Application and method of tomato slcml39 gene in improving tomato high temperature tolerance

Abstract

This invention discloses the application and method of the tomato SlCML39 gene in improving the high-temperature tolerance of tomatoes. The nucleotide sequence of the tomato SlCML39 gene is shown in SEQ ID NO. 1; the amino acid sequence of the protein encoded by the SlCML39 gene is shown in SEQ ID NO. 2. This invention reports for the first time the use of the SlCML39 gene in negatively regulating the resistance of tomatoes to high-temperature stress. Functional verification of this gene using CRISPR / Cas9 gene editing technology revealed that mutant plants with the loss of function of the SlCML39 gene showed significantly enhanced resistance to high temperatures. This invention utilizes CRISPR technology to innovate tomato germplasm resources through gene editing, obtaining high-temperature tolerant plants without introducing exogenous genes, which has significant theoretical and practical application value.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology engineering, and in particular to the application and method of the tomato SlCML39 gene in improving the high-temperature tolerance of tomatoes. Background Technology

[0002] Tomato (Solanum lycopersicum L.) is an annual herbaceous plant belonging to the genus Solanaceae. It originated in Peru, Ecuador, and other regions in the Andes Mountains of South America.

[0003] Tomatoes are rich in nutrients and have a great flavor. They are one of the world's top 30 highest-yielding crops and are very popular with consumers. Tomatoes prefer warm climates but are not tolerant of high temperatures. The optimal daytime growing temperature is 25℃~28℃, and the optimal nighttime growing temperature is 15℃~22℃.

[0004] The optimal temperature range for tomato seed germination and photosynthesis is 25-30℃, with the most suitable temperature during flowering and fruit setting being around 25℃. When the ambient temperature reaches 35℃, tomato seed viability decreases, plant growth slows, fruit setting rate decreases, and fruit quality declines; photosynthesis is inhibited, and reactive oxygen species accumulate in large quantities.

[0005] In recent years, with global warming, tomatoes grown in the south of my country have often been affected by high temperatures in summer and autumn. Therefore, it is of great significance to study the mechanism of high temperature tolerance regulation of tomatoes and to cultivate new high temperature tolerant tomato varieties.

[0006] To adapt to high-temperature stress, plants form a complex regulatory network of related genes. Transcriptional regulation is an important component of this gene regulatory network. Transcription factors promote the expression of downstream stress-resistance genes through different signal transduction pathways, thereby enhancing plant heat tolerance.

[0007] Calcium ions, as important second messengers in eukaryotic cells, mediate the responses of eukaryotes to almost all stimuli related to development and environmental adaptation. Calcium decoders in plants contain EF-chiral domains. The binding proteins mainly include calmodulins (CaMs) and calmodulin-like proteins (CMLs), calcineurin B-like proteins (CBLs) and their interacting protein kinases (CBL-interacting protein kinases, CIPKs), as well as calcium-dependent protein kinases (Ca... 2+ -dependent protein kinases, CDPKs / CPKs).

[0008] Among them, CAMs / CMLs are one of the most conserved protein families in eukaryotes. Their structure contains only the EF-chiral domain and no other functional domains, playing a crucial role in sensing, transmitting and decoding calcium signals.

[0009] In recent years, the physiological functions of CaMs / CMLs in regulating plant growth and development, abiotic stress, and biotic stress resistance have been increasingly revealed. These functions mainly include: participating in the regulation of developmental processes such as pollen germination, pollen tube elongation, flowering period regulation, root growth, leaf epidermal hair development, root nodule development, and nitrogen fixation symbiosis; participating in the regulation of plant resistance to abiotic stresses such as salt, drought, cold, heat, abscisic acid (ABA), light, and mechanical contact; and regulating immunity against bacterial, fungal, oomycete, viral, and herbivorous insect infections.

[0010] Although the functions of many CML family members are still unknown, they play important roles in different growth and development processes and environmental conditions, and have high research value.

[0011] Therefore, to address the aforementioned technical challenges, researching the role of CML39 in regulating tomato's high-temperature tolerance can not only directly promote the development of the tomato industry but also provide experimental ideas and theoretical basis for studying the response mechanisms of other plants to high temperatures. Furthermore, it offers a theoretical foundation and practical guidance for enhancing the high-temperature tolerance of tomatoes and other crops using genetic methods. Summary of the Invention

[0012] In view of this, the present invention provides the application and method of the tomato SlCML39 gene in improving the high temperature tolerance of tomatoes. The purpose of the present invention is to mine genes that regulate the plant's resistance to high temperature stress from the tomato genome, and to provide gene resources for the creation of new high temperature tolerant tomato germplasm.

[0013] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0014] Application of the tomato SlCML39 gene in improving the high-temperature tolerance of tomatoes, the nucleotide sequence of the tomato SlCML39 gene is shown in SEQ ID NO. 1.

[0015] Preferably, the amino acid sequence of the protein encoded by the SlCML39 gene is shown in SEQ ID NO.2.

[0016] Preferably, the indicators of resistance to high temperature stress include at least one of the following: plant phenotype, antioxidant enzyme activity, hydrogen peroxide accumulation, relative conductivity (REL), malondialdehyde (MDA) content, chlorophyll content, and PSII maximum photochemical quantum yield (Fv / Fm).

[0017] Preferably, biological techniques are used to downregulate or eliminate the function of the SlCML39 gene in tomato plants to enhance the resistance of tomato plants to high-temperature stress.

[0018] The method of improving the high temperature tolerance of tomatoes by knocking out the SlCML39 gene in tomatoes using CRISPR / Cas9 gene editing technology to obtain homozygous mutant tomato plants.

[0019] The expression of the SlCML39 gene was suppressed in the target plant.

[0020] Preferably, the suppression of SlCML39 gene expression in the target plant is achieved by constructing CRISPR / Cas9 vectors of the gene and then infecting them with Agrobacterium.

[0021] The construction method includes the following steps:

[0022] (1) Select a target fragment containing a PAM structure in the protein coding region of the tomato SlCML39 gene. Based on the first 20 bases of the PAM structure of the target fragment, design primers and construct a CRISPR / Cas9 vector.

[0023] The PAM structure is NGG, where N represents any base;

[0024] The 20 bp sequence preceding NGG is defined as sgRNA, and sgRNA sequences that are located in the gene protein coding region and have high specificity are selected.

[0025] (2) Then, Agrobacterium-mediated genetic transformation technology was used to transform the tomato recipient tissue, and the homozygous mutant line with the loss of function of the SlCML39 gene was obtained by culturing and screening.

[0026] Preferably, the nucleotide sequence of the first 20 bases of the target fragment PAM structure is as shown in SEQ ID NO.3.

[0027] Preferably, the nucleotide sequences of the primer pairs used to construct the CRISPR / Cas9 vector are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0028] Preferably, the vector used to inhibit SlCML39 gene expression is the pHSbdcas9i-tRNA(k5) vector; and the Agrobacterium genetically engineered strain is Agrobacterium EHA105.

[0029] Preferably, the homozygous mutant line with loss of SlCML39 gene function is a homozygous mutant line in which a base deletion occurs at the sgRNA position, causing premature termination of SlCML39 gene translation, which does not contain exogenous Cas9 protein and is stably inherited.

[0030] The present invention achieves the following technical effects compared to the prior art:

[0031] (1) The plant’s resistance to high temperature damage is significantly improved: Under the same high temperature conditions, the slcml39 mutant tomato obtained by knocking out SlCML39 has significantly lower relative conductivity and malondialdehyde content than the wild type plant, and the high temperature damage to the plant is significantly reduced.

[0032] (2) The photosynthetic performance of the plant was effectively maintained under high temperature: the maximum photochemical quantum yield Fv / Fm and chlorophyll content of PSⅡ of the slcml39 mutant were significantly higher than those of wild tomato, ensuring that the plant could maintain a high photosynthetic capacity under high temperature stress and maintain the basis for normal growth and development.

[0033] (3) Enhanced activity of plant antioxidant system and reduced oxidative damage: The activity of antioxidant enzymes such as peroxidase POD and catalase CAT in slcml39 mutant was significantly upregulated, which can more efficiently remove reactive oxygen species, resulting in less accumulation of hydrogen peroxide (H2O2) in the plant and a lower degree of oxidative damage caused by high temperature.

[0034] (4) The breeding materials are highly safe and have outstanding application value: This invention clarifies that SlCML39 is a negative regulator of tomato high temperature resistance. New germplasm is created using CRISPR gene editing technology. Stable heat-resistant tomato plants can be obtained without the introduction of exogenous genes. This not only clarifies the molecular mechanism of tomato high temperature response and provides a clear target gene for heat-resistant tomato breeding, but also obtains safe and reliable breeding materials. It has important theoretical research value and practical production application value in the field of tomato heat resistance breeding. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the gene editing sites of the T1 generation mutant plant obtained in Example 2 of the present invention;

[0036] In this diagram, A represents the tomato plant tissue culture process; B represents the identification of positive tomato seedlings; and C represents the two mutant lines slcml39#12 and slcml39#44 obtained. Compared with the unedited common AC tomato (hereinafter referred to as wild-type WT), the gene-edited mutants have base deletions at the sgRNA position. Tomato slcml39#12 and slcml39#44 have deletions of 58 and 2 bases, respectively, compared with the control, which prematurely formed stop codons in the translation region, causing premature termination of translation.

[0037] Figure 2 The images show the phenotypic figures of tomato slcml39 mutant and wild-type plants after being treated at 42°C for 6 hours in Example 3 of this invention.

[0038] The higher the degree of leaf wilting, the more severe the damage caused by high temperature stress.

[0039] Figure 3 The results of physiological state detection of tomato slcml39 mutant and wild-type plants after high temperature treatment at 42℃ for 6 hours in Example 4 of this invention;

[0040] Where A is the color image of tomato leaves corresponding to the maximum photochemical efficiency Fv / Fm of photosystem II;

[0041] B is a color image of a tomato leaf corresponding to the chlorophyll index;

[0042] C represents the Fv / Fm value;

[0043] D represents the relative value of chlorophyll content;

[0044] The data shown in C and D are the average of three repetitions, with the standard error indicated by a vertical line;

[0045] The Tukey test was used, and ** indicates that the differences between different treatments are significant at the p-value ≤ 0.001 level;

[0046] Figure 4 The image shows the cell membrane damage detection results of leaves of tomato slcml39 mutant and wild-type plants after high-temperature treatment at 42℃ for 6 h in Example 5 of this invention.

[0047] In this figure, A represents the results of MDA content determination; B represents the results of conductivity REL determination.

[0048] The Tukey test was used, and ** indicates that the differences between different treatments were significant at the p-value ≤ 0.01 level;

[0049] Figure 5 The results of DAB staining and antioxidant enzyme activity detection of leaves of tomato slcml39 mutant and wild-type plants after high-temperature treatment at 42℃ for 6 h in Example 6 of this invention;

[0050] In the figure, A represents the DAB staining result; B represents the SOD enzyme activity assay result; and C represents the POD enzyme activity assay result.

[0051] ** represents p≤0.01. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] This invention discloses the application of the tomato SlCML39 gene in improving the high-temperature tolerance of tomatoes. The nucleotide sequence of the tomato SlCML39 gene is shown in SEQ ID NO. 1.

[0054] The amino acid sequence of the protein encoded by the SlCML39 gene is shown in SEQ ID NO.2.

[0055] Indicators of resistance to high temperature stress include at least one of the following: plant phenotype, antioxidant enzyme activity, hydrogen peroxide accumulation, relative conductivity (REL), malondialdehyde (MDA) content, chlorophyll content, and maximum photochemical quantum yield of PSII (Fv / Fm).

[0056] Biological techniques were used to downregulate or eliminate the function of the SlCML39 gene in tomato plants, thereby enhancing the resistance of tomato plants to high-temperature stress.

[0057] This invention also discloses a method for improving the high-temperature tolerance of tomatoes by knocking out the SlCML39 gene in tomatoes using CRISPR / Cas9 gene editing technology to obtain homozygous mutant tomato plants.

[0058] The expression of the SlCML39 gene was suppressed in the target plant.

[0059] This invention also discloses a method for improving the high-temperature tolerance of tomato using the SlCML39 gene. The inhibition of SlCML39 gene expression in the target plant is achieved by constructing CRISPR / Cas9 vectors of the gene and then infecting them with Agrobacterium.

[0060] The construction method includes the following steps:

[0061] (1) Select a target fragment containing a PAM structure in the protein coding region of the tomato SlCML39 gene. Based on the first 20 bases of the PAM structure of the target fragment, design primers and construct a CRISPR / Cas9 vector.

[0062] The PAM structure is NGG, where N represents any base;

[0063] The 20 bp sequence preceding NGG is defined as sgRNA, and sgRNA sequences that are located in the gene protein coding region and have high specificity are selected.

[0064] (2) Then, Agrobacterium-mediated genetic transformation technology was used to transform the tomato recipient tissue, and the homozygous mutant line with the loss of function of the SlCML39 gene was obtained by culturing and screening.

[0065] The nucleotide sequence of the first 20 bases of the target fragment PAM structure is shown in SEQ ID NO.3.

[0066] The nucleotide sequences of the primer pairs used to construct the CRISPR / Cas9 vector are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0067] The vector used to suppress SlCML39 gene expression is the pHSbdcas9i-tRNA(k5) vector; the Agrobacterium genetically engineered strain is Agrobacterium EHA105.

[0068] The homozygous mutant line of SlCML39 gene loss of function is a homozygous mutant line in which a base deletion occurs at the sgRNA position, causing premature termination of SlCML39 gene translation, which does not contain exogenous Cas9 protein and is stably inherited.

[0069] The tomato variety used in the following examples is the conventional variety "Ailsa Craig", and unedited ordinary tomatoes are used as a control.

[0070] The nucleotide sequence runs from the 5' end to the 3' end from left to right.

[0071] Main materials: Catalase (POD) activity assay kit (colorimetric method), superoxide dismutase (SOD) activity assay kit, malondialdehyde (MDA) content assay kit, and chlorophyll content assay kit (colorimetric method), Sangon Biotech (Shanghai) Co., Ltd.

[0072] Catalase (CAT) activity assay kit, ammonium molybdate colorimetric method, Beijing Box Biotechnology Co., Ltd.; all other reagents were domestically produced analytical grade.

[0073] HS-800D constant temperature water bath, Taicang Science and Education Equipment Factory; IMS-20 fully automatic snowflake ice maker, Changshu Xueke Electric Appliance Co., Ltd.; PRX-1608 intelligent artificial climate chamber, KSPL-1100 intelligent artificial climate chamber, Ningbo Kesheng Experimental Instrument Co., Ltd.; CT15RE benchtop micro-volume high-speed refrigerated centrifuge, Hitachi Koki Co., Ltd.; PlantExplorer V1.0 plant phenotyping and imaging analysis system, PhenoVation, Netherlands; VL0LATD0 Varioskan™ LUX multi-functional microplate reader, Thermo Fisher Scientific (China) Co., Ltd.; BX51TRF research-grade upright fluorescence microscope, Olympus Corporation. Primers were synthesized by Sangon Biotech (Shanghai) Technology Co., Ltd.

[0074] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0075] Example 1: Construction of a CRISPR / Cas9 vector containing SlCML39-specific sgRNA

[0076] The DNA sequence of the SlCML39 (Solyc11g071740) gene was found in the Slycopersicum_ITAG4.0 genome database of the Solanaceae Genomics Network website (https: / / www.solgenomics.net / ), as shown in SEQ ID NO.1. Using the website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR, a 20 bp base sequence AAGTGTCACCTGCAGAGTTG (SEQ ID NO.3) was identified that has a high Onscore score, GC content ≥40%, Offtargetscore score ≤0.4, and is located in the protein coding region before the PAM structure.

[0077] Design CRISPR primers as follows:

[0078] CRISPR pre-primer: cagtGGTCTCatgcactaccaccattgtcactcacgttttagag (SEQ IDNO.4);

[0079] CRISPR post-primer: cattGGTCTCaaaaccaactctgcaggtgacactttgcac (SEQ ID NO.5);

[0080] The above primers were used to perform PCR amplification with pHSbdcas9i-tRNA(k5) plasmid as template. After the product was purified by a conventional DNA purification kit, the amplified fragment was ligated to the pHSbdcas9i-tRNA(k5) plasmid with BsaI enzyme. The product was transformed into E. coli plate at 42℃ with kanamycin as the antibiotic.

[0081] Single colonies were selected and verified by PCR using the universal front primer Pbw2+:GCAACGCTCTGTCATCGTTACAAT (SEQ ID NO. 6) and the universal back primer Pbw2-:GCGATTAAGTTGGGTAACGCCAGGG (SEQ ID NO. 7) for the pHSbdcas9i-tRNA(k5) vector.

[0082] The bacterial culture with the correct band size was sent to a sequencing company for sequencing. The sequencing results showed that the vector contained one sgRNA sequence. After plasmid extraction, it was electroporated into Agrobacterium EHA101 competent cells. After two days of incubation at 28°C, the plasmids were picked for PCR verification, and Agrobacterium strains that can be used to construct CRISPR / Cas9 gene editing materials were obtained.

[0083] Among them, SEQ ID No. 1:

[0084] ATGGTATCATCAGTGTCTGTTTCCACAGCTGAGAAAGAGTCTTTTTTCTCGAGATTACGAAATATGTTTCATTTCAGAAGGAATGAAGATGAGAAGAAGACAACGACAACAACAACAACGAGAGCTGCTGCTGCTGCAGCAACTACTACTACTAACTGTGTTCCTGTGAGTGACAATGGTGGTAGTAGTAACAAGGGAGAGTTAGAGAGGGTGTTTACGTACTTTGATGAGAATGGAGATGGAAAAGTGTCACCTGCAGAGTTGAGGAAGTGTGTGAAGGCGGTAGGAGGTGAGCTGACGGTGGAGGAGGCGGAGATGGCAGTGAGGCTATCGGATTCTGATGGGGACGGATTATTGGGCATTGAGGATTTTACGAAACTGATGGAAGGAATGGAGGAAGAGAGGAATAAAGAGGGTGAGTTGATGGGAGCATTTGGAATGTATGAAACAGAAGGATACATTACTCCTAAGAGCTTGAAGAACATGTTGAGTCGACTAGGTGAATCAACCTCCATTGATAACTGCAAAGCTATGATTCGAAGGTTTGATCTCAATGGAGATGGAGTCCTCAGCTTTGATGAGTTCAAAGTTATGATGACAACTTAG

[0085] SEQ ID No. 2:

[0086] MVSSVSVSTAEKESFFSRLRNMFHFRRNEDEKKTTTTTTTRAAAAAATTTTNCVPVSDNGGSSNKGELERVFTYFDENGDGKVSPAELRKCVKAVGGELTVEEAEMAVRLSDSDGDGLLGIEDFTKLMEGMEEERNKEGELMGAFGMYETEGYITPKSLKNMLSRLGESTSIDNCKAMIRRFDLNGDGVLSFDEFKVMMTT

[0087] Example 2: Preparation and identification of tomato slcml39 mutant material

[0088] 1. Seed disinfection: Wash with sterile water for 2 minutes, disinfect with 75% alcohol for 40 seconds, wash with 84 disinfectant for 7 minutes, wash with sterile water 3 times, and soak in sterile water for 1 hour.

[0089] 2. Sowing: Sow the sterilized tomato seeds on the germination medium and allow them to germinate for 7-9 days.

[0090] 3. Preparation and pre-culture of explants: Germinated tomato seedlings, after the cotyledons are fully expanded, the cotyledon petioles and cotyledon tips are removed with a scalpel, leaving the middle part, cut into 2-3 segments, and inoculated into pre-culture medium, pre-cultured at 23±2 ℃ for 2-3 days.

[0091] 4. Agrobacterium infection and co-culture: Agrobacterium was picked into the infection solution and OD was prepared. 600 = 0.1% Agrobacterium resuspension; infect for 10-15 min, then inoculate the dried explants into co-culture medium and incubate in the dark at 23±2 ℃ for 2 days.

[0092] 5. Screening and Differentiation Rooting: The recovered callus was inoculated onto a screening medium and screened for 15-30 days. The screened callus was then inoculated onto a differentiation medium and differentiated for 30-40 days. When the seedlings to be differentiated grew to about 2-3 cm, they were removed from the callus and inoculated onto a rooting medium. Rooting culture was carried out for 10-15 days to obtain the T0 generation gene-edited tomato.

[0093] 6. Detection: Genomic DNA was extracted from T0 generation tomato plants using the CTAB method. Primers were designed approximately 150-200 bp before and after the DNA sequence containing sgRNA for PCR amplification and sequencing verification.

[0094] Pre-seedling primer: CCTCCATTCCTTCCATCAG (SEQ ID NO. 8);

[0095] Seedling verification primer: TGTCTGTTTCCACAGCTGAGAC (SEQ ID NO. 9);

[0096] The obtained PCR products were sent to a sequencing company for sequencing.

[0097] The sequencing results were compared with the original gene sequence using Snapgene software. Plants with base deletions in the sgRNA sequence and single-peak sequencing were selected for self-pollination to obtain T0 generation seeds.

[0098] The T0 generation seeds were planted in a growth chamber to obtain T1 generation plants. The sgRNA sequence editing status of the T1 generation plants was detected using the same method as above. Simultaneously, PCR amplification of the DNA of the T1 generation plants was performed using Cas9 gene primers to detect the presence of the Cas9 sequence. T1 generation plants with mutated sgRNA and lacking the Cas9 protein were selected and identified as gene-edited lines, named slcml39#12 and slcml39#44, with their gene editing sites as follows: Figure 1 As shown.

[0099] Compared to the control plant, slcml39#12 lacks 58 base pairs, and slcml39#44 lacks two base pairs. After sowing seeds of the T1 generation of this line, stable T2 generation plants without the exogenous Cas9 gene and exhibiting sgRNA variation were obtained.

[0100] The following examples all used the above-mentioned homozygous T2 generation plants as materials for the experiment.

[0101] Example 3: High-temperature resistance phenotype of tomato slcml39 mutant

[0102] 1. Tomato seeds (slcml39 and AC) were soaked in 55℃ warm water for 15 minutes, then germinated at 28℃ for 2-3 days. Once the seeds showed signs of germination, they were sown in a mixture of peat moss, vermiculite, and perlite in a 3:1:1 (volume ratio) substrate and cultured in an artificial climate chamber. The photoperiod was 12 / 12h (day / night), the average day / night temperature was 25 / 20℃, and the light intensity was approximately 600 μmol / m². -2 s -1 When the tomato seedlings have grown to the stage of four leaves and one bud, healthy plants of similar size are selected and randomly divided into two groups.

[0103] The control group was placed in an artificial climate chamber for continued cultivation, while the experimental group was moved into an artificial climate chamber for high-temperature treatment at 42℃. After 6 hours, the high-temperature damage to the plants was observed and measured.

[0104] The results are as follows Figure 2 As shown, after treatment at 42℃ for 6 h, the WT wild-type plants showed obvious wilting, with all leaves drooping, most leaves severely curled, and the growing point bent. They could not recover to normal growth after being placed at room temperature for 12 h. In contrast, the wilting degree of the slcml39 mutant was much weaker than that of the WT. The plant leaves drooped slightly, with only the edges curled. The degree of impact from high temperature stress was significantly lower than that of the wild-type plants, and the plants recovered to normal growth after being placed at room temperature for 12 h.

[0105] Example 4: Determination of photosynthetic physiological status of tomato slcml39 mutant and wild-type plants

[0106] 1. After dark treatment of tomato plants before and after high temperature treatment for 0.5 h, functional leaves of the second and third nodes of plants under different treatments were placed in the PlantExplorer Pro multifunctional plant photosynthetic phenotyping system (PhenoVation, Netherlands) for irradiation to detect the minimum fluorescence Fo and maximum fluorescence Fm of the leaves. Fv / Fm was obtained by the following formula.

[0107] Fv / Fm = (Fm-Fo) / Fm.

[0108] The results are as follows Figure 3 As shown in A and C, before the high-temperature treatment, the Fv / Fm ratios of the two plants were similar and there was no significant difference. After 6 hours of high-temperature treatment at 42℃, the Fv / Fm ratios of both WT and slcml39 plants decreased and were significantly lower than those of the untreated plants. Among them, the Fv / Fm ratio of the slcml39 mutant was significantly higher than that of the WT wild-type plant, indicating that the photosystem II light energy conversion efficiency of the slcml39 mutant was significantly higher than that of the wild type under high-temperature stress, and it could still maintain high photosynthetic performance under high-temperature stress conditions.

[0109] 2. Tomato plants before and after high-temperature treatment were dark-treated for 0.5 h. Chlorophyll fluorescence imaging was performed on the tomato plants using the PlantExplorerPro multifunctional plant photosynthetic phenotyping system. Data Analysis Software automatically calculated and generated a chlorophyll index distribution map based on the spectral reflectance ratio of a single pixel or region of interest (ROI).

[0110] The results are as follows Figure 3 As shown in Figure B, the chlorophyll indices of the two plants were similar before the high-temperature treatment, with no significant difference. After 6 hours of high-temperature treatment at 42℃, the chlorophyll content of both WT and slcml39 plants decreased, significantly lower than that of the untreated plants. However, the chlorophyll index of the slcml39 mutant plant was significantly higher than that of WT, indicating that the chlorophyll of the slcml39 mutant was less damaged by heat stress than that of WT and had stronger heat resistance.

[0111] 3. After high-temperature treatment (42℃, 6h), the detached leaves of tomato plants were decolorized with acetone, and the chlorophyll content was measured. The results are as follows: Figure 3 As shown in Figure D, the chlorophyll content of the detached leaves of the treated SlCML39 gene knockout tomato plants was higher than that of the wild type, indicating that SlCML39 gene knockout helps maintain photosynthetic capacity under high temperature stress.

[0112] Example 5: Determination of membrane damage in tomato slcml39 mutant and wild-type plants under high temperature stress

[0113] 1. Following Example 3, after treating cultivated slcml39 mutant and control tomato WT at 42℃ for 6 h, 0.1 g (W) of tomato leaves were taken, 6 mL of 10% TCA solution was added, and the mixture was ground in an ice bath. After centrifugation at 12000g for 20 min, the resulting supernatant was the extract (V). 2 mL (V2) of the supernatant was added to 2 mL of 0.6% TBA reaction solution, and after boiling in a water bath for 15 min, it was immediately cooled in an ice bath. After centrifugation at 15000g and 4℃ for 10 min, the supernatant (V1) was taken and its absorbance values ​​at 532 nm, 600 nm, and 450 nm were measured. The MDA content was calculated according to the following formula.

[0114] MDA content (nmol g-1FW) = [6.452×(A532-A600)-0.56×A450] ×(V1×V) / (V2×W).

[0115] The results are as follows Figure 4 As shown in Figure A, after treatment at 42℃ for 6 h, the MDA content of WT plants was significantly higher than that of the slcml39 mutant, indicating that the degree of membrane lipid peroxidation in WT plants was more severe and the damage was greater than that in the slcml39 mutant, and that they were more severely affected by high temperature stress.

[0116] 2. Following Example 3, after treating cultivated slcml39 mutant and control tomato WT at 42℃ for 6 h, the leaves of the second and third nodes of the functional leaves were taken, rinsed with deionized water, and dried. The leaves were punched into leaf discs using a 5 mm diameter punch. 0.1 g of the leaf discs were soaked in 10 mL of ddH2O to completely submerge the leaves in ultrapure water. Three replicates were taken for each treatment.

[0117] The above samples were placed in a shaker at 28℃ and shaken at 200 rpm for 2-3 hours. The EC1 value was measured using a digital conductivity meter (DDS-307 digital conductivity meter, Shanghai Yueping). Then, the samples were placed in a water bath at 95℃ for 20 minutes. After cooling to room temperature, the total conductivity value EC2 was measured.

[0118] Calculate the conductivity REL (%) using the following formula;

[0119] REL(%) = EC1 / EC2 × 100%.

[0120] The results are as follows Figure 4 As shown in Figure B, after high-temperature treatment, the REL of WT plants was significantly higher than that of the slcml39 mutant, indicating that the cell membrane damage of WT plants was stronger than that of the slcml39 mutant, and that they were more severely affected by high-temperature stress.

[0121] Example 6: Determination of antioxidant capacity of tomato slcml39 mutant and wild-type plants under high temperature stress

[0122] 1. Select wild-type WT and slcml39 mutant plants with uniform growth at the four-leaf stage. After treatment at 42℃ for 6 hours, immerse an appropriate amount of leaves in DAB staining solution and stain by slow shaking in the dark for 8-10 hours. Discard the staining solution, add bleaching solution, heat in a 95℃ water bath for 15 minutes, replace the bleaching solution, and shake to decolorize. Observe the phenotype after complete decolorization. Each group has 3 biological replicates.

[0123] The results are as follows Figure 5 As shown in Figure A: After high-temperature treatment, the stained areas of the leaves of the slcml39 mutant tomato plants were less and the staining was lighter than that of the wild-type plants. The results indicate that when the slcml39 mutant tomato plants and WT tomato plants are subjected to the same high-temperature stress, the oxidative damage to the leaves of the slcml39 mutant tomato plants is less than that to the leaves of the WT tomato plants.

[0124] 2. Healthy SlCML39 gene knockout and wild-type four-leaf stage tomato plants were treated at 42℃ for 6 hours, and samples (approximately 3 leaves) were taken for triple biological replicates. 1.6 mL of enzyme extraction buffer was added, and the mixture was ground in liquid nitrogen, then centrifuged at 12000×g for 30 min at 4℃. The supernatant was collected as the crude tomato enzyme extract. The activities of SOD and POD, two antioxidant protective enzymes, were measured at the appropriate wavelengths, with three replicates.

[0125] The results are as follows Figure 5 As shown in B and C, the activities of SOD and POD in WT tomato plants were significantly lower than those in slcml39 mutant tomato plants. The results indicate that under high temperature stress of 42℃, the overall activity of antioxidant protective enzymes in slcml39 mutant tomato plants was higher than that in WT tomato plants.

[0126] In summary, tomato mutants with the SlCML39 gene knocked out exhibited significantly less wilting under high-temperature stress than the wild type, with significantly lower REL and MDA content in leaves. Their Fv / Fm ratio, chlorophyll index, and antioxidant capacity remained at higher levels, demonstrating overall stronger high-temperature resistance. These results demonstrate that the SlCML39 gene negatively regulates tomato high-temperature resistance, and that knocking out the SlCML39 gene is a scientifically efficient technique for creating heat-resistant tomato germplasm resources.

[0127] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The application of the tomato SlCML39 gene in improving the heat tolerance of tomatoes, characterized by, The nucleotide sequence of the tomato SlCML39 gene is shown in SEQ ID NO.

1.

2. The application of the tomato SlCML39 gene according to claim 1 in improving the high-temperature tolerance of tomatoes, characterized in that, The amino acid sequence of the protein encoded by the SlCML39 gene is shown in SEQ ID NO.

2.

3. The application of the tomato SlCML39 gene according to claim 1 in improving the high-temperature tolerance of tomatoes, characterized in that, The indicators of resistance to high temperature stress include at least one of the following: plant phenotype, antioxidant enzyme activity, hydrogen peroxide accumulation, relative conductivity (REL), malondialdehyde (MDA) content, chlorophyll content, and PSII maximum photochemical quantum yield (Fv / Fm).

4. The application of the tomato SlCML39 gene according to claim 1 in improving the high-temperature tolerance of tomatoes, characterized in that, Biological techniques were used to downregulate or eliminate the function of the SlCML39 gene in tomato plants, thereby enhancing the resistance of tomato plants to high-temperature stress.

5. A method for improving the heat tolerance of tomatoes using the tomato SlCML39 gene, characterized in that, Homozygous mutant tomato plants were obtained by knocking out the SlCML39 gene in tomatoes using CRISPR / Cas9 gene editing technology. The expression of the SlCML39 gene was suppressed in the target plant.

6. The method for improving the high-temperature tolerance of tomatoes using the tomato SlCML39 gene according to claim 5, characterized in that, Suppressing the expression of the SlCML39 gene in the target plant was achieved by constructing CRISPR / Cas9 vectors of the gene and then infecting them with Agrobacterium. The construction method includes the following steps: (1) Select a target fragment containing a PAM structure in the protein coding region of the tomato SlCML39 gene. Based on the first 20 bases of the PAM structure of the target fragment, design primers and construct a CRISPR / Cas9 vector. The PAM structure is NGG, where N represents any base; The 20 bp sequence preceding NGG is defined as sgRNA, and sgRNA sequences that are located in the gene protein coding region and have high specificity are selected. (2) Then, Agrobacterium-mediated genetic transformation technology was used to transform the tomato recipient tissue, and the homozygous mutant line with the loss of function of the SlCML39 gene was obtained by culturing and screening.

7. The method for improving the high-temperature tolerance of tomatoes using the tomato SlCML39 gene according to claim 6, characterized in that, The nucleotide sequence of the first 20 bases of the target fragment PAM structure is shown in SEQ ID NO.

3.

8. The method for improving the high-temperature tolerance of tomatoes using the tomato SlCML39 gene according to claim 6, characterized in that, The nucleotide sequences of the primer pairs used to construct the CRISPR / Cas9 vector are shown in SEQ ID NO.4 and SEQ ID NO.

5.

9. The method for improving the high-temperature tolerance of tomatoes using the tomato SlCML39 gene according to claim 6, characterized in that, The vector used to inhibit SlCML39 gene expression is the pHSbdcas9i-tRNA(k5) vector; the Agrobacterium genetically engineered strain is Agrobacterium EHA105.

10. The method for improving the high-temperature tolerance of tomatoes using the tomato SlCML39 gene according to claim 6, characterized in that, The homozygous mutant line with loss of function of the SlCML39 gene is a homozygous mutant line in which a base deletion occurs at the sgRNA position, causing premature termination of SlCML39 gene translation, which does not contain exogenous Cas9 protein and is stably inherited.

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