Application of tomato SlCOMT1 gene in regulating and controlling heat resistance of tomato anther

Abstract

The invention belongs to the field of biotechnology engineering, and particularly relates to application of a tomato SlCOMT1 gene in regulation and control of tomato anther heat resistance. The invention discloses a nucleotide sequence SEQ ID NO.1 of a tomato SlCOMT1 gene and an amino acid sequence SEQ ID NO.2 of a protein coded by the gene, a slcomt1 knockout mutant plant is obtained by utilizing a CRISPR-Cas9 technology, and under high temperature stress, compared with a wild plant, the tomato pollen viability and the fruit setting rate of the SlCOMT1 knockout mutant plant are remarkably improved. The invention proves that SlCOMT1 mutation can cause accumulation and increase of endogenous 5-hydroxytryptamine in tomato flowers at high temperature, so that the high-temperature resistance of tomato anther development is improved, and the yield of tomato anther is stabilized. According to the method, the new germplasm of the heat-resistant tomato can be quickly created, support is provided for high-temperature-resistant production of the tomato, and the method has great application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology engineering, specifically relating to tomatoes. SlCOMT1 Application of genes in regulating heat tolerance of tomato anthers. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes are one of the most widely cultivated and valuable vegetable crops in the world. They are highly favored by consumers due to their rich nutrition, unique flavor, and dual use as both fruit and vegetable. my country is the world's largest producer and consumer of tomatoes. Tomatoes prefer warm temperatures but are not heat-tolerant. Summer and autumn cultivation of tomatoes often encounters temperatures of 32-35°C or even higher, nearly 10°C higher than the optimal growth and reproduction temperature for tomatoes. Heat stress is particularly harmful to the reproductive process of tomatoes, affecting pollen and stigma viability, pollination and flowering, pollen tube elongation, and early embryonic development (Lamaoui et al., 2018). High temperatures not only reduce viable pollen and fruit set but also damage fruit growth and ripening, reducing crop yield (Alsamir et al., 2021). Heat stress-induced pollen abortion is a major limiting factor for high-efficiency tomato production in summer and autumn. To ensure normal fruit production in tomatoes during the high-temperature cultivation season, auxin-like hormones are often used to protect flowers and fruits, thereby achieving high yields under sub-optimal conditions in greenhouse cultivation. However, hormone use not only increases costs but also often causes hormone-related harm, resulting in fruit deformities and decreased product quality. Currently, the sensitivity of tomato male reproductive development to high-temperature stress and the adaptive regulatory mechanisms are still unclear (Katano et al., 2018; Zhang et al., 2021). Therefore, identifying the key regulatory factors of tomato male reproductive development in response to high temperatures, and improving the temperature adaptability of tomato anthers, would reduce reliance on hormone application during fruit setting and development, undoubtedly greatly improving tomato fruit setting stability and quality. This would be a significant breakthrough in the efficient production of tomatoes in greenhouses during the high-temperature season.

[0003] Previous studies have shown that serotonin (5-HT) is an important plant growth regulator that can regulate plant growth and development and plays a crucial role in responses to abiotic stresses such as salt and drought (Byeon & Back, 2014; Kaur et al., 2015). However, its function in the male reproductive development response to high temperatures remains unclear. Previous studies have demonstrated that exogenous 5-HT can improve the heat tolerance of tomato anthers and the fruit set rate under high-temperature stress (Yu Fangjie, 2021), but the relationship between endogenous 5-HT metabolism and the heat tolerance of tomato anthers is still unknown.

[0004] In recent years, influenced by the global warming trend, the duration and intensity of high temperatures in summer and autumn have significantly increased. High temperatures have had a severe negative impact on tomato production. High temperature stress leads to reduced pollen viability and sterility in tomatoes, which in turn hinders pollination, significantly reduces fruit set, and ultimately causes a sharp decline in tomato yield and quality. Therefore, it is urgent to discover heat-resistant genes during the tomato anther development stage to provide key gene resources for breeding resistant varieties during the critical period of tomato reproductive development (i.e., the heat-sensitive stage), and to explore effective genetic improvement techniques. Summary of the Invention

[0005] In view of the shortcomings of existing technologies, the purpose of this invention is to provide a gene for controlling the heat resistance of tomato anthers. SlCOMT1 Genes provide technical support for revealing the molecular mechanisms regulating heat tolerance in tomatoes and improving heat tolerance during reproductive and developmental stages.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides tomatoes SlCOMT1 The application of genes in regulating the heat tolerance of tomato anthers specifically involves: obtaining CRISPR / Cas9 knockout. SlCOMT1 The mutant exhibits the same vegetative growth, flowering, and fruiting characteristics as the wild type under normal temperatures. However, under high-temperature stress, its pollen viability and fruit set rate are significantly higher than those of the wild type, resulting in a significant increase in tomato yield under high-temperature cultivation conditions. The increase in its heat tolerance is closely related to the increase in the endogenous 5-hydroxytryptamine content of the plant.

[0007] The nucleotide sequence of the SlCOMT1 gene is shown in SEQ ID NO.1, and the tomato... SlCOMT1 The gene negatively regulates the heat tolerance of tomato anthers; the amino acid sequence of the protein encoded by the SlCOMT1 gene is shown in SEQ ID NO.2.

[0008] Furthermore, the application involves using CRISPR / Cas9 gene editing technology to obtain knockouts. SlCOMT1 Mutant tomato plants.

[0009] Furthermore, the method for preparing the mutant tomato plant includes the following steps: S1. Design primer pairs based on gRNA. The sequences of the gRNA primer pairs are shown in SEQ ID NO.3 and SEQ ID NO.4. S2. Using pCAMBIA1301 as a template, PCR amplification, purification, and enzyme digestion were performed using the primer pair described in S1, followed by ligation with the vector to obtain the recombinant plasmid. S3. The recombinant plasmid was transformed into Agrobacterium GV3101, screened and verified, and an expression vector with the SlCOMT1 gene knocked out was obtained. S4. The expression vector obtained in step S3 is transformed into tomato cotyledons, and mutant tomato plants are obtained after verification.

[0010] Furthermore, the verification process included the identification and detection sequencing of the mutant tomato plants; the identification used sequence pairs as shown in SEQ ID NO.5 and SEQ ID NO.6 and SEQ ID NO.7 and SEQ ID NO.8; the detection sequencing used sequence pairs as shown in SEQ ID NO.9 and SEQ ID NO.10.

[0011] The present invention also provides a biomaterial comprising gRNA primer pairs as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0012] Furthermore, the biomaterial includes an expression cassette, a vector, and engineered bacteria.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention verifies SlCOMT1 Genes are closely related to the heat tolerance of tomato anthers, namely SlCOMT1 This invention negatively regulates tomato pollen viability and germination rate under high temperatures, and affects fruit set rate. It provides genetic resources and application value for a deeper understanding of the regulatory mechanisms of tomato male reproductive response to high-temperature stress and for finding new strategies to improve tomato anther heat tolerance. Using CRISPR / Cas9 technology, site-specific editing of the nucleotide sequence shown in SEQ ID No. 1 can yield new tomato germplasm with significantly enhanced heat tolerance, independent of genetic background, thus showing significant application potential in tomato heat tolerance breeding. Attached Figure Description

[0014] Figure 1 shows the transfer of pCAMBIA1301-CRISPR / Cas9-sg SlCOMT1 Detection of mutant tomato plants of the vector, where Figure A is an electrophoresis image of T-DNA insertion detection in transgenic plants, and Figure B is... SlCOMT1 Electrophoresis images of PCR products for knockout type detection; Figure C shows the sequencing results of the PCR products; in Figure C, 1 represents the tomato shoot line. CR-slcomt1-1 The gene is missing 2 bp; 2 represents the tomato bud lineage. CR-slcomt1-2 The gene is missing 5 bp; the red box indicates the sgRNA location, M: Marker.

[0015] Figure 2 shows the morphological observations of floral organs in SlCOMT1 knockout and overexpression tomato plants. Figure A shows sepal length; Figure B shows petal length; Figure C shows stamen length; and Figure D shows pistil length. Values ​​represent mean ± standard deviation; *p < 0.05, **p < 0.01 (Student)t - test).

[0016] Figure 3 shows the pollen development characteristics of SlCOMT1 knockout and overexpression tomato plants. Figure A shows the Alexander staining analysis, scale bar = 100 μm. Figure B shows the pollen viability statistics, the values ​​represent the mean ± standard deviation of 3 biological replicates, each with at least 10 fields of view. Pollen viability frequency represents pollen viability.

[0017] Figure 4 shows the wild type and... SlCOMT1 The characteristics of mature fruit and fruit set rate of knockout and overexpression tomato plants were studied. Figure A shows mature tomato fruit with a scale bar of 10 mm. Figure B shows the fruit set rate statistics. The data represent the mean ± standard deviation of three biological replicates. At least 15 plants were counted for each type. The fruit setting percentage represents the fruit set rate.

[0018] Figure 5 shows the wild type and SlCOMT1 Pollen activity of knockout and overexpression tomato plants before and after high-temperature stress, where Figure A shows the pollen activity under high-temperature stress. CR-slcomt1-1 / 2, OECOMT1-26 Alexandrine staining analysis of pollen, scale bar = 100 μm; Figure B shows the results at normal and high temperatures. CR-slcomt1-1 / 2, OECOMT1-26 Pollen viability was compared with that of wild-type plants; WT represents wild-type, NT and HT represent normal temperature and high temperature treatment groups, respectively; pollen viability frequency represents pollen viability. Data bars and error bars represent mean ± standard deviation, with 3 biological replicates and at least 10 fields of view per replicate; an asterisk indicates a significant difference from the high temperature treatment group (HT) WT, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student) t -test).

[0019] Figure 6 shows the wild type and... SlCOMT1 Fruit set rate of knockout and overexpression transgenic lines before and after high temperature stress. Figure A shows the fruit set of different tomato types in the control group (NT) under normal temperature; Figure B shows the fruit set of plants after high temperature treatment (HT); Figure C is a statistical graph of fruit set rate before and after high temperature treatment; WT represents wild type, NT and HT represent normal temperature and high temperature treatment groups, respectively; data bars and error bars represent mean ± standard deviation, respectively, with 3 biological replicates, each with more than 15 plants; asterisks indicate significant differences from the high temperature group (HT) WT; * p < 0.05, ** p < 0.01, ***p <0.001 (Student) t -test).

[0020] Figure 7 For high temperature SlCOMT1 Changes in serotonin (A) and its metabolite melatonin (B) levels in the flowers of knockout mutant and overexpressing tomato plants. Figure A shows the serotonin content results for each group; Figure B shows the melatonin content results. Data represent the mean ± standard deviation of three biological replicates. p <0.01 (Student) t- test). Detailed Implementation

[0021] The exemplary embodiments of the present invention will now be described in detail. The description of the exemplary embodiments is for illustrative purposes only and is not intended to limit the invention or its applications or uses.

[0022] The tomato variety 'Mirco-Tom' used in this invention originated from the U.S. National Germplasm Bank and was bred and preserved by the Vegetable Research Institute of Zhejiang University.

[0023] MS medium was purchased from Beijing SolarBio. Plasmid pMD18T was purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0024] All primers were synthesized by the company.

[0025] Unless otherwise specified, other reagents, instruments, and equipment mentioned are all commonly used in this field and can be purchased commercially.

[0026] Example 1 CRISPR / Cas9 system-mediated SlCOMT1 Gene knockout.

[0027] gRNA target selection Log in to the Plant Genome Database (https: / / phytozome-next.jgi.doe.gov / ) to search. SlCOMT1 The gene sequence, nucleotide sequence is shown in SEQ ID NO.1, and amino acid sequence is shown in SEQ ID NO.2. It was selected using the online gRNA design website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). SlCOMT1 The gRNA sequence pair, as shown in SEQ ID NO.3 and SEQ ID NO.4, is located in the fourth exon of the SlCOMT1 gene.

[0028] Construction of pCAMBIA1301-SlCOMT1-KO knockout vector The gRNA with the target linker GATT(g) was synthesized into a sense and complementary strand using primers. A concentration of 100 µM was used, and the sense and antisense gRNA strands were annealed to form double strands. The procedure was as follows: 1 µL each of the sense and antisense strands were added to 8 µL of water, incubated at 37 °C for 30 min, then at 95 °C for 5 min, followed by cooling to room temperature at a rate of 5 °C / min. The resulting double-stranded sgRNA was diluted 100-fold with sterile water and ligated with T4 ligase. Bpi The linearized vector of pAtU6-sgRNA-pAtUBQ-Cas9-pMD18T, digested with enzyme I, was ligated to obtain the recombinant vector pMD18T-sgSlCOMT1. The pAtU6-sgRNA-pAtUBQ-Cas9-pMD18T plasmid was constructed in our laboratory. Hind III and Kpn I. The pMD18T-gSlCOMT1 and pCAMBIA1301 vectors were double-digested with enzymes. The pMD18T-gSlCOMT1 vector was cloned into the pCAMBIA1301 vector via T4 ligation to obtain the pCAMBIA1301-SlCOMT1-KO knockout vector.

[0029] pCAMBIA1301-SlCOMT1-KO plasmid was transformed into Agrobacterium GV3101 The 1 μL recombinant vector pCAMBIA1301-SlCOMT1-KO from step 2 was introduced into 30 μL of Agrobacterium GV3101 competent cells by heat shock and named GV3101-SlCOMT1-KO.

[0030] Tomato genetic transformation Tomato variety 'Mirco-Tom' was pre-sown in MS medium and cultured in a tissue culture room with a photoperiod of 16 h / 8 h and a temperature of 25℃. All operations were performed in a clean bench. The culture medium formulation is shown in Table 1.

[0031] Table 1. Tomato genetic transformation medium formulation .

[0032] (1) Pre-culture of tomato cotyledons When the two cotyledons of the tomato are fully unfolded but the true leaves have not yet grown, cut the middle part of the cotyledons of the sterile tomato seedlings, with the back side facing up, and place them on the pre-culture medium. Place the tissue culture room with the back side of the cotyledons facing up and culture for 48 hours.

[0033] co-culture of infected leaves Preparation of recombinant Agrobacterium GV3101-SlCOMT1-KO bacterial culture, OD 600 =0.6, using the leaf disc method, 30 ml of recombinant Agrobacterium GV3101-SlCOMT1-KO was used to infect tomato cotyledons. The cotyledons were placed back into the original pre-medium with the underside facing up and cultured at 25°C in the dark for 48 h.

[0034] Differentiation culture Cotyledons were inserted into differentiation medium and cultured upside down in a tissue culture room. The differentiation medium was changed every 15-20 days until the explants differentiated into sufficient shoots.

[0035] (4) Subgeneration Browned callus tissue was removed from the explants, and the differentiated green shoots were transferred to a subculture medium with a zeatin concentration of 1 mg / L. The medium was changed every 15-20 days until the shoots differentiated into independent individual plants. The individual plants were then cut off and transferred to a rooting medium for further culture.

[0036] (5) Rooting culture Single bud lines were transferred to rooting medium. After approximately 20 days of cultivation, once roots had formed, they were transplanted into the substrate. Plastic wrap was used to cover the seedlings to maintain moisture. The plastic wrap was removed after 14 days to allow the transgenic seedlings to acclimatize to the external environment. The obtained transgenic plants were named... SlCOMT1 Mutant plant.

[0037] Identification of transgenic positive seedlings and detection of mutation types The mutant plants obtained by transplanting from the culture medium were designated as the T0 generation. DNA was extracted from the T0 generation transgenic plants using the CTAB method. The T-DNA insertion detection primers M13-F and sgSlCOMT1-R were used, with primer sequences as follows: M13-F: 5′-GTAAAACGACGGCCAGT-3′ (SEQ ID NO.5) and sgSlCOMT1-R: 5′-AAACTCCGGATTCGTATATCGTGC-3′ (SEQ ID NO.6). The target fragment was amplified by PCR, and transgenic positive plants were screened.

[0038] according to SlCOMT1 Gene sequence was obtained, and mutation detection primers SlCOMT1-F and SlCOMT1-R were designed. The mutation type of the target site was determined by sequencing the PCR amplification products. The primer sequences used for amplification were: SlCOMT1-F: 5′-AGATGGGGCAAACGGTC-3′ (SEQ ID NO.7) and SlCOMT1-R: 5′- AGAGGCTGG AAATACGGATAG-3′ (SEQ ID NO.8).

[0039] The PCR amplification system is shown in Table 2.

[0040] Table 2 PCR amplification system

[0041] The PCR reaction procedure is as follows: First, initial denaturation is performed at 95°C for 3 minutes to completely denature the DNA template; then, the cycling phase is entered, with each cycle consisting of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C at a rate of 15 seconds per kilobase; a total of 30 to 35 cycles are performed; finally, a final extension is performed at 72°C for 5 minutes.

[0042] Obtained through T1 generation seeding and separation SlCOMT1 For homozygous mutant plants, when T1 generation seeds developed two true leaves, 0.5 g of fresh tomato leaves were collected. Genomic DNA was extracted from the leaves of 23 plants using the CTAB method. The DNA was amplified by PCR and subjected to 1% agarose gel electrophoresis to determine whether the plants had successfully inserted T-DNA. A clear band at 750 bp was considered a successful insertion. Figure 1 A). Using knockout-type detection primers det- SlCOMT1 -F: 5′-TTCAAAAAGATGGGTTCAACAAGCC-3′ (SEQ IDNO.9) and det- SlCOMT1 -R: 5′- GAAACACCATCGGCATTCTTAGTCA-3′ (SEQ ID NO.10) was used for PCR amplification. After electrophoresis, the PCR product with the correct band was selected. Figure 1 B) Sequencing was performed, and the sequencing results, when compared with wild-type genes, showed the isolation of two homozygous knockout lines with deletions of 2 bp and 5 bp. Figure 1 C), respectively named CR- slcomt1-1 With CR-s lcomt1-2 .

[0043] Example 2 SlCOMT1 Observation of knockout mutant phenotype.

[0044] (1) Growth of transgenic plants under normal temperature T1 generation sown at the same time CR-slcomt1-1 and CR-slcomt1-2 Mutant strains OECOMT1-26 Overexpression lines and 'Micro-Tom' wild-type plants (WT) were cultured under a 16 h / 8 h (light / dark) photoperiod and a 25℃ / 20℃ (day / night) temperature cycle for observation of floral organ morphology and size. Results were found... CR-slcomt1 / 2 mutants and overexpression lines OECOMT1-26The floral organs were morphologically normal and showed no significant difference in size. Further observation revealed no significant difference in stamen and pistil length between knockout and overexpression plants, but differences existed in sepal and petal length. CR-slcomt1-1 The mutant plants had significantly shorter petals and sepals than the control plants, while the overexpression lines... OECOMT1-26 Only the sepals showed a significant shortening compared to the control plants. At normal temperatures (25℃ / 20℃), there was no significant difference in pollen viability between wild-type and transgenic plants. Figure 2 A and 3B). Furthermore, there were no significant differences in fruit development and fruit set rate between wild-type and control plants (A and 3B). Figure 3 ).

[0045] (2) Figure 4 Changes in gene expression significantly affect tomato pollen viability under high temperature stress. For tomatoes WT, SlCOMT1 mutant lines and overexpression lines CR-slcomt1 During the tetrad stage of pollen development (5-6 weeks of growth), plants underwent a 4-day high-temperature treatment (33℃ / 28℃). After the treatment, they were returned to normal culture conditions. Pollen viability was assessed when the marked flower buds were fully open, and pollen viability was observed using Alexandrine staining. The results showed that, compared to normal temperatures (25℃ / 20℃), all plants exhibited varying degrees of significant pollen abortion under the high-temperature treatment. OECOMT1-26 A). Pollen viability in WT plants decreased from 99.04% to 14.53%, significantly lower than that under normal growth conditions (P < 0.001). Knockout lines Figure 5 and CR-slcomt1-1 The pollen viability of the mutants decreased from 97.69% and 98.96% at normal temperatures to 35.63% and 31.13%, respectively; the overexpression lines CR-slcomt1-2 The pollen viability decreased from 99.04% to 58.63%. OECOMT1-26 B).

[0046] Although the pollen viability of the knockout and overexpression lines was significantly reduced at high temperatures compared to normal temperatures, the decrease was significantly smaller than that at WT, and both were significantly higher than the pollen viability of wild-type plants under high-temperature stress. Figure 5 The changes in expression significantly affected the viability of tomato pollen under high temperature, indicating that it plays a regulatory role in the response to high temperature stress during tomato anther development.

[0047] (3) SlCOMT1 Changes in gene expression significantly affect the fruit set rate of tomatoes under high temperature stress. Under normal temperature conditions, WT, SlCOMT1 Knockout mutants and CR-slcomt1-1 / 2The fruit setting rates of the overexpression lines were 62.24%, 65.71%, 64.63%, and 58.28%, respectively. OECOMT1-26 (B) No significant difference. After high-temperature treatment, the fruit set rate of all plant types decreased to varying degrees, with the WT plant showing a highly significant decrease of approximately half in fruit set rate. Figure 4 CR- The decrease in fruit set rate after high-temperature treatment was significantly less in the knockout mutant compared to the WT mutant, and the fruit set rate was significantly higher in the knockout mutants (32.92%, 53.52%, and 43.59%, respectively). This indicates that... slcomt1-1 / 2 Changes in expression also had a significant impact on the fruit set rate of tomatoes under high temperatures. SlCOMT1 (4) Figure 6 Gene knockout significantly affected the 5-hydroxytryptamine content in tomato flowers under high temperature stress. The 5-hydroxytryptamine content of the open flowers (grade VI) after high-temperature treatment was detected, and the results are as follows: SlCOMT1 A. After high-temperature treatment ， The 5-hydroxytryptamine content in the flowers changed significantly compared to the WT level. Figure 6 The 5-hydroxytryptamine content in the flowers of the 1 / 2 knockout mutant was significantly increased to approximately 2-fold, and the overexpression lines... CR-slcomt1- No significant difference; and compared with normal temperature, at WT and OECOMT1-26 The 5-hydroxytryptamine content in the flowers of the plants decreased by 40.08% and 46.29%, respectively, while... OECOMT1-26 In the -1 / 2 mutant flowers, the serotonin content decreased by only 12.69% and 9.20% after high-temperature treatment, a significantly smaller decrease than in the wild-type plants. This indicates that knockout... CR-slcomt1 The gene can significantly alleviate the effects of high temperature on serotonin metabolism in tomato flowers, further verifying that... SlCOMT1 The role of genes in regulating heat tolerance in tomatoes. It is worth noting that after high-temperature treatment, SlCOMT1 CR- The levels of melatonin, a downstream metabolite of serotonin, were not significantly altered in the flowers of the 1 / 2 knockout mutant. slcomt1- Figure 6 B) indicates that 5-hydroxytryptamine, independently of the endogenous melatonin metabolic pathway, alleviates high-temperature stress damage to tomato anthers, increases fruit set rate, and improves yield and quality.

[0048] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0049] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Solanum lycopersicum SlCOMT1 application of the gene in regulating heat tolerance of anthers in tomato, characterized in that, The SlCOMT1 gene nucleotide sequence is shown as SEQ ID NO. 1, and the tomato SlCOMT1 gene negatively regulates the heat tolerance of anther of tomato.

2. Use according to claim 1, characterized in that, The SlCOMT1 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, The application is to obtain a knockout of the gene using CRISPR / Cas9 gene editing technology SlCOMT1 Mutant tomato plants of the gene.

4. Use according to claim 3, characterized in that, The method for preparing the mutant tomato plant comprises the following steps: S1. Designing a primer pair according to gRNA; S2. Using the primer pair in S1 to perform PCR amplification, purification and enzyme digestion on pCAMBIA1301 as a template, and then connecting the vector to obtain a recombinant plasmid; S3. Transform the recombinant plasmid into Agrobacterium, screen and verify, obtain the knockout SlCOMT1 expression vector of the gene; S4. Transforming the expression vector obtained in step S3 into tomato cotyledons, and obtaining a mutant tomato plant through verification.

5. Use according to claim 3, characterized in that, In the step S1, the sequence of the gRNA primer pair is shown in SEQ ID NO. 3 and SEQ ID NO.

4.

6. Use according to claim 3, characterized in that, In the step S3, the agrobacterium is GV3101.

7. Use according to claim 3, characterized in that, In the step S4, the verification process comprises identification and detection sequencing of the mutant tomato plant; The identification adopts the sequence pairs shown in SEQ ID NO. 5 and SEQ ID NO. 6, and SEQ ID NO. 7 and SEQ ID NO. 8; The detection sequencing adopts the sequence pairs shown in SEQ ID NO. 9 and SEQ ID NO.

10.

8. A biomaterial, characterized by, The biological material comprises the gRNA primer pair shown in SEQ ID NO. 3 and SEQ ID NO.

4.

9. The biomaterial of claim 8, wherein, It comprises an expression cassette, a vector and an engineering bacterium.

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