Method for knocking out tomato slprmt4b gene to improve tomato high temperature resistance and application

Abstract

The application belongs to the field of tomato stress resistance genetic engineering technology, and discloses a method for knocking out tomato SlPRMT4b gene to improve tomato high-temperature resistance and application, the method is to knock out the SlPRMT4b gene in tomato to significantly improve the resistance of tomato to high-temperature stress, the coding sequence of the SlPRMT4b gene is shown in SEQ ID NO. 1. The method discloses the negative regulation of the SlPRMT4b gene in tomato high-temperature resistance for the first time, and it is clear that knocking out the gene is an effective technical means to enhance the high-temperature resistance of tomato. The related achievements not only lay a theoretical foundation for analyzing the molecular mechanism of tomato resisting high-temperature stress, but also provide core gene resources for cultivating new tomato varieties resistant to high temperature. At the same time, the method has practical application value of energy saving and consumption reduction in tomato production in high-temperature seasons, and can help to alleviate the influence of high-temperature stress on tomato yield and quality, and has important theoretical and production practice significance for guaranteeing the long-term stable and balanced supply of vegetable products in China.

Description

Technical Field

[0001] This application relates to the technical fields of genetic engineering, molecular biology, and plant physiology, and in particular to a knockout tomato. SlPRMT4b Methods and applications of using genes to improve the high-temperature resistance of tomatoes. Background Technology

[0002] tomato( Solanum lycopersicum L. Tomato is an important vegetable crop in greenhouse cultivation in my country, with an optimal growth temperature of 20–28℃. High-temperature stress has become a key factor restricting its growth and development. Currently, affected by global warming and frequent extreme heat events, coupled with low levels of facility equipment and insufficient temperature control capabilities, the problem of high-temperature stress in tomato cultivation is becoming increasingly prominent, leading to frequent problems such as stunted plant growth, reduced yield, and deteriorated quality. Therefore, in-depth research into the physiological and molecular mechanisms of tomato response to high-temperature stress and the identification of key heat-resistant genes is expected to improve tomato heat tolerance, mitigate high-temperature damage, and thus effectively reduce economic losses caused by high temperatures.

[0003] Epigenetic modification is a class of gene expression regulation mechanisms that do not involve changes in DNA sequence, among which the protein arginine methyltransferase (PRMT) family plays an important role. PRMTs are responsible for catalyzing arginine methylation and participate in various cellular processes such as transcriptional regulation, RNA processing, signal transduction, and cell differentiation. In Arabidopsis, AtPRMT4b is a type I histone arginine methyltransferase that catalyzes monomethylation and asymmetric dimethylation of arginine. The AtPRMT4a / 4b double mutant exhibits a distinct late-flowering phenotype and regulates flowering time through the FLOWERING LOCUS C (FLC)-dependent pathway (Niu L et al., Redundant requirement for a pair of PROTEIN ARGININE METHYLTRANSFERASE4homologs for the proper regulation of Arabidopsis flowering time. Plant Physiol. 2008, 148, 490-503). AtPRMT4a / 4b also participates in the regulation of key genes in photomorphogenesis (Hernando CE et al., Genome wide comparative analysis of the effects of PRMT5 and PRMT4 / CARM1 arginine methyltransferases on the Arabidopsis thaliana transcriptome. BMC Genomics. 2015, 16, 192).

[0004] Numerous studies have shown that PRMTs are deeply involved in abiotic and biotic stress responses. AtPRMT4a / 4b and AtPRMT5 positively regulate salt stress tolerance (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Arabidopsis protein arginine methyltransferases and their encoding genes and applications: CN200610088825.9[P]. 2007-01-03; Hernando CE et al., 2015). Rice OsPRMT5 enhances rice resistance to bacterial blight by methylating OsPAL1 (Sheng C et al., OsPRMT5 methylates OsPAL1 to promote rice resistance, hindered by a Xanthomonas oryzae effector. J Integr Plant Biol. 2025, 67, 1599-1613). Under high temperature conditions, OsPRMT6a mediates arginine methylation of OsJAZ1, maintaining normal spikelet development in rice (Dong K et al. OsPRMT6a-mediatedarginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice. Mol Plant. 2024, 17, 900-919). The expression level of the banana MaPRMT4 gene is higher at 28℃ than at 4℃ (Fan Zhengyang et al. Identification of the whole genome of the banana PRMT family and evolutionary analysis of PRMT5 [J]. Journal of Jiangxi Agricultural University, 2022, 44(03), 569-582). In tomatoes, the expression level of the SlPRMT4a / 4b gene was significantly downregulated after treatment at 40℃ for 8 hours compared to the control at room temperature (Yuke Shao et al. Identification, systematicevolution and expression analysis of PRMT gene family in Solanum lycopersicum,2023, Preprint (Version 1) available at Research Square). Heat shock proteins (HSPs) pathways play a crucial role in the response to high-temperature stress (Zubair A et al. Protein networks: integrating pathways for plant heat stress adaptation. Funct IntegrGenomics. 2025, 25(1), 183), and the expression intensity of some HSP genes is positively correlated with plant heat tolerance. SlHSP17.4 / 20 / 70 The SlPRMT4b gene is a key gene for heat tolerance in tomatoes and is widely used as a molecular indicator to assess the heat tolerance of plants. However, it is still unknown whether the tomato SlPRMT4b gene positively or negatively regulates heat resistance, and its specific mechanism in heat stress has not yet been reported. Elucidating the function of the SlPRMT4b gene in the tomato response to heat stress is of great significance for breeding heat-resistant tomato varieties.

[0005] In recent years, CRISPR / Cas9 gene editing technology has developed rapidly. Compared with traditional breeding methods, this technology can precisely knock out target genes in the genome, achieving targeted improvement of crop traits and significantly shortening the breeding cycle. For example, knocking out the SlPRMT4b gene in tomatoes using CRISPR / Cas9 technology can effectively enhance the tomatoes' resistance to high-temperature stress, while also providing a powerful tool for discovering gene resources related to abiotic stress. Summary of the Invention

[0006] Therefore, the purpose of this application is to provide a method and application for knocking out the SlPRMT4b gene in tomatoes to improve their high-temperature resistance.

[0007] According to an embodiment of this application, a method for knocking out the tomato SlPRMT4b gene to improve tomato heat resistance is provided. The method involves knocking out the SlPRMT4b gene in the tomato... SlPRMT4b Genes designed to improve the resistance of tomatoes to high-temperature stress, the aforementioned SlPRMT4b The coding sequence of the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the aforementioned SlPRMT4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 2.

[0009] Furthermore, the gene knockout technology is specifically as follows: In tomatoes SlPRMT4bThe protein-coding region of the gene (Solyc05g054240) was selected from a target fragment containing a PAM motif adjacent to the pre-interstitial region sequence. Based on the first 20 bases of this fragment, corresponding primers were designed to construct a CRISPR / Cas9 vector. The CRISPR / Cas9 vector was introduced into host cells, and then used to infect the cotyledons of the target tomato. Positive gene-edited plants were screened to obtain heat-resistant gene-edited plants. SlPRMT4b .

[0010] Furthermore, the aforementioned SlPRMT4b Gene knockout mutant plants enhance the expression of key heat-resistant genes SlHSP17.4 , SlHSP20 , SlHSP70 The level of gene expression can be increased, thereby enhancing the plant's resistance to high-temperature stress.

[0011] Furthermore, the host cell is an Escherichia coli cell or an Agrobacterium cell.

[0012] Furthermore, the host cell is preferably a GV3101 Agrobacterium cell.

[0013] Furthermore, the nucleotide sequence containing the first 20 bases of the PAM structure adjacent to the preinterspace sequence is shown in SEQ ID NO: 3.

[0014] The specific ways in which the method described in this invention improves the high-temperature resistance of tomatoes include a decrease in relative electrical conductivity, an increase in maximum photochemical efficiency (Fv / Fm), an increase in survival rate, a decrease in malondialdehyde (MDA) content, and an increase in key heat-resistant genes. SlHSP17.4 / 20 / 70 An increase in the amount of expression.

[0015] tomato SlPRMT4b The application of genes in improving the heat resistance of tomatoes, wherein the application is by knocking out the gene in tomatoes. SlPRMT4b Genes enhance the resistance of tomatoes to high-temperature stress. SlPRMT4b The coding sequence of the gene is shown in SEQ ID NO.1.

[0016] The beneficial effects of this invention are as follows: 1. This invention is the first to demonstrate that the SlPRMT4b gene plays a negative regulatory role in the tomato's response to high temperature stress. It successfully constructed a homozygous tomato line with the SlPRMT4b gene knockout without exogenous Cas9 fragments, filling the research gap in the regulation of tomato PRMT family genes in the field of high temperature resistance and laying a solid theoretical foundation for elucidating the molecular mechanism of tomato's high temperature tolerance.

[0017] 2. This invention can significantly improve the high-temperature resistance of tomatoes by knocking out a single gene. The technical approach is precise and efficient, and the breeding cycle is greatly shortened compared to traditional methods. It provides a brand-new core gene resource and molecular breeding target for breeding new heat-resistant tomato varieties.

[0018] 3. This gene-editing technology can be directly applied to greenhouse cultivation and open-field production of tomatoes during the high-temperature season without the need for additional energy consumption for environmental control. It can effectively alleviate the problems of reduced tomato yield and quality deterioration caused by high-temperature stress, and has important practical value and application prospects for ensuring stable production of my country's vegetable industry and achieving a balanced supply of vegetable products.

[0019] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0020] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0021] Figure 1 For tomatoes SlPRMT4b Nucleic acid sequences of the T2 generation (homozygous, without exogenous Cas9 fragments) of the gene knockout strain. The location and sequence of the sgRNA in the genome are identified, and PAM sites are boxed.

[0022] Figure 2 The tomato in Example 3 of this invention SlPRMT4b Phenotype of gene knockout lines after high-temperature treatment; Figure 3 The tomato in Example 3 of this invention SlPRMT4b Maximum photochemical efficiency (Fv / Fm) of PSII in gene knockout lines after high-temperature treatment; Figure 4 The tomato in Example 3 of this invention SlPRMT4b Relative electrical conductivity of gene knockout lines after high-temperature treatment; Figure 5 The tomato in Example 3 of this invention SlPRMT4b Survival rate of gene knockout lines after high-temperature treatment; Figure 6 The tomato in Example 3 of this invention SlPRMT4b Malondialdehyde (MDA) content in gene knockout lines after high-temperature treatment; Figure 7 Wild-type (WT) and wild-type (WT) in Embodiment 3 of the present invention SlPRMT4b Changes in the expression levels of key heat-tolerant genes in gene knockout lines after 1 hour of high-temperature treatment at 45℃, where A represents tomato. SlHSP17.4Expression level of gene (Solyc08g062340), B represents tomato. SlHSP20 Expression level of gene (Solyc08g062450), C represents tomato SlHSP70 Expression level of gene (Solyc11g020040). Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The present invention is intended to cover other modifications and variations within the scope and spirit of the present invention.

[0024] Unless otherwise stated, the present invention will be practiced using conventional botanical techniques, tissue culture, molecular biology, biological physiology and biochemistry, DNA recombination, and bioinformatics techniques that are readily apparent to those skilled in the art. These techniques are fully explained in the literature.

[0025] Example 1: Construction of the SlPRMT4b CRISPR / Cas9 gene knockout vector; SlPRMT4b The gene mutation vector was constructed using the CRISPR-P2.0 website. SlPRMT4b The gene target sequence, specifically shown in SEQ ID No: 3, is TTTGGACAGGAGTCTCAATC. The primer sequence is Target1-F: CTGGTCTCTATTGaacaaagcaccagtggtctagtg. Using the CRISPR-P2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), the U6 promoter was selected for screening. SlPRMT4b The sgRNA of the gene. Among them, SlPRMT4b The CRISPR / Cas9 vector uses sgRNA from the exon of the gene itself. The sgRNA has one forward primer and one reverse primer for PCR to obtain fragments containing sgRNA. The forward primer of the sgRNA used in the vector is a universal forward primer. The PCR primer sequences are shown in Table 1. Figure 1 Indicating gRNA in tomatoes SlPRMT4bGenomic location and sequence. The specific construction process was as follows: using a tRNA vector as a template, PCR was performed on two target sites using KOD high-fidelity enzyme, and the PCR products were purified to a fragment size of approximately 200 bp. Multi-fragment recombination was performed using the Golden Gate Assembly Kit (BsaIHFv2) (NEB, E1601). The recombination system included Golden Gate Assembly Mix, PHEE401 vector, target PCR purified products, and T4 DNA ligase buffer, with ddH2O added to a final volume of 20 μL. The PCR program settings followed the kit instructions. The recombinant products were heat-shocked and transformed into trans5α E. coli competent cells (TransGen, CD201). After activation at 37°C and 200 rpm for 1 h, the supernatant was discarded by centrifugation, leaving 100–150 μL of liquid. The precipitate was resuspended and evenly spread on solid LB medium containing 50 mg / L kanamycin, and incubated overnight at 37°C. Single-spot small-scale shaking was performed, and positive clones were identified by bacterial culture PCR using universal M13-F and M13-R primers. The positive plasmids with correct sequencing alignment were named pHEE401-SlPRMT4b.

[0026] Table 1: PCR primer sequences for constructing the CRISPR / Cas9 vector (pHEE401-SlPRMT4b); Example 2: Tomato SlPRMT4b Construction and Detection of Gene Editing Materials Gene-editing vectors were transformed into Agrobacterium GV3101 and infected with tomato cotyledons. Callus induction, hygromycin resistance induction differentiation, and rooting culture were then performed to obtain tissue culture seedlings. Positive results were verified using PCR and next-generation sequencing. SlPRMT4b Gene-edited mutant plants were propagated for two generations until they were homozygous, and no Cas9 residues were found. SlPRMT4b #1 is missing 4 bases. SlPRMT4b #2 is missing 1 base ( Figure 1 ).

[0027] Example 3: SlPRMT4b High-temperature resistance testing of gene-editing materials WT and the gene-editing material obtained in Example 2 SlPRMT4b #1, SlPRMT4b#2 seedlings were germinated at 28℃ and then sown in seedling trays containing a mixture of peat moss and vermiculite (v / v=3:1). Hoagland's nutrient solution was applied regularly to keep the substrate moist. When the seedlings reached the three-leaf stage, they were transplanted into plastic pots. Subsequently, WT tomato seedlings with five leaves and one bud, along with the two gene-edited materials of SlPRMT4b obtained in Example 2, were treated at 25℃ (room temperature) and 45℃ (high temperature) in an artificial climate incubator. Four treatment groups were established, comparing wild-type (WT) and the two SlPRMT4b gene-edited materials (#1 and #2) under room temperature and high temperature conditions. One hour after high-temperature treatment, RNA was extracted from the second leaf of each group, and the expression changes of key heat-resistant genes were analyzed by qRT-PCR. After 10 h of high-temperature treatment, the high-temperature resistance index of the high-temperature stress treatment group (treated at 45℃) and the control group (treated at 25℃) under the same conditions without high-temperature treatment was measured. The changes in phenotype, maximum PSII photochemical efficiency (Fv / Fm), relative conductivity, and survival rate of WT and gene-edited tomato plants were observed, and the MDA content in leaves was measured.

[0028] The maximum photochemical efficiency of PSII (Fv / Fm) is a core parameter characterizing the health status of photosynthetic organs and the degree of photoinhibition. High temperatures directly damage PSII reaction centers, leading to a decrease in Fv / Fm. The method for determining Fv / Fm is as follows: tomato plants are dark-acclimated for more than 30 minutes, leaves from the same functional parts are placed on a test tray, and the maximum photochemical efficiency of PSII of tomatoes (Fv / Fm) after 10 hours of high-temperature treatment is measured using a chlorophyll fluorescence imaging spectrometer (IMAG-PAM).

[0029] High temperatures can ultimately damage cell membrane structures. Therefore, relative conductivity (REL) is widely used as a benchmark to assess the extent of cell membrane damage caused by high temperatures. The method for determining the relative conductivity of leaves is as follows: 0.20 g of tomato leaf sample (with veins removed) is placed in a 50 ml sealed culture tube containing 20 ml of ddH2O. The sample is shaken for 2 h on a rotary shaker (200 rpm) at 28°C. The conductivity R1 of the extract is then measured using a conductivity meter. The sample is then incubated in a water bath at 95°C for 15 min. After cooling to room temperature, the conductivity R2 of the extract is measured. Relative conductivity = R1 / R2 × ​​100%.

[0030] As an outcome indicator, survival rate comprehensively reflects a plant's overall ability to repair, recover, and continue growing after suffering high-temperature damage, and is the ultimate manifestation of its heat resistance. Survival rate = (Number of plants with surviving apical meristems / Total number of plants treated with high temperatures) × 100%.

[0031] High temperatures disrupt the balance of reactive oxygen species (ROS) metabolism within cells, leading to a large accumulation of micronutrients (MDA) that attack membrane systems. Therefore, a significant increase in MDA content at high temperatures is a marker of cellular oxidative stress. Reducing MDA accumulation thus becomes a key objective in enhancing heat resistance. MDA content in leaves was determined using an MDA content detection kit (Boxbio, AKFA013M): 0.10 g of fresh tomato leaves (with veins removed) was weighed, 1 ml of extraction buffer was added, the sample was treated, homogenized in an ice bath, and centrifuged at 8000 g for 10 min at 4℃. The supernatant was used as the test sample. Then, 150 μL of the test sample and 50 μL of reagent 1 were added sequentially to a well-sealed screw-cap centrifuge tube, mixed thoroughly, treated in a boiling water bath for 5 min, cooled to room temperature in an ice bath, and centrifuged at 8000 g for 10 min. The supernatant was collected, and 250 μL of working solution and 100 μL of reagent 3 were added to 150 μL of the supernatant. The mixture was mixed thoroughly, treated in a boiling water bath for 60 min, cooled to room temperature in an ice bath, and centrifuged at 8000 g for 10 min. The supernatant was collected, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance at 450 nm, 532 nm, and 600 nm was measured using a microplate reader.

[0032] qRT-PCR experiments were used to reveal the strength of active molecular protection mechanisms initiated by plants under high temperatures at the gene transcription level. The specific method of qRT-PCR experiments is as follows: 0.20 g of leaf sample was taken for RNA extraction. RNA was extracted using the Trizol lysis method with a total RNA extraction kit (Tiangen, DP419) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using HiScript II QRTSuperMix (Vazyme Biotech, China), followed by qRT-PCR experiments. Real-time quantitative PCR (qRT-PCR) was performed using the 480II RealTimePCR detection system (Roche, Swiss) and AceQ qPCR SYBR Green Master Mix kits (Vazyme Biotech, China), with housekeeping genes as the primary target. SlACTIN2 and SlUBI3 This was used as an internal control. The qRT-PCR reaction conditions were: 95℃ for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, for 40 cycles. Fluorescence data were collected at the end of the extension phase of each cycle. Based on the tomato... SlHSP17.4 / 20 / 70 Gene sequence-specific primers: SlHSP17.4 Pre-primer: 5'GATGGAAGCTGCTGGTGAAG (SEQ ID NO. 4); SlHSP17.4 Gene post-primer: 5'ACTCTTTCACGTCCACTGGT (SEQ ID NO. 5); SlHSP20 Pre-primer: 5'TGTTGTGGATATGCCAGGGT (SEQ ID NO. 6); SlHSP20 Post-gene primer: 5'CCCAACCCTTCTCTCCATCC (SEQ ID NO. 7); SlHSP70 Pre-primer: 5'AGTAATGCAGCTGGGACAGT (SEQ ID NO. 8); SlHSP70 Post-gene primer: 5'TGTACCAGCACCAGGAGAAG (SEQ ID NO. 9); In the figure, lowercase letters a, b, and c indicate that the differences between different plants are significant at the 5% level. The statistical method used is Turkey's test.

[0033] The results showed that after treatment at 45℃ for 10 hours, the leaves of wild-type tomato plants severely wilted, while SlPRMT4b Gene knockout tomato plants showed reduced leaf wilting and significantly improved heat tolerance. Figure 2 ); Fv / Fm of gene-edited plants ( Figure 3 The relative conductivity was also significantly higher than that of the WT plant, indicating that the SlPRMT4b-edited plant effectively mitigated the damage of high temperature to the core photosynthetic structure, and the photoinhibition of photosystem II was significantly reduced, thus maintaining a stronger photosynthetic capacity under stress. This is a key physiological basis for its heat-resistant phenotype. Furthermore, the relative conductivity was significantly lower than that of the WT plant, meaning that the cell membrane electrolyte permeability of the gene-edited material was reduced, and the damage was less severe. Figure 4 In addition, the number of surviving growth points increased significantly compared to WT, meaning the survival rate increased significantly. Figure 5 The MDA content was significantly lower than that of WT ( ); Figure 6 This indicates SlPRMT4b Gene knockout tomato leaves showed less oxidative damage to their cell membranes at high temperatures, exhibiting stronger heat resistance. RNA was extracted from leaves treated at 25℃ and 45℃ after 1 h of treatment, and quantitative PCR was performed using reverse transcription. The results showed that high temperature-induced... SlHSP17.4 ( Figure 7 A) SlHSP20 ( Figure 7 (B) SlHSP70 ( Figure 7 The transcriptional level of C in the WT was significantly higher than that of WT. Figure 7This indicates that knocking out the SlPRMT4b gene under high-temperature stress can improve the expression of key heat-resistance genes in tomato plants. SlSlHSP17.4 / 20 / 70 The gene expression level was adjusted, and the gene-edited plants initiated a stronger heat stress protection mechanism. This indicates that... SlPRMT4b Gene-edited materials are more heat-resistant, namely tomatoes. SlPRMT4b Genes negatively regulate the heat tolerance of plants. Knockout. SlPRMT4b Genes can significantly enhance the high-temperature resistance of tomatoes.

[0034] Based on the above embodiments, the nucleotide sequence shown in SEQ ID NO: 1 is mutated by gene editing technology by substituting, deleting and / or adding one or more nucleotides without changing the function of the original nucleotide sequence. Then, it is transformed into Agrobacterium, and then the transformed Agrobacterium is used to infect tomato cotyledons and carry out plant tissue culture. Positive mutant plants are screened, and heat-resistant mutant tomatoes can also be obtained.

[0035] Based on the above embodiments, the nucleotide sequence with the same function obtained by hybridization with the sequence shown in SEQ ID NO: 1 under strict conditions is mutated in tomatoes using gene editing technology, then transformed into Agrobacterium, and then the transformed Agrobacterium is used to infect tomato cotyledons and plant tissue culture is carried out. Positive mutant plants are screened, and heat-resistant mutant tomatoes can also be obtained.

[0036] Based on the above embodiments, nucleotide sequences that have more than 90% homology with nucleotide sequences of 1), 2), or 3) and encode the same functional protein are mutated in tomatoes using gene editing technology, then transformed into Agrobacterium, and then the transformed Agrobacterium is used to infect tomato cotyledons and plant tissue culture is carried out. Positive mutant plants are screened, and heat-resistant mutant tomatoes can also be obtained.

[0037] Although the present invention has been described in detail above with general descriptions and specific embodiments, the present invention is not limited to the above embodiments, and many modifications or improvements are possible, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A type of knockout tomato SlPRMT4b A method for improving the heat resistance of tomatoes using genes, characterized by: The method involves knocking out the tomato... SlPRMT4b Genes enhance the resistance of tomatoes to high-temperature stress. SlPRMT4b The coding sequence of the gene is shown in SEQ ID NO.

1.

2. The method as described in claim 1, characterized in that, The SlPRMT4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that, The gene knockout technology is as follows: In tomatoes SlPRMT4b The protein-coding region of the gene was selected from a target fragment containing a PAM motif adjacent to the pre-intercalation region. Based on the first 20 bases of this fragment, corresponding primers were designed to construct a CRISPR / Cas9 vector. The CRISPR / Cas9 vector was introduced into host cells, and then used to infect the cotyledons of the target tomato. Positive gene-edited plants were screened to obtain heat-resistant gene-edited plants. SlPRMT4b .

4. The method as described in claim 1, characterized in that, The SlPRMT4b Gene knockout mutant plants enhance the expression of key heat-resistant genes SlHSP17.4 , SlHSP20 , SlHSP70 The level of gene expression can be increased, thereby enhancing the plant's resistance to high-temperature stress.

5. The method according to claim 3, characterized in that, The host cell is either an Escherichia coli cell or an Agrobacterium cell.

6. The method according to claim 3, characterized in that, The host cell is preferably GV3101 Agrobacterium cells.

7. The method according to claim 3, characterized in that, The nucleotide sequence containing the first 20 bases of the PAM motif adjacent to the preinterspace sequence is shown in SEQ ID NO:

3.

8. Tomato SlPRMT4b The application of genes in improving the heat resistance of tomatoes is characterized by, The application involves knocking out [the pollutants] in tomatoes. SlPRMT4b Genes enhance the resistance of tomatoes to high-temperature stress. SlPRMT4b The coding sequence of the gene is shown in SEQ ID NO.1.

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