Application of SlCAS gene and protein coded by SlCAS gene in regulation and control of tomato blossom-end rot

Abstract

The invention is applicable to the fields of molecular biology and biotechnology, and provides an application of an SlCAS gene and a protein coded by the SlCAS gene in regulation and control of tomato blossom-end rot. Two S1CAS knockout strains (S1CAS-CR-4 and S1CAS-CR-6) are successfully obtained through a gene editing technology, and compared with a wild type MM, the S1CAS knockout strains have the advantages that the plant height, stem diameter and root system of the S1CAS knockout strains in the seedling stage are obviously reduced, flowers are easy to fall in the flowering stage, and obvious blossom-end rot occurs under normal culture conditions. According to the discovery, the CAS-mediated calcium signal pathway is associated with the tomato blossom-end rot, and the association between the occurrence of the tomato blossom-end rot and the CAS-mediated calcium signal is preliminarily proved. The invention not only discloses the effect of the calcium receptor protein SlCAS and a calcium signal pathway coupled with the calcium receptor protein SlCAS in regulation and control of the tomato blossom-end rot, but also provides an important basis for cultivating a blossom-end-rot-resistant tomato variety and improving the quality and yield of tomatoes.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and biotechnology, and particularly relates to application of S1CAS gene and protein encoded thereby in regulating tomato navel rot. Background Art

[0002] Tomato (Solanum lycopersicum) is popular among the public because of its rich carotenoids, strong antioxidant properties, unique flavor and dual-use characteristics of fruits and vegetables. It plays an important role in the global vegetable industry. However, with global warming and frequent extreme weather, tomato agricultural production faces a series of problems such as frequent diseases and quality decline. Among them, blossom end rot (BER) on tomato fruits is particularly prominent, which seriously reduces the yield and quality of tomatoes. The cause of BER in tomato agricultural production is often difficult to predict. It affects the global tomato production by about 15% each year, and in areas with high incidence of the disease, this impact is even as high as more than 50%, causing huge economic losses. Although a large number of studies have been conducted on BER internationally, and a large amount of data support has been obtained from environmental, physiological and genetic aspects, its pathogenesis has not yet been fully clarified and remains at the hypothesis stage.

[0003] Regarding the cause of tomato navel rot, foreign scholars have basically reached a consensus that NH4 + Environmental factors such as concentration, salt content of soil or nutrient solution, temperature, and humidity affect the incidence of tomato BER. However, there is still controversy as to whether calcium is the root cause of tomato BER. Existing studies have shown that external environmental factors may only indirectly affect the occurrence of BER, while physiological lesions caused by calcium deficiency in tomatoes may be the direct cause, but it is not yet completely clear how calcium deficiency causes physiological lesions in tomatoes. Based on calcium ions (Ca 2+ ) and its distribution characteristics, most researchers support that BER is a physiological disease caused by the uneven absorption and distribution of calcium ions between cells. How calcium deficiency causes tomato BER may involve multiple aspects: calcium deficiency affects the activity of antioxidant enzymes in tomato plants, leading to the accumulation of hydrogen peroxide in tomatoes, which in turn aggravates lipid peroxidation and inhibits the function of other enzymes, causing cell membrane damage and ultimately causing tomato BER.

[0004] At present, basic research on the causes of BER in tomatoes faces many difficulties. Since there are too many factors that induce or aggravate the incidence of BER, and the synergistic effects and mechanisms are complex, it is often difficult to achieve ideal results by controlling a single factor or macroscopically. Therefore, some foreign scholars have started from the perspective of molecular biology, first studying the changes in cell structure, function, and physical and chemical properties when BER occurs, and then looking for factors that can produce or aggravate these changes, and then find the real cause of BER.

[0005] Calcium sensing receptor (CAS) is a phosphorylated protein on the thylakoid membrane of plant chloroplasts. It is a single copy in most plants, with a conserved sequence and no homologous sequence in animals. As an important calcium signal receptor in plants, CAS can sense the level of extracellular calcium ions and participate in the regulation of the extracellular high calcium-induced intracellular calcium increase signal pathway. It plays an important regulatory role in plant growth and development, abiotic stress (such as drought, carbon dioxide concentration, etc.) and biotic stress (such as wheat Fusarium graminearum, Sclerotinia sclerotiorum, Botrytis cinerea, etc.). Based on the above research background, the present invention proposes the application of SlCAS gene and the protein encoded by it in regulating tomato navel rot. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the SlCAS gene and the protein encoded by it in regulating tomato blossom rot, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] Application of SlCAS gene or the protein encoded by it in regulating tomato blossom rot.

[0009] Furthermore, the nucleotide sequence of the S1CAS gene is shown in SEQ ID NO.1.

[0010] Furthermore, the amino acid sequence of the protein encoded by the S1CAS gene is shown in SEQ ID NO.2.

[0011] Furthermore, a knockout mutant of the SlCAS gene is obtained by knocking out the SlCAS gene, and the knockout mutant is cultured to obtain a SlCAS knockout strain.

[0012] Furthermore, the SlCAS knockout strains include SlCAS-CR-4 and SlCAS-CR-6.

[0013] Furthermore, the specific performance of the SlCAS knockout strain is that after the SlCAS gene is knocked out, compared with the wild-type MM, the plant height, stem diameter and root system are all reduced, and under normal culture conditions, the flower drop phenomenon is exhibited and navel rot occurs.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention successfully obtained two SlCAS knockout strains (SlCAS-CR-4, SlCAS-CR-6) through gene editing technology. Compared with the wild-type MM, the plant height, stem thickness and root system of the SlCAS knockout strains in the seedling stage were significantly reduced, and the flowers were easily dropped during the flowering period, and under normal culture conditions, they were also accompanied by obvious navel rot. This discovery links the CAS-mediated calcium signaling pathway with tomato navel rot, and preliminarily proves that the occurrence of tomato navel rot is associated with CAS-mediated calcium signals. The present invention not only reveals the role of the calcium receptor protein SlCAS and its coupled calcium signaling pathway in the regulation of tomato navel rot, but also provides an important basis for cultivating navel rot-resistant tomato varieties and improving tomato quality and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the T0 generation detection of SlCAS knockout plants; in the figure, M represents DL-2000 Marker, and 1-8 represent different T0 generation plants.

[0017] Figure 2 The figures are the mutation map and phenotype of SlCAS knockout plants; (a) is the mutation map of SlCAS knockout plants, and (b) is the phenotype of 5-week-old SlCAS knockout plants (SlCAS-CR-4, SlCAS-CR-6).

[0018] Figure 3 Analysis of plant height, root system and stem diameter of SlCAS knockout plants (SlCAS-CR-4, SlCAS-CR-6) and wild-type MM; (a) is the plant height phenotype of SlCAS knockout plants and wild-type MM, (b) is the root system phenotype of SlCAS knockout plants and wild-type MM, (c) is the plant height statistics of SlCAS knockout plants and wild-type MM, and (d) is the stem diameter statistics of SlCAS knockout plants and wild-type MM.

[0019] Figure 4 The occurrence of navel rot in SlCAS knockout plants (SlCAS-CR-4, SlCAS-CR-6) and wild-type MM.

[0020] Figure 5 Flower drop in SlCAS knockout plants (SlCAS-CR-4, SlCAS-CR-6) and wild-type MM. DETAILED DESCRIPTION

[0021] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0022] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0023] Example 1: Obtaining S1CAS gene knockout transgenic plants;

[0024] The nucleotide sequence of the S1CAS gene is shown below:

[0025]

[0026]

[0027] (as shown in SEQ ID NO.1).

[0028] The amino acid sequence of the SlCAS protein encoded by the SlCAS gene is as follows:

[0029] MALRASATAKSPLPLPPPPPSSSSSTSSPPKVFSLPKLTQKPVSVSFSTSTALFLFPLFTATHEARAINLPKEDIVSSLNQVESVVNQAQEVGSNIFDTVSGVIGPVIEFVKPGIDAALPLVKQAGEEVLKNASPVISDATKKAQEAMQSAGMDSEPVMTAAKTVVDAAQQTSKVIEGAKPIATSTVETISSTDPAVI AVAGGTLFLAYLLLPPVFSALSFSFRGYKGELTPAQTLDNMCSKNYVLIDIRTEKDKDKAGIPRLPSSAKNKMIQIPLEDLPSKVKSLVRNPKKVEAEIVALKISFLKKINKGSNIVIMDSYSDSAKTVAKSLTSFGFNNCWIMTDGFSGGKGWLQSRLGTDSYNFSFAEILSPSRVIPGRRFGTTGTVKLLSD (such as SEQ ID NO.2 shown).

[0030] The nucleotide sequence of the S1CAS-CR-4 gene is shown below:

[0031] ATGGCACTTAGAGCTTCAGCCACCGCTAAATCACCTCTTCCTCTGCCCCCTCCTCCTCCTTCTCTTCTTCTTCTACTTCATCACCACCTAAAGTTTTTAGTCTTCCTAAACTTACTCAAAAACC TGTATCAGTATCATTCTCTACATCCACTGCACTTTTCCTCTTTCCACTTTTCACTGCGACCCATGAAGCAAGAGCAATCAACTTCCCAAGGAAGACATCGTCTCTTCCCTTAATCAGGTAG (such as SEQ ID NO.17).

[0032] The nucleotide sequence of the S1CAS-CR-6 gene is shown below:

[0033]

[0034] (as shown in SEQ ID NO.18).

[0035] Note: The double-underlined sequence is the designed target, and the single-underlined base is the PAM sequence.

[0036] 1. Materials;

[0037] The pYLCRISPR / Cas9-DH CRISPR / Cas9 knockout binary vector used was from Liu Yaoguang's laboratory at South China Agricultural University; the wild-type tomato Moneymaker (MM) was preserved in the laboratory; the Escherichia coli competent DH5α and the Agrobacterium competent GV3101 were purchased from Shanghai Bioengineering Co., Ltd.

[0038] 2. Primers;

[0039] The primer names and sequences are shown in Table 1 and were synthesized by Jilin Kumei Biotechnology Co., Ltd.

[0040] Table 1 Primer sequences

[0041]

[0042]

[0043] 3. Construction of SlCASCRISPR / Cas9 knockout vector;

[0044] This experiment used the pYLCRISPR / Cas9-DH CRISPR / Cas9 knockout binary vector method, designed two target sites of 20bp and 19bp respectively for the SlCAS gene, and designed target primers corresponding to the target sites. The gRNA was connected to the target linker primer by the cutting and ligation method to obtain the gRNA expression cassette connection product. The gRNA expression cassette was amplified by two rounds of PCR reaction, and the amplified product was recovered after agarose gel electrophoresis detection to obtain the gRNA expression cassette. The pYLCRISPR / Cas9-DH plasmid was digested with BsaⅠ, and the digested product was Golden Gate connected (cut and ligated) with the recovered gRNA expression cassette, and the connection product was transformed into Escherichia coli DH5α competent for amplification. The plasmid was extracted from the amplified bacterial solution to obtain the constructed pYLCRISPR / Cas9-DH SlCAS knockout vector. The vector construction process is as follows:

[0045] 1) Design of CRISPR / Cas9 target sites;

[0046] In order to obtain a good mutation effect, CRISPR / Cas9 target sites are usually designed on exon sequences that are relatively forward. Use the online website https: / / crispor.tefor.net / , select the PAM mode, and design the target site on the exon of the SlCAS gene. Select two targets with an interval of 150-200bp based on the target score and perform a specificity check.

[0047] The specific process of specificity check: perform BLAST on the candidate target sequence and dozens of base pairs upstream and downstream of the target genome to avoid similarity with the sequences of other genes in the genome.

[0048] 2) Construction of gRNA expression cassette;

[0049] According to the expression vector construction strategy, the target linker was connected to the restriction-cut gRNA vector, and the ligation product was subjected to two rounds of nested PCR.

[0050] a. Preparation of adapters: dilute adapter primers to 10 μM, take 1 μL of each pair of adapter primers, add 8 μL ddH2O (two sets in total), mix well, incubate at 90°C for 30s, and cool at room temperature to complete annealing.

[0051] b. Connection of gRNA expression cassette: Perform connection reaction according to the system in Table 2.

[0052] Table 2 Connection system

[0053]

[0054] Reaction conditions: room temperature (20-28°C) for 10-15 min.

[0055] c. First round of RNA expression cassette PCR amplification (2 PCR reactions for each gRNA expression cassette, 4 reactions in two groups, 20 μL each): Amplify according to the system in Table 3.

[0056] Table 3 The first round of PCR amplification system

[0057]

[0058] Among them, UF and SlCASgRNA-R downstream of the adapter primer constitute a primer pair, gRNA-R and SlCASgRNA-F upstream of the adapter primer constitute a primer pair, with a total of two targets, 4 pairs of primers and 4 groups of reactions, namely: ①U-F+SlCASgRNA-R1; ②gRNA-R+SlCASgRNA-F1; ③U-F+SlCASgRNA-R2; ④gRNA-R+SlCASgRNA-F2.

[0059] Reaction conditions: 95°C for 10 s, 58°C for 15 s, 68°C for 20 s, for 28 cycles.

[0060] Take 4-5 μL of PCR product for agarose gel electrophoresis (1.5% agarose gel), and the amplified length is about 140 bp. If the amplified product is weak, you can continue with the second round of PCR.

[0061] d. Second round of PCR amplification and recovery of RNA expression cassette (two sets of reactions, 50 μL each);

[0062] The specific primer pairs were pre-mixed into a 10 μM working solution, and the two target primer combinations were: ①Uctcg-B1'+gRctga-B2; ②Uctga-B2'+gRcggt-BL.

[0063] Amplify according to the system in Table 4:

[0064] Table 4 Second round PCR amplification system

[0065]

[0066] Reaction conditions: 95°C for 10 s, 58°C for 15 s, 68°C for 60 s, for 25 cycles.

[0067] The PCR product was detected by agarose gel electrophoresis (1% agarose gel). The amplified length was about 500 bp and the electrophoresis band was bright and could be recovered.

[0068] 3) Connect the gRNA expression cassette to the plasmid (cut and ligate method);

[0069] Add ingredients according to Table 5:

[0070] Table 5 Enzyme digestion system

[0071]

[0072] After mixing evenly, enzyme digestion was performed at 37°C for 10 min.

[0073] After 10 minutes, immediately prepare the following system for reaction:

[0074] Table 6 Connection system

[0075]

[0076] Reaction conditions: 37°C for 2 min, 10°C for 3 min, 20°C for 5 min, 37°C for 2 min (lid temperature 40°C), 15 cycles.

[0077] 4) The ligation product was transformed into E. coli DH5α competent cells and coated on Kan resistance plates.

[0078] 5) After a single colony grows out, pick a single colony for PCR verification (primers: SP-DL+SP-R), expand the positive colony, extract the plasmid and send it for sequencing, and compare the sequencing results.

[0079] 6) Obtain the CRISPR / Cas9 vector plasmid with correct sequencing.

[0080] 7) The correctly sequenced CRISPR / Cas9 vector plasmid is transformed into Agrobacterium, and the bacterial solution is stored for use in subsequent tomato genetic transformation.

[0081] 4. Transform the SlCASCRISPR / Cas9 knockout expression vector into tomato (Agrobacterium-mediated method);

[0082] 1) Sterilization and sowing of seeds;

[0083] Preparation: Prepare sterile tissue culture glass bottles, 1 / 2MS seeding medium, sterile water, 75% alcohol and 10% NaClO solution in advance.

[0084] Seed cleaning: Pour an appropriate amount of MM seeds into a sterilized tissue culture bottle, wash the seeds 2-3 times with sterile water, and shake for 2 minutes each time.

[0085] Disinfection: Add 75% alcohol and shake for 2 minutes; pour out the alcohol and wash once with sterile water, then pour in an appropriate amount of 10% NaClO and shake for 10 minutes in the dark. Pour out the NaClO solution and wash 4 to 5 times with sterile water, 2 minutes each time, to ensure that the NaClO solution is completely washed.

[0086] Sowing and cultivation: Sow the disinfected and cleaned seeds evenly on the sowing medium, dark-treat them for 5 days (24°C), and then place them in a light incubator for cultivation (light cultivation for 16 hours, 25°C, dark cultivation for 8 hours, 20°C).

[0087] 2) Explant preparation and pre-culture;

[0088] After the seeds have been cultured for 9-10 days, select the seedlings that are about to grow true leaves, cut off the petiole and leaf tip of the cotyledon with a surgical blade, and cut the middle part of the explant into 2-3 pieces. Try to ensure that the wounds at both ends of the explant are flat after cutting.

[0089] The cut explants were transferred to the pre-culture medium, with about 30 explants placed on each plate, and cultured at 24°C for 48 hours under light.

[0090] 3) Explant infection and co-cultivation;

[0091] Agrobacterium propagation: Take 100 μL of the preserved Agrobacterium and inoculate it into LB medium, add antibiotics Rif and Kan, and culture it at 28°C and 220 rpm until OD600=0.6, take an appropriate amount of bacterial solution, centrifuge at 5000 rpm for 5 minutes, and remove the supernatant. Wash the bacteria with a small amount of MS liquid medium, centrifuge at 5000 rpm for 5 minutes again, remove the supernatant, add MS liquid medium to resuspend the bacteria, and adjust the infection solution to OD600=0.1.

[0092] Explant infection: Pour the infection solution into a sterile culture bottle, and add 2-3 plates of cultured explants for every 50 ml of infection solution. Make sure that the infection solution fully covers the explants and that the wound part of the explants is in full contact with the infection solution. Gently shake the culture bottle and infect for 10-15 minutes.

[0093] Co-culture: The infected explants were gently picked out with forceps, placed on co-culture medium lined with sterile round filter paper, and cultured in the dark at 24°C for 2 days.

[0094] 4) Differentiation culture of explants;

[0095] Differentiation culture: After 2 days of dark culture, the explants were transferred to callus culture medium and cultured at 24°C under long-day conditions (light culture for 16 h, 25°C, dark culture for 8 h, 20°C).

[0096] Culture medium replacement: Depending on the differentiation of the explants, transfer the explants to new callus culture medium every 8-10 days.

[0097] Subculture: After the explants have grown differentiated buds, they are transferred to subculture medium. Similarly, depending on the production of the explants, they are transferred to new subculture medium every 8-10 days.

[0098] 5) Rooting culture of explants;

[0099] Rooting culture: After the explant grows an independent main stem on the subculture medium, the main stem bud is cut off and inserted into the rooting medium.

[0100] Hardening and transplanting: After the explant grows roots longer than 3 cm, open the cap of the tissue culture bottle halfway, harden the seedlings for 1 day, then open the cap of the tissue culture bottle completely, harden the seedlings for another day, remove the regenerated seedlings from the tissue culture bottle, wash the roots with clean water, transplant them into vermiculite, apply Hoagland nutrient solution regularly, place them in a light culture room, and cover them with plastic wrap to maintain high humidity. After about 7-10 days, move the regenerated plants into nutrient soil (nutrient soil: vermiculite = 3:1) and carry out normal plant material cultivation management.

[0101] 5. Identification of SlCASCRISPR / Cas9 knockout plants;

[0102] 1) Extract leaf DNA;

[0103] Material preparation: Cut the leaves and add them into 2mL centrifuge tubes and mark them accordingly.

[0104] Grinding and lysis: Add 200 μL of 2% CTAB buffer to each centrifuge tube and grind. After grinding, add 400 μL of CTAB buffer to each tube and mix well.

[0105] DNA purification: Add 600μL chloroform-isopropanol (24:1) and mix well; centrifuge the mixture at 10,000rpm for 10 minutes. At the same time, add 400μL isopropanol to another new centrifuge tube; after centrifugation, use a pipette to aspirate the supernatant and transfer it to the centrifuge tube containing isopropanol. Shake the centrifuge tube up and down for 30 seconds until flocs can be seen. After centrifugation at 10,000rpm for 10 minutes, immediately invert the liquid and use the tip of the pipette to aspirate the remaining liquid.

[0106] Washing and dissolving: add 800 μL 75% ethanol; centrifuge at 12000 rpm for 3 min, pour out the liquid, aspirate the remaining ethanol, air-dry for several minutes; add 40 μL ddH2O, and store at -20°C for later use.

[0107] 2) Detection and identification;

[0108] Prepare the following PCR amplification system:

[0109] Table 7 PCR amplification system

[0110]

[0111] PCR reaction conditions: 94°C for 3 min, 94°C for 30 s, 55°C for 30 s, 72°C for 60 s, for 32 cycles.

[0112] Take 5 μL of PCR product for agarose gel electrophoresis (1% agarose gel). The amplified length is about 750 bp. If the electrophoresis band size is correct, use the forward primer for sequencing.

[0113] Example 2: Phenotypic analysis of MM and SlCAS knockout mutants and observation of navel rot phenotype;

[0114] 1. Sowing and culture conditions of plant materials;

[0115] Place tomato seeds in a culture dish lined with two absorbent filter papers, add an appropriate amount of distilled water, and culture in the dark in the culture room for 2 to 3 days for germination. After germination, sow the seeds in a nutrient pot with a mixture of peat soil and vermiculite (volume ratio of 3:1) as the matrix, and cover the top with the same thickness of matrix. Place the nutrient pot in the culture room, and the growth environment is: temperature 25 / 20℃ (day / night), photoperiod 16 / 8h light-dark cycle, light intensity 600mol·m -2 ·s -1 , prepare nutrient solution with 1g nutrient fertilizer and 1L distilled water for cultivation.

[0116] 2. Observation of MM phenotypes of SlCAS knockout plants and wild-type plants;

[0117] Construction of SlCAS knockout plants: In order to study the function of the SlCAS gene, a knockout vector was constructed. Using CRISPR / Cas9 technology, two knockout sites were selected to construct a genetic expression vector, which was transformed into tomatoes through Agrobacterium-mediated transformation and tissue culture to obtain transgenic plants. The T0 generation plants obtained were tested, and the plant DNA with amplified band size of 750 bp was amplified and sequenced (see Figure 1 ). The sequencing results were compared with the wild-type genome sequence, and it was found that the sequencing results of two strains (4 and 6) showed overlapping peaks, indicating that the target sites of these two strains may have mutated. Therefore, the PCR product was connected to the T vector, a single clone was constructed and sequenced, such as Figure 2 The sequencing results shown in (a) show that the target sites of the two strains have mutated. Compared with the wild-type MM plants, the height of the two gene knockout strains at the seedling stage is significantly lower than that of the wild-type ( Figure 2 (b)). The two strains were planted and propagated to the T1 generation. Each plant of the T1 generation was sequenced and the homozygous mutants were selected for subsequent experiments. Finally, two SlCAS knockout plants were obtained, named SlCAS-CR-4 and SlCAS-CR-6, which were missing 1bp and 23bp, respectively, resulting in premature termination of protein translation.

[0118] Phenotypic observation of SlCAS knockout plants and wild-type MM: Phenotypic observation of SlCAS knockout plants and wild-type MM was carried out at the five-leaf stage, and their plant height and stem thickness were measured. Tomato plants with consistent growth under the same growth environment were selected for each group. The plant height was measured from the position of the cotyledon node to the top part of the inflorescence, in cm; the stem thickness was based on the diameter of the first internode, measured with a vernier caliper, in mm. During the growth of the plants, it was found that there were significant differences in plant height between the wild-type MM and SlCAS knockout T2 generation plants in the same growth period, and the wild-type MM was significantly taller than the SlCAS knockout plants. Specifically, the two SlCAS knockout plants developed relatively slowly in the early vegetative growth stage, and the plants were shorter than the wild-type MM. In addition, their root system was significantly sparser than that of the wild-type MM, and the stem thickness was also significantly smaller than that of the wild-type MM (see Figure 3 (a)-(d)).

[0119] 3. Observation of navel rot phenotypes of SlCAS knockout plants and wild-type MM;

[0120] SlCAS is a light-regulated calcium receptor protein located in the thylakoid membrane of chloroplasts. It can sense the signal process of increased intracellular calcium caused by high extracellular calcium. The loss of its function will lead to disorder of intracellular calcium signal.

[0121] Seeds of uniform size and full appearance were selected and sown in 24-hole plug trays. When they grew to three leaves and one heart, they were transplanted into large flower pots filled with peat soil and vermiculite (volume ratio of 3:1) for normal culture and observation of navel rot. The experiment showed that the loss of SlCAS gene function caused the two knockout mutants SlCAS-CR-4 and SlCAS-CR-6 to show flower drop even under normal culture conditions (see Figure 5 ), and accompanied by the occurrence of navel rot, which indicates that SlCAS protein plays an important regulatory role in the formation and occurrence of tomato navel rot (see Figure 4 ).

[0122] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Application of SlCAS gene or the protein it encodes in regulating tomato navel rot.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the S1CAS gene is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the S1CAS gene is shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: A knockout mutant of the SlCAS gene is obtained by knocking out the SlCAS gene, and the knockout mutant is cultured to obtain a SlCAS knockout strain.

5. The use according to claim 4, characterized in that: The SlCAS knockout strains include SlCAS-CR-4 and SlCAS-CR-6.

6. The use according to claim 5, characterized in that: The specific performance of the SlCAS knockout strain is that after the SlCAS gene is knocked out, compared with the wild-type MM, the plant height, stem diameter and root system are all reduced, and under normal culture conditions, the flower drop phenomenon is exhibited and navel rot occurs.

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