellular osmotic pressure and pH, damage to cell structure and plasma membrane system, and effects on the functions of organelles such as chloroplasts and mitochondria will occur, ultimately leading to abnormal plant growth and development (Yamazaki et al., 2009; Yin et al., 2014; Lohani et al., 2020).

[0003] Tomato (Solanum lycopersicum) is a perennial herbaceous plant of the Solanum subgenus of the Solanaceae family. It is rich in vitamins and has high nutritional and medicinal value. In addition, the tomato genome is small and easy to transform. It is a commonly used model plant and occupies an important position among fruits and vegetables. The optimal growth temperature of tomatoes is around 28°C, and their growth and development are easily affected by high temperatures. When tomatoes are affected by high temperatures, many adverse consequences will occur, such as: affecting the normal development of the plant and causing premature aging of the plant; the leaves of the plant are burned by high temperatures, destroying the plant's own structural tissues; causing pollen abortion, fertilization failure, and low fruit setting rate, thus affecting yield.

[0004] During the long evolution process, plants have acquired a series of mechanisms to respond to external stress. The mitogen activated protein kinase (MAPK) cascade is an important member of this mechanism. The MAPK cascade pathway is an important signaling module that is highly conserved in eukaryotes. This pathway can amplify the signals that plants respond to the external environment and identify and transduce them into the cell (Meng and Zhang 2013). It is an important hub for signal transduction in plant cells. Many studies have shown that the MAPK cascade not only plays an important role in the growth and development of plants, but also plays a pivotal role in the process of plants adapting to various adverse environments.

[0005] The biological functions of the MAPK cascade pathway are mainly determined by the downstream target proteins phosphorylated by MAPKs. MAPK targets specific downstream substrates including other kinases, enzymes and transcription factors (Joo et al., 2008; Meng et al., 2013; Zhang et al., 2018). The MAPK cascade is an important signal transduction module involved in the entire growth and development process of plants. The diversity of its downstream substrates enables the MAPK cascade to regulate different biological processes. Many studies have shown that MAPK substrates play an important role in the growth and development of plants, including cytokinesis, reproduction and leaf senescence (Soyano, et al. 2003; Wang et al., 2008; Zhou et al., 2009). At the same time, MAPK substrates also play an important role in abiotic stress signal transduction, including plant tolerance to low temperature environment, resistance to high temperature and drought, salt tolerance, etc. (Li et al., 2017; Evrard et al., 2013; Zhang et al., 2012; Manuka et al., 2018). Therefore, studying MAPK targeting specific downstream substrates to improve tomato's tolerance to high temperature can not only directly promote the development of the tomato industry, but also provide experimental ideas and theoretical basis for studying the response mechanism of other plants to high temperature. At the same time, it provides a theoretical basis for using genetic means to enhance the tolerance of crops such as tomatoes to high temperature and has practical guiding significance. Summary of the invention

[0006] Technical problem to be solved: The present invention provides an application of the tomato SlSPRH2 gene in improving the heat tolerance of tomatoes. The gene is knocked out by CRISPR / Cas9 to further realize the application of SlSPRH2 in improving the heat tolerance of tomatoes.

[0007] Technical solution: A protein capable of regulating the heat resistance of tomatoes, comprising an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 95% homology thereto, and the sequence retains the functional characteristics of regulating the heat resistance of tomatoes.

[0008] The SlSPRH2 gene encodes the above protein and has a nucleic acid sequence as shown in SEQ ID NO: 1, or a nucleic acid sequence having at least 95% homology thereto, and the gene can encode a protein having the function of regulating the heat tolerance of tomatoes.

[0009] The above protein is used as a negative regulatory factor to regulate the heat tolerance of tomatoes.

[0010] The above genes are used as negative regulatory factors to regulate the heat tolerance of tomatoes.

[0011] A method for improving heat resistance of tomatoes comprises reducing the expression level of the above protein, wherein the method for reducing the expression comprises gene silencing, gene mutation, gene knockout or antisense RNA technology.

[0012] A method for breeding heat-resistant tomato varieties comprises screening out plants with reduced gene expression, or introducing overexpression vectors and knockout vector plasmids into tomato plants through genetic transformation.

[0013] A kit is used for detecting the presence or expression level of the above protein or gene in tomatoes, and the kit contains an antibody capable of specifically recognizing the protein or a probe capable of specifically binding to the gene.

[0014] The above protein is used as a detection marker in the preparation of biological preparations for detecting and regulating the heat resistance of tomatoes.

[0015] The above gene is used as a detection marker in the preparation of transgenic tomatoes for detecting and regulating heat resistance, or other biological preparations.

[0016] Beneficial effects: The present invention selected the wild type M14 in the tomato variety Ailsa Craig (AC) as the research basis and successfully constructed SlSPRH2, SlSPRH2 S42A SlSPRH2 S42D Three overexpression transgenic tomato materials were used, and the mutant plants of the SlSPRH2 gene were accurately obtained by using CRISPR / Cas9 gene editing technology. Through heat tolerance evaluation, including but not limited to plant phenotype observation under high temperature environment, phenotypic analysis of detached leaves, DAB and NBT staining experiments of tomato leaves, and detailed analysis of changes in antioxidant enzyme activity, this study strongly demonstrated the key role played by the SlSPRH2 gene in significantly improving the heat tolerance of tomatoes. Furthermore, this study preliminarily revealed the core function and action pathway of the SlSPRH2 gene and its specific Ser-42 phosphorylation site in the complex mechanism of tomato coping with high temperature stress, providing insights for a deeper understanding of the molecular basis of tomato heat tolerance. This discovery not only enriches our understanding of the heat tolerance mechanism of plants, but also opens up a new gene resource approach for the future breeding of new tomato varieties that can withstand high temperature adversity and maintain high yield and quality, which has important theoretical guidance significance and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Overexpression vector plasmid map (A: pBI121-35S:SlSPRH2 plasmid map; B: pBI121-35S:SlSPRH2 S42A Plasmid map; C:pBI121-35S:SlSPRH2S42D Plasmid map).

[0018] Figure 2 For some SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE positive identification results of three overexpression transgenic tomatoes (1-6: SlSPRH2-OE positive plant identification; 7-12: SlSPRH2 S42A -OE positive plant identification; 13-15: SlSPRH2 S42D -OE positive plant identification; - represents wild type; + represents positive plasmid).

[0019] Figure 3 For some SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE three kinds of overexpression transgenic tomato relative expression level determination (OE-S2109, OE-S2105, OE-S255 are overexpression SlSPRH2 plant phenotype, OE-2A61, OE-2A25, OE-2A35 are overexpression SlSPRH2 S42A Plant phenotypes, OE-2D30, OE-2D118, and OE-2D101 overexpress SlSPRH2 S42D Plant phenotype; analysis of difference: n=3, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001).

[0020] Figure 4 The wild type and SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE tomato growth phenotype (OE-S2 W 109. OE-S2 W 53. OE-S2 W 55 is the phenotype of plants overexpressing SlSPRH2, OE-2 A 6. OE-2 A 25. OE-2 A 35 for overexpression of SlSPRH2 S42A Plant phenotype, OE-2 A 102. OE-2 A 118. OE-2 A 101 is overexpression of SlSPRH2 S42D plant phenotype).

[0021] Figure 5The wild type and SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE detached leaf phenotype (A) and chlorophyll content changes (B) (OE-S2109 and OE-S255 are phenotypes of plants overexpressing SlSPRH2, OE-2A25 and OE-2A35 are phenotypes of plants overexpressing SlSPRH2 S42A Plant phenotypes, OE-2D118 and OE-2D101 overexpress SlSPRH2 S42D plant phenotype).

[0022] Figure 6 The wild type and SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE tomato leaf DAB (A) and NBT (B) staining results. (OE-S2109, OE-S255, OE-S2105 are overexpressed SlSPRH2 plants, OE-2A61, OE-2A25, OE-2A35 are overexpressed SlSPRH2 S42A Plant phenotypes, OE-2D30, OE-2D118, and OE-2D102 overexpress SlSPRH2 S42D plant phenotype).

[0023] Figure 7 SlSPRH2-OE and SlSPRH2 under high temperature stress S42A -OE and SlSPRH2 S42D -Changes in the activities of antioxidant enzymes (CAT, APX, GR, POD and SOD) in leaves of OE and wild-type tomatoes. (OE-S2109, OE-S255, OE-S2105 are phenotypes of plants overexpressing SlSPRH2, OE-2A61, OE-2A25, OE-2A35 are phenotypes of plants overexpressing SlSPRH2 S42A Plant phenotypes, OE-2D30, OE-2D118, and OE-2D101 overexpress SlSPRH2 S42D Plant phenotype. Control: control group without high temperature treatment; HT: 45℃, 6h; * represents p<0.05, ** represents p<0.01, *** represents p<0.001).

[0024] Figure 8 Designed for SlSPRH2 gene knockout target.

[0025] Fig. 9 Identification of SlSPRH2 homozygous and mutant materials.

[0026] Fig.10The growth phenotypes of wild-type and SlSPRH2 knockout mutant tomatoes under medium- and long-term high temperature stress.

[0027] Fig.11 Phenotype (A) and changes in chlorophyll content (B) of detached leaves of wild-type and SlSPRH2 knockout mutant tomatoes under high temperature stress.

[0028] Fig.12 NBT (left) and DAB (right) staining results of wild-type and SlSPRH2 knockout mutant tomato leaves under high temperature stress.

[0029] Fig.13 Changes in the activities of antioxidant enzymes (CAT, APX, POD and SOD) in leaves of SlSPRH2 knockout mutant and wild-type tomato under high temperature stress (Control: control group without high temperature treatment; HT: 45℃, 6h; * represents p<0.05, ** represents p<0.01, *** represents p<0.001).

[0030] Fig.14 Schematic diagram of the overlap extension PCR amplification steps.

[0031] Fig.15 Sequencing results of pBI121-SlSPRH2 vector (A) pBI121-SlSPRH2 S42A Vector sequencing results (B) pBI121-SlSPRH2 S42D Vector sequencing results (C). DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific embodiments.

[0033] Example 1: Overexpression of SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D Tomato Plants Acquired

[0034] The specific operation method is as follows:

[0035] (1) According to the ID of SlSPRH2 gene (Solyc09g089650), the corresponding CDS sequence information was found in the Solanaceae database (Sol Genomics Network), and full-length sequence primers were designed using Primer 5.0. BamH I and Sac I were used as restriction sites and homology arms were added at the 5' end of the upstream and downstream primers, respectively. Primer 1: SlSPRH2-pBI121-F: gatgacgatgacaagggatccATGTCAATTAACCACCGGACTTCC (SEQ ID NO.9); Primer 6: SlSPRH2-pBI121-R: atcggggaaatcatagagctcTCACCGTCTATCGGCGGTT (SEQ ID NO.10).

[0036] Note: The lowercase letters in the primer sequences represent the vector homology arms.

[0037] (2) After the primers were designed, they were compared with the NCBI database to determine their specificity. The cDNA of wild-type M14 tomato was used as a template, and the PCR amplification program (Table 2) corresponding to the high-fidelity amplification system (Table 1) was used to amplify the target fragment. The complete gene of SlSPRH2 was obtained.

[0038] Table 1 High-fidelity amplification system

[0039]

[0040] Table 2 PCR amplification program

[0041]

[0042] (3)SlSPRH2 S42A and SlSPRH2 S42D Gene point primer design: Based on the CDS sequence of SlSPRH2 gene, TCA (124-126) → GCA (124-126) were used to obtain SlSPRH2 S42A ; TCA(124-126)→GAT(124-126) to obtain SlSPRH2 S42D The single point mutation primers for SlSPRH2 and SlSPRH3 were designed by using the Novizan single point mutation primer design website (https: / / crm.vazyme.com / cetool / singlepoint.html). S42A and SlSPRH2 S42D Primers for amplifying single point mutations.

[0043] Primer 3: SlSPRH2 S42A-pBI121-mu-F:5'-CTCTTTTAgcaCCGTTGATTACTTCTCCGGTT T-3'(SEQ ID NO.11)

[0044] Primer 2: SlSPRH2 S42A -pBI121-mu-R:5'-CAACGGtgcTAAAAGAGGTATAGCCACATTCC AAT-3'(SEQ ID NO.12)

[0045] Primer 4: SlSPRH2 S42D -pBI121-mu-F:5'-CTCTTTTAgatCCGTTGATTACTTCTCCGGTT T-3'(SEQ ID NO.13)

[0046] Primer 5: SlSPRH2 S42D -pBI121-mu-R:5'-CAACGGatcTAAAAGAGGTATAGCCACATTCC AAT-3'(SEQ ID NO.14)

[0047] Note: The capital letters in the primer sequence represent the vector homology arms, and the lowercase letters represent the punctate sites.

[0048] (4) After designing the single point mutation amplification primers, overlap extension PCR was performed. Fig.14 As shown, the first round of PCR was performed with primers 1 / 2 and 3 / 6; primers 1 / 5 and 4 / 6, and the required mutation sites were introduced into primers 2 / 3 and 4 / 5. After the first round of PCR, the two PCR products were recovered by gel recovery, and after being evenly mixed, the second round of PCR was performed with primers 1 / 4, and the product was SlSPRH2 S42A and SlSPRH2 S42D Point gene.

[0049] (5) Overexpression vector construction: pBI121 (overexpression vector, direct source and original source: plasmid stored in the laboratory) was double-digested with BamH I and Sac I restriction endonucleases. The double-digestion system is shown in Table 3. After electrophoresis gel recovery, the SlSPRH2 and SlSPRH2 mentioned above were S42A and SlSPRH2 S42D The gene was homologously recombined and connected. The recombination connection reaction system is shown in Table 4. The connection was carried out at 50°C for 30 minutes. After the connection was completed, the competent E. coli DH5α cells (Nanjing Qingke Biotechnology Co., Ltd.) were transformed and spread on the plate. The cells were grown overnight. The next day, single clones were picked and the bacterial solution was identified. The positive bacterial solution that was successfully identified was sequenced. The sequencing results are shown in Fig.15After the bacterial solution was sequenced and confirmed to be correct, the bacterial solution was amplified and the plasmid was extracted.

[0050] Table 3 Double enzyme digestion reaction system

[0051]

[0052] Table 4 Recombination ligation reaction system

[0053]

[0054] (6) Agrobacterium-mediated genetic transformation of tomato: pBI121-SlSPRH2, pBI121-SlSPRH2 S42A pBI121-SlSPRH2 S42D The recombinant plasmid was transformed into Agrobacterium competent cells GV3101 (Nanjing Novozyme Biotechnology Co., Ltd.), spread on the plate, wait for the bacteria to grow, pick the single clone colony on the third to fourth day, identify the bacterial solution, and the positive bacterial solution successfully identified was used as the Agrobacterium species for subsequent infection of tomato cotyledons. Finally, the genetic transformation of tomatoes was carried out through relevant plant tissue culture experiments.

[0055] (7) Positive identification: Design specific primer 35S-3'-F: 5'-GACGCACAATCCCACTATCC-3' (SEQ ID NO.15). Use 35S-3'-F and SlSPRH2-pBI121-R to identify tissue culture seedlings by PCR, screen positive plants, and some SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE overexpression transgenic tomato positive identification results Figure 2 shown.

[0056] Example 2: Overexpression of SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D Relative expression analysis in tomato plants.

[0057] The specific operation method is as follows:

[0058] (1) Fresh overexpressed SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D The transgenic tomato leaf samples were cut and wrapped in tin foil, marked, and quickly cooled in liquid nitrogen and stored in a -80°C refrigerator. Total RNA was extracted from the samples (total RNA of tomato samples was extracted according to the instructions of RNA isolater Total RNA Extraction Reagent (Nanjing Novozyme Biotechnology Co., Ltd.)).

[0059] (2) Reverse transcription synthesis: In the first step, extract RNA from overexpressed tomato leaves (positive plants that have been identified). Prepare the mixed solution in an RNase-free centrifuge tube according to Table 5. After the preparation is completed, gently blow and mix with a pipette, and react at 42°C for 2 minutes. In the second step, prepare the reverse transcription reaction system: add 4μL of 5×HisScriptⅢQrtSuperMix to the reaction solution in the first step. Perform the reverse transcription reaction: 37°C, 15min; 85°C, 5min. In the third step, obtain cDNA.

[0060] Table 5 Genomic DNA removal system

[0061]

[0062] (3) qRT-PCR was performed to detect SlSPRH2-OE and SlSPRH2 according to the tomato qRT-PCR primers in Table 6, the qRT-PCR amplification system in Table 7, and the qRT-PCR amplification program in Table 8. S42A -OE and SlSPRH2 S42D -OE transgenic tomato relative expression. Figure 3 shown.

[0063] Table 6 Tomato qRT-PCR primer design

[0064]

[0065] Table 7 qRT-PCR amplification system

[0066]

[0067] Table 8 qRT-PCR amplification program

[0068]

[0069] (4) The relative expression results showed that the expression levels in the overexpression positive plants were significantly higher than those in the wild-type plants. S42A -OE and SlSPRH2 S42D -OE Tomato materials are all built successfully.

[0070] Example 3: Overexpression of SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D Analysis of heat tolerance in tomato plants.

[0071] The specific operation method is as follows:

[0072] (1) Tomato soil culture phenotype: Wild-type and overexpressing tomato plants that have grown to the five-leaf stage were selected and treated with 42℃ / 38℃ (16h / 8h) for one week. After that, they were restored to normal growth conditions and their phenotypes were observed. Figure 4 As shown in the results, after high temperature treatment, SlSPRH2-OE and SlSPRH2 S42D -OE plants grew worse than wild-type plants, and SlSPRH2 S42A -OE had no significant difference compared with wild-type plants.

[0073] (2) Phenotype of detached leaves: The detached leaves that had recovered from high temperature treatment (45°C, 3h) for 5 days were decolorized with acetone and the chlorophyll content was measured. The results were as follows: Figure 5 As shown, overexpression of SlSPRH2-OE and SlSPRH2 S42D -OE tomato plants had lower chlorophyll content in detached leaves than the wild type, while SlSPRH2 S42A The chlorophyll content of -OE plants was not much different from that of the wild type.

[0074] (3) DAB and NBT staining of tomato leaves: Select wild-type and overexpression positive plants at the 5-leaf stage with consistent growth and treat at 45°C for 6 hours before staining. The experiment was divided into two groups: control group and high temperature treatment group, with 3 biological replicates in each group. Figure 6 As shown: The NBT staining results are consistent with the DAB staining results. After high temperature treatment, SlSPRH2-OE and SlSPRH2 S42D -OE tomato plants had more and darker staining areas on their leaves than wild-type plants. S42A -OE has a similar color intensity to the wild type.

[0075] (4) Determination of antioxidant enzyme activity: Samples (about 3 leaves) were taken from well-growing overexpression positive and wild-type five-leaf tomato plants after high temperature treatment at 0 h and 45 °C for 6 h, respectively, with 3 biological replicates. 1.6 mL of enzyme extract was added, and the mixture was ground in liquid nitrogen. The mixture was centrifuged at 4 °C and 12,000 × g for 30 min, and the supernatant was taken as the crude tomato enzyme extract. The activities of five antioxidant enzymes, APX, CAT, GR, SOD and POD, were measured at the corresponding wavelengths and repeated 3 times. The results are shown in the figure. Figure 7 Shown: SlSPRH2-OE and SlSPRH2 S42D -OE tomato plants were treated with high temperature and the antioxidant enzyme activities were compared with those of wild type and SlSPRH2 S42A -OE tomato plants showed a greater decline.

[0076] (5) After overexpression of SlSPRH2 and SlSPRH2S42A and SlSPRH2 S42D Analysis of heat tolerance in tomato plants revealed that SlSPRH2-OE and SlSPRH2 S42D -OE tomato plants compared to wild type and SlSPRH2 S42A -OE tomato plants suffered more severe oxidative damage, indicating that SlSPRH2 is a negative regulator of high temperature tolerance in tomatoes, and Ser-42 is a key phosphorylation site.

[0077] Example 4: Obtaining SlSPRH2 gene knockout tomato plants

[0078] The specific operation method is as follows:

[0079] (1) Design of sgRNA for SlSPRH2 and construction of knockout vector: The target sequence sgRNA for SlSPRH2 gene was designed using the CRISPR-P2 website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR): target sequence 1 (sgRNA-1): 5'-GGACTTCCACCGGAGACCGT-3' (SEQ ID NO.3); target sequence 2 (sgRNA-2): 5'-CCACATTCCAATCGGTCGGT-3' (SEQ ID NO.4). The knockout target site of SlSPRH2 gene was designed as shown in the following figure. Figure 8 shown.

[0080] (2) Using the kit, first synthesize oligo, then prepare oligo dimer: Buffer Anneal 18 μL, UPOligo 1 μL, Low Oligo 1 μL, H2O to 20 μL; after mixing, heat at 95°C for 3 minutes, then slowly decrease to 20°C at about 0.2°C / second; wherein the primer sequence is:

[0081] UP-1Oligo:gtttGGACTTCCACCGGAGACCGT(SEQ ID NO.5)

[0082] Low-1 Oligo: aaacACGGTCTCCGGTGGAAGTCC (SEQ ID NO.6)

[0083] UP-2Oligo:gtttCCACATTCCAATCGGTCGGT(SEQ ID NO.7)

[0084] Low-2 Oligo:aaacACCGACCGATTGGAATGTGG(SEQ ID NO.8)

[0085] (3) The oligo dimer was connected to the vector BGK015 to obtain the knockout vector: CRISPR / Cas Vector 2μL, oligo dimer 1μL, Enzyme Mix 1μL, H2O supplemented to 10μL; the components were mixed on ice and reacted at room temperature at 20°C for 1 hour. The ligation product was then transformed into Escherichia coli competent cells DH5α (Nanjing Qingke Biotechnology Co., Ltd.), spread on the plate, and grown overnight. The next day, a single clone colony was picked for bacterial liquid identification. The positive bacterial liquid that was successfully identified was sequenced, and the subsequent genetic transformation of tomatoes was carried out after the bacterial liquid sequencing was confirmed to be correct.

[0086] (4) The genetic transformation method of SlSPRH2 gene knockout tomato plants was consistent with that in Example 1. The results of identification of pure and mutant materials of SlSPRH2 gene knockout tomato lines were as follows: Fig. 9 shown.

[0087] Example 5: Analysis of heat tolerance of SlSPRH2 gene knockout tomato plants.

[0088] (1) Tomato soil culture phenotype: Wild-type and SlSPRH2 knockout tomato plants grown to the five-leaf stage were selected and treated with 42℃ / 38℃ (16h / 8h) for 5 days. After recovery for 2 days, the phenotype was observed. Fig.10 As shown in the results, it can be seen that after high temperature treatment, the SlSPRH2 gene knockout tomato plants grew better than the wild-type plants.

[0089] (2) Phenotype of detached leaves: The detached leaves that had recovered from high temperature treatment (45°C, 6 h) for 7 days were decolorized with acetone and the chlorophyll content was measured. The results are as follows: Fig.11 As shown, the chlorophyll content of detached leaves of SlSPRH2 gene knockout tomato plants after treatment was higher than that of wild type.

[0090] (3) DAB and NBT staining of tomato leaves: Leaves of wild-type and SlSPRH2 knockout tomato plants at the 5-leaf stage with consistent growth were selected and stained after being treated at 45°C for 6 h. The experiment was divided into two groups: a control group and a high temperature treatment group, with 3 biological replicates in each group. Fig.12 As shown: The NBT staining results were consistent with the DAB staining results. After high temperature treatment, the leaves of SlSPRH2 gene knockout tomato plants had fewer stained areas than those of wild-type plants, and the staining was lighter.

[0091] (4) Determination of antioxidant enzyme activity: SlSPRH2 knockout and wild-type five-leaf tomato plants with good growth were treated with high temperature at 0h and 45℃ for 6h, and samples (about 3 leaves) were taken, with 3 biological replicates. 1.6mL of enzyme extract was added, and the mixture was ground in liquid nitrogen. The mixture was centrifuged at 4℃ and 12000×g for 30min, and the supernatant was taken as the crude enzyme solution for tomato extraction. The activities of five antioxidant enzymes, APX, CAT, SOD and POD, were measured at the corresponding wavelengths and repeated 3 times. The results are as follows: Fig.13 As shown: The antioxidant enzyme activity of SlSPRH2 gene knockout tomato plants decreased less after high temperature treatment than that of the wild type.

[0092] (5) After analyzing the heat tolerance of SlSPRH2 gene knockout tomato plants, it was found that wild-type tomatoes suffered more severe oxidative damage under high temperature stress than SlSPRH2 gene knockout plants. The results corroborated the results in Example 3, further showing that SlSPRH2 is a negative regulator of high temperature tolerance in tomatoes. This indicates that the SlSPRH2 gene plays a significant role in improving the heat tolerance of tomatoes and can provide new gene resources for breeding new varieties of high temperature resistant tomato crops.

[0093] The present invention uses the wild type M14 of tomato Ailsa Craig (AC) background as material to construct SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D Overexpression transgenic tomato materials were obtained, and mutant plants of the SlSPRH2 gene were obtained by CRISPR / Cas9 gene editing technology. Through relevant heat resistance analysis (plant phenotypic analysis under high temperature stress, in vitro leaf phenotypic analysis, tomato leaf DAB and NBT staining results and analysis of changes in antioxidant enzyme activity), this study strongly demonstrated the potential value of the SlSPRH2 gene in improving the heat resistance of tomatoes. At the same time, it preliminarily revealed the core function and action pathway of the SlSPRH2 gene and its unique Ser-42 phosphorylation site in the complex mechanism of tomato coping with high temperature stress, opening up a new genetic resource approach for the future breeding of new tomato varieties that can withstand high temperature adversity and maintain high yield and high quality.

[0094] The above embodiments are descriptions of preferred embodiments of the present invention, and are not intended to limit the present invention. Without departing from the spirit or scope of the present invention, technical improvements and equivalent substitutions made by relevant technical personnel to the present invention are within the protection scope of the present invention.

Claims

1. A protein capable of regulating the heat resistance of tomatoes, characterized in that: The invention comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 95% homology thereto, and the sequence maintains the functional characteristics of regulating the heat tolerance of tomatoes.

2. The gene of S1SPRH2, encoding the protein according to claim 1, characterized in that: The invention relates to a gene having a nucleic acid sequence as shown in SEQ ID NO: 1, or a nucleic acid sequence having at least 95% homology thereto, and the gene can encode a protein having the function of regulating the heat tolerance of tomatoes.

3. Use of the protein according to claim 1 as a negative regulatory factor in regulating the heat tolerance of tomatoes.

4. Use of the gene according to claim 2 as a negative regulatory factor in regulating the heat tolerance of tomatoes.

5. A method for improving the heat tolerance of tomatoes, characterized in that: The method comprises reducing the expression level of the protein of claim 1, wherein the method of reducing the expression comprises gene silencing, gene mutation, gene knockout or antisense RNA technology.

6. A method for breeding heat-resistant tomato varieties, characterized in that: The method comprises screening out plants with reduced gene expression as claimed in claim 2, or introducing overexpression vectors and knockout vector plasmids into tomato plants through genetic transformation.

7. A kit for detecting the presence or expression level of the protein according to claim 1 or the gene according to claim 2 in tomatoes, the kit comprising an antibody capable of specifically recognizing the protein or a probe capable of specifically binding to the gene.

8. Use of the protein according to claim 1 as a detection marker in the preparation of a biological agent for detecting and regulating the heat resistance of tomatoes.

9. Use of the gene according to claim 2 as a detection marker in the preparation of transgenic tomatoes or other biological preparations for detecting and regulating heat tolerance.

Images