Application of SlCT1 gene and molecular marker thereof in regulation and control of botrytis cinerea resistance

Abstract

The invention provides an application of an SlCT1 gene and a molecular marker thereof in regulation and control of botrytis cinerea resistance, a nucleotide sequence of the SlCT1 gene is shown as SEQ ID NO.1, the botrytis cinerea resistance can be remarkably improved by over-expressing the SlCT1 gene in a tomato plant, and further, the SlCT1 gene can be used for regulating and controlling the botrytis cinerea resistance. The molecular marker designed according to the natural variation of the SlCT1 promoter region can distinguish the susceptibility resistance of the tomato to the gray mold. The invention provides a more economical, direct and effective breeding method by researching the SlCT1 gene and utilizing a gene editing technology, so that the problem of shortage of germplasm resources is solved, and the breeding period is greatly shortened.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology and specifically relates to SlCT1 Application of genes and their molecular markers in regulating tomato gray mold resistance. Background Art

[0002] The necrotrophic pathogen Botrytis cinerea ( Botrytis cinerea Gray mold, caused by the fungus Botrytis cinerea, poses a serious threat to the sustainable development of the tomato industry. This disease typically reduces tomato yields by 10-20%, and in severe cases, by 30-60%. Botrytis cinerea has a wide host population, infecting over 200 commercial crops and causing typical symptoms such as soft rot and black rot. Current control strategies that rely on chemical agents face a dual dilemma: pesticide residues pose environmental and food safety concerns; coupled with long-term selective pressure leading to the evolution of pesticide resistance in pathogens, this ultimately reduces the effectiveness of gray mold control.

[0003] The publication of the tomato genome and the development of bioinformatics have opened up new avenues for the study of disease resistance mechanisms. Based on reverse genetic analyses of transcriptomes, proteomes, and epigenomes, researchers have screened a series of key genes that respond to gray mold. However, compared with biotrophic / semi-biotrophic pathogen systems, the study of the interaction mechanism of necrotrophic pathogens still faces special challenges: GWAS analysis in Arabidopsis thaliana revealed that Arabidopsis resistance to gray mold presents typical quantitative trait characteristics, coordinated by thousands of minor effect loci, which is in sharp contrast to the "gene-to-gene" model dominated by major effect resistance genes in most pathogen systems. Although Davis et al. located multiple gray mold resistance QTLs through introgression lines, the cloning and functional verification of key genes have not yet been achieved, reflecting that there are still bottlenecks in the analysis of molecular mechanisms in this field.

[0004] Therefore, further research on the genetic mechanism of tomato resistance to gray mold can not only reveal the molecular mechanism of interaction between plants and necrotrophic pathogens, but also provide a theoretical basis for breeding disease-resistant varieties, which is conducive to the development of new disease prevention and control strategies. Summary of the Invention

[0005] In view of this, the present invention provides a gene that positively regulates tomato gray mold resistance: SlCT1 , the gene can significantly enhance the gray mold resistance of tomatoes after overexpression. SlCT1 Primers designed based on the 290 bp natural variation in the promoter region can be used to distinguish the gray mold resistance and susceptibility of GWAS population materials. SlCT1 Whether the gene promoter contains 290bp, detect or assist in detecting the resistance of tomato gray mold; provide a new method for breeding gray mold resistant tomatoes. In addition, knocking out the region containing 290bp in susceptible materials can make SlCT1The expression level increases, which enhances the gray mold resistance of susceptible materials.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide SlCT1 Application of genes in regulating resistance to tomato gray mold, the SlCT1 The nucleotide sequence of the gene is shown in a or b: a: the sequence shown in SEQ ID NO.1; b: A nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added to the sequence shown in SEQ ID NO.1 and which can encode the amino acid sequence shown in SEQ ID NO.2.

[0007] Among them, SEQ ID NO.1: ATGGCATCTTGGAATTCAATTCCTCTTGAAGTTGTGTACACTAGTTTGGGATGGATCGCATTTTTCTCTTGGTCTATTAGCTTTTACCCTCAAGTAATCTTGAATTTCAGACGCAAAAGTGTGGTGGGGTTGAATTTTGATTTCGTGGTGTTGAATTTGACAAAACACTCGTCTTATCTCATTTATAATGCTTCCATGTTCTTTAGTTCTGTTGTTCAGAAGCAGTACCGTGAGAAATATGGTCTGAATGAGATGATACCTGTGGCTGCCAATGATGTTGCTTTCTCAATCCATGCAGTTCTATTGACAGCATTTACCCTGTTTCAAATTGCTATTTATGATCGTGGAAGCCAGAAGGTCTCAAGGACTTCGATTGGGATCGTGTGTGTTGCTTGGTTAACTGTTGCAGTTTGTGTGTTTATAGCTCTCCCCAGCCATTCCTGGCTTTGGCTGATATCCTGCTTCAACACGTTACAGGTTGTCATGACAGTCATCAAGTACATTCCTCAGGCTATTATGAACTTTCAGCGGAAGAGCACTATTGGATTTAGTATTGGCAACATTTTGCTGGACTTGCTTGGAGGTGTCACAAATTATGGTCAGATGGCAGTGCAATCTATAGATCAGAATTCGTGGGTGAACTTTTATGGAAACATAGGCAAGACATTATTGTCATTGGTTTCGATATTCTTTGACATTCTCTTCATTTTGCAACATTATGTGCTCTATCCTGCTTGGAAGATTGGGACCTGTCGAGATGTTGATGTGATAGGCAAGAAGCCGTTACTTAAAACTTCTGATCACGAAAACAAGGAAGATGTATAA。

[0008] SEQ ID NO.2: MASWNSIPLEVVYTSLGWIAFFSWSISFYPQVILNFRRKSVVGLNFDFVVLNLTKHSSYLIYNASMFFSSVVQKQYREKYGLNEMIPVAANDVAFSIHAVLLTAFTLFQIAIYDRGSQKVSRTSIGIVCVAWLTV AVCVFIALPSHSWLWLISCFNTLQVVMTVIKYIPQAIMNFQRKSTIGFSIGNILLDLLGGVTNYGQMAVQSIDQNSWVNFYGNIGKTLLSLVSIFFDILFILQHYVLYPAWKIGTCRDVDVIGKKPLLKTSDHENKEDV.

[0009] The second object of the present invention is to provide a method for regulating tomato gray mold resistance, comprising: overexpressing tomato SlCT1 Genes to improve gray mold resistance in tomatoes; or, by knocking out SlCT1 Genes and their homologous genes SlCT2 , reducing the gray mold resistance of tomatoes, the SlCT2 The gene nucleotide sequence is shown in SEQ ID NO.3; or, by editing SlCT1 Gene promoter sequence, so SlCT1 Gene overexpression improves gray mold resistance in tomatoes, the SlCT1 The gene promoter sequence is shown in SEQ ID NO.4.

[0010] Among them, SEQ ID NO.3: ATGGCTTCTTGGAATTCCGTTCCACTGGAAGTGTTGTATAATGTTTTGGGATGGATTGCGTTTGTGTCATGGTCTATTAGCTTTTATCCTCAGGTCATTTTGAATTTTCGTCGGAAAAGTGTGGTGGGATTGAATTTTGATTTCGTGGTGTTGAATTTGACAAAGCACTCGTCTTATCTCATCTACAATGCTTCCATGTTCTTCAGTTGTGCTGTTCAAAGGCAATATCATAATCGATATGGCAAAAATGAGATGATACCTGTAGCTGCCAATGATGTGGCTTTCTCTACTCATGCTCTTCTTTTAACAGCCTTTACCTTGTTTCAGATCTCTATCTATGATCGTGGAAATCAGAAGGTATCGAAAATTGCTATAGCAATTGTTTCTGTTGCTTGGTTAAGTGTTGCAGTTTGTGTGTTTGTGGCTATCCCTAAGCATTCCTGGCTATGGCTAGTGTCCTGTTTCAACGGATTGCAGGTTGCTATGACAGTAACCAAGTACATTCCCCAGGCTGTTATGAACTTCCGGAGAAAGAGCACCGTTGGTTTTAGTATTGGTAACATTTTGCTGGATTTGTTTGGAGGTTTAACAAATTACGGGCAGATGGCAGTGCAATCTATAGATCAACATTCGTGGGTGAACTTTTATGGAAACATCGGCAAGACTTTGTTGTCATTGGTGTCGATATTCTTTGACATTCTCTTCATTCTGCAACATTATGTGCTATATCCTTCAACGAAAGAAGTGGTTTCTCCTAAATTTGATGTGGAGGAACCAAGGGACATTCAGGGACGTTGA。

[0011] SEQ ID NO. 4: TCCTTTATTAAAACTATTCAAATGAACTCTCCTTTCTAATTGAATCTTGTAGAGTACGAGTTCATATTTCATTGAAGATTTTAGAATTTTTATTCTATGAGTTTGAAAAGAAAATATAGTGCATGAAATTTTAATTTGAAAAAAGTATTCAAAGTGTATTTTTGAACCTCCTTTATTATTGAGCCTTCTCTTATATTTAAGGAATTCAAAATCTCTCTCTATATATAAATGATATTTTTTTATAGAGAGAATTTCGATAAACACGAACCCTAAATATACCCCAACTCCAATATTAGCGACCCGCATTTACCAACAATTGGTCTGGTCCATGATAAATGACATGTTGG GCCTTGTCATTCAAGCATCCCTAGGAATTGCTTTATCAAGGCTTTTAAGTAATGCTTGAATTTGGAATTTTATCATATAATAATGTTTGGAAGAAACTACATCATTAGATATATTATAATATCATATATATCTATATTATTTATATTTTAAAGATATTACGGATTTTATTATTAATTAAGAGTTTTGTATCATATATTAAAAAAATATTTAGATACATGTATTTTGTTATTAGATATACTAAAGTACGAATAGAGTTGAGCGAAATGGGGAGGGAGGCGAGCGAGATTTGTTATGTGCCACAGATACATGCAAATCACAATATATCTATTGTGGTTTCCATGTACCTGATATACT GAATATATAGAAGAGCGGTGAACGAGATGGGAGGAA GACGAGTGAGATTGTTATGTATCCCATCACTTAGACAAAGTGTGTCTAGAAGAAATTACACATAATTTTGATTCGT GTATCTCAAAATACACATATCTCAACGTATTTAAATGTATCTAGATGAATCAAAATTTAATAAGATACATAATATT GTAAACTAGATTATATGTATACAATTTACTCCTAGATTAGTAAAATTTATGTAAGTTTCTCTATATTTTTTAAGTT TATTATATTCAAAATTTATCAATTAT

[0012] In some specific embodiments, preferably, by overexpressing tomato SlCT1 The specific steps for genetically improving tomato gray mold resistance are as follows: construct an overexpression vector and transfect tomatoes with Agrobacterium to obtain overexpression plants, thereby improving tomato gray mold resistance. The primers used to construct the overexpression vector are as follows: 35S- SlCT1 -OE-F:CATTTGGAGAGGACACGCTCGAG ATGCAAGGCTCAATTCATTAC; 35S- SlCT1 -OE-R:TCTCATTAAAGCAGGACTCTAGACACAAGGCCAGGCATGGGGC.

[0013] In some specific embodiments, preferably, by simultaneously knocking out SlCT1 Genes and their homologous genes SlCT2 The specific steps to reduce the gray mold resistance of tomatoes are as follows: construct a CRISPR / Cas9 multi-target knockout vector and transfect tomatoes with Agrobacterium to obtain SlCT1 / 2 Double mutant plants can reduce the gray mold resistance of tomatoes. The target fragments for constructing the CRISPR / Cas9 multi-target knockout vector are as follows: SlCT1 Gene target 1: TTGTGTACACTAGTTTGGGATGG; SlCT1 Gene target 2: GGGGTTGAATTTTGATTTCG; SlCT2 Gene target 3: TTCTTGGAATTCCGTTCCAC; SlCT2 Gene target 4: ATGATGTGGCTTTCTCTACT.

[0014] In some specific embodiments, preferably, by editing SlCT1 The specific steps of improving the gray mold resistance of tomatoes by using gene promoter sequences are as follows: constructing a CRISPR / Cas9 editing system and transfecting tomatoes with Agrobacterium to create a SlCT1 The mutant plants of the promoter can improve the gray mold resistance of tomatoes; the target fragments of CRISPR / Cas9 are constructed as follows: SlCT1 promoter target 1: GAGCGGTGAACGAGATGGG; SlCT1 promoter target 2: AGATTAGTAAAATTTATGT.

[0015] The third object of the present invention is to provide a method for identifying tomato plants [[ID= The molecular marker of gene expression, the molecular marker nucleotide sequence is shown in SEQ ID NO.5 (i.e. ​ Gene promoter sequence (sequence in italics in SEQ ID NO.4).

[0016] SEQ ID NO.5: GAATATATAGAAGAGCGGTGAACGAGATGGGAGGAAGACGAGTGAGATTGTTATGTATCCCATCACTTAGACAAAGTGTGTCTAGAAGAAATTACACATAATTTTGATTCGTGTATCTCAAAATACACATATCTCAACGTATT TAAATGTATCTAGATGAATCAAAATTTAATAAGATACATAATATTGTAAACTAGATTATATGTATACAATTTACTCCTAGATTAGTAAAATTTATGTAAGTTTCTCTATATTTTTTAAGTTTATTATATTCAAAATTTATCAATTAT.

[0017] A fourth object of the present invention is to provide a primer set for detecting the above-mentioned molecular markers, wherein the primer set is as follows: ​ -jc-F:AGGCTTTTAAGTAATGCTTG; ​ -jc-R:GTGGGTGGTCCATCATCCTC.

[0018] A fifth object of the present invention is to provide a kit containing the primer set for detecting the above-mentioned molecular markers.

[0019] A sixth object of the present invention is to provide a method for using the primer set or the kit for detecting the molecular markers in breeding tomato varieties resistant to gray mold.

[0020] The seventh object of the present invention is to provide a ​ The application of gene vectors and engineered bacteria in breeding tomato varieties resistant to gray mold.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention first identified a candidate gene regulating tomato resistance to gray mold through GWAS analysis ​ ; Further, according to ​Molecular markers designed from natural variations in the promoter region can distinguish the resistance of tomatoes to gray mold, providing information for molecular marker-assisted selection breeding. The use of CRISPR / Cas9 gene editing technology to obtain resistant plants is more economical, direct and effective than traditional breeding methods, solving the problem of scarce germplasm resources and greatly shortening the breeding period. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] ​ In Example 4 of the present invention ​ -OE T0 generation strains SlCT1 gene overexpression level analysis.

[0023] ​ In Example 5 of the present invention ​ -DKO homozygous strain ​ 、 ​ Sequencing peak diagram of gene editing.

[0024] ​ In Example 6 of the present invention ​ -OE and ​ -DKO homozygous tomato plant leaves inoculated with Botrytis cinerea 72 hours after infection.

[0025] ​ In Example 6 of the present invention ​ -OE and ​ -DKO homozygous tomato plants were inoculated with Botrytis cinerea for 72 h.

[0026] ​ The gray mold resistant material and the gray mold susceptible material in Example 7 of the present invention are ​ Gel electrophoresis diagram of promoter 290bp detection.

[0027] ​ The gray mold resistant material and the gray mold susceptible material in Example 7 of the present invention are ​ Statistical results of promoter 290bp detection.

[0028] ​ For example 8 of the present invention, the AC is edited ​ Sequencing peak alignment of the transgenic material p290-KO-19 with a promoter of 290 bp.

[0029] ​ For p290-KO-19 and control materials in Example 9 of the present invention ​ Expression level detection.

[0030] ​ The photos (A) and the statistics of the lesion area (B) were taken for the leaves of p290-KO-19 and the control material in Example 9 of the present invention after being inoculated with gray mold for 72 hours. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0032] Example 1 This embodiment provides ​ The identification of genes and the preparation of overexpression vectors are as follows: 1.1. ​ Identification of genes By conducting three independent in vitro inoculation experiments with Botrytis cinerea on more than 300 tomato materials and performing GWAS analysis together with the resequencing results, the SlCT1 gene of the present invention was found to be a major candidate gene for GWAS positioning.

[0033] 1.2. ​ Preparation of excess vector (1) Search on the SGN website (https: / / solgenomics.net / ) ​ The DNA sequence of the gene is shown in SEQ ID NO. 1. The artificially synthesized primers are as follows: 35S- ​ -OE-F:CATTTGGAGAGGACACGCTCGAG ATGCAAGGCTCAATTCATTAC; 35S- ​ -OE-R:TCTCATTAAAGCAGGACTCTAGACACAAGGCCAGGCATGGGGC.

[0034] Full-length amplified from tomato material Alisa Craig (AC) ​ The amplification method is to first extract RNA from young tomato leaves (Trizol one-step method), then reverse transcribe and synthesize cDNA according to the instructions of the two-step reverse transcription kit (purchased from Nanjing Novozymes Co., Ltd., see the instructions for the specific procedures), and use the designed primers 35S- ​ -OE-F+35S- ​ -OE-R was obtained by PCR amplification using high-fidelity Phanta enzyme (purchased from Nanjing Novozymes Co., Ltd., see the instructions for specific procedures) ​ The full-length gene was detected by 0.8% agarose gel, and the target fragment was recovered using a recovery kit (purchased from OMEGA, see the instructions for specific procedures).

[0035] (2) Double-digest the pHellsgate8 vector with XhoI and XbaI, digest at 37 degrees for 3 hours, and detect with 1.0% agarose gel. The amplified fragment and the digested pHellsgate8 vector are mixed with the optimal volume ratio required by the homologous recombination kit (purchased from Nanjing Novozymes, see the instructions for specific procedures), add 5×CE II Buffer 2μL and Exnase II 1μL, and add sterile water to the volume of 10μL. After reacting at 37℃ for 30 minutes, quickly transfer to ice water and place for 5 minutes. Heat shock at 42℃ to transform the homologous recombination product into Escherichia coli DH5α strain, screen positive clones with Spec resistance plates, and send the bacterial solution to a sequencing company for sequencing after PCR detection. Compare the sequencing results with the known sequence to determine the correct Escherichia coli bacterial solution and then shake the bacteria to extract its plasmid. ​ Overexpression vector.

[0036] Example 2 This embodiment provides ​ and its homologous genes ​ The specific steps for constructing a CRISPR / Cas9 multi-target knockout vector are as follows: 2.1 Target and primer design The construction of CRISPR / Cas9 multi-target knockout vectors was mainly based on the method of Xie et al. (Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system[J]. ​ ​ , 2015, 112(11): 3570-3575.) Search on the SGN website (https: / / www.sgn.cornell.edu / search / locus) ​ Homologous genes ​ The DNA sequence of the gene is SEQ ID NO.2, according to the SGN website. ​ and ​ The sequence identity of ​ and ​ The DNA sequence was input into the CRISPR design website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), and the off-target rate was calculated. ​ and ​ Two appropriate targets were selected on each exon. The target sequences are as follows: ​ Gene target 1: TTGTGTACACTAGTTTGGGATGG; ​ Gene target 2: GGGGTTGAATTTTGATTTCG; ​ Gene target 3: TTCTTGGAATTCCGTTCCAC; ​ Gene target 4: ATGATGTGGCTTTCTCTACT.

[0037] Gene synthesis amplification and primers capable of tandemly connecting four targets. The primer sequences are as follows: pG1-SlCT2-F: ATATATGGTCTCGTTTGTTCTTGGAATTCCGTTCCACGTTTTAGAGCTAGAAATAGC; pG1-SlCT2-R:ATTATTGGTCTCGTACTTGCACCAGCCGGGAATCGAA; pG2-SlCT2-F: ATATATGGTCTCGAGTAGAGAAAGCCACATCATGTTTTAGAGCTAGAAATAGC; pG2-SlCT2-R: ATTATTGGTCTCGACAATGCACCAGCCGGGAATCGAA; pG3-SlCT1-F: ATATATGGTCTCGTTGTGTACACTAGTTTGGGAGTTTTAGAGCTAGAAATAGC; pG3-SlCT1-R:ATTATTGGTCTCGAAACCGAAATCAAAATTCAACCCCTGCACCAGCCGGGAATCGAA.

[0038] 2.2 Amplification and transformation into Escherichia coli by ​ Using the plasmid as a template, phanta high-fidelity enzyme was used to amplify and purify each target fragment. Multiple amplified fragments were concatenated using the Golden Gate ligation method. The reaction system and steps are shown in Tables 1 and 2, respectively. The reaction products were heat-shock transformed into Escherichia coli DH5α, and the correct single colony was detected and shaken to extract the plasmid.

[0039] Table 1 Golden Gate reaction system

[0040] Table 2 Golden Gate reaction steps

[0041] Example 3 This example provides the preparation of gene-edited tomato plants, comprising the following steps: 3.1 Recombinant vectors were transformed into Agrobacterium The recombinant vectors prepared in Example 1 and Example 2 were respectively transformed into Agrobacterium, and the operation method was as follows: Correctly identified recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method. Overexpression recombinant vectors were screened using LB solid plates containing 50 mg / L Rif and 100 mg / L Spec. CRISPR / Cas9 knockout recombinant vectors were screened using LB solid plates containing 50 mg / L Rif and 100 mg / L Kana. Positive clones were selected by shaking and culturing overnight at 28°C and 200 rpm. 1 µL of the Agrobacterium solution was used as a template. Primers were 35S: CTATCCTTCGCAAGACCCTTC and 35S- ​ -OE-R: TCTCATTAAAGCAGGACTCTAGACACAAGGCCAGGCATGGGGC, PCR detection of overexpression recombinant monoclones; vector primers PTX-FW: AGCGGATAACAATTTCACACAGGA plus PTX-RV: GCAGGCATGCAAGCTTATTGG were used for PCR detection of multi-target knockout recombinant monoclones.

[0042] 3.2 Agrobacterium infection of tomato cotyledons The two Agrobacterium strains obtained in the previous step were used to infect tomato cotyledons respectively. The operation method is as follows: Tomato seeds Ailsa Craig (AC or A57, a product of the American Tomato Genetic Resource Center) were sterilized with sodium hypochlorite for 15 minutes (2% available chlorine) and sown on 1 / 2 MS medium (pH = 5.8). They were cultured at 25 ± 2°C in the dark until the seeds germinated. The seeds were then cultured under a light intensity of 1800 lx and a photoperiod of 16 h light / 8 h dark. Cotyledons of 7-8 day old sterilized seedlings were cut and pre-cultured for 2 days in MS medium (pH = 5.8). The seeds were resuspended to an OD of 0. 600Inoculate with ≈0.5% Agrobacterium solution for 3-5 minutes. Remove excess liquid with sterile filter paper and place back on the pre-culture medium. Incubate in the dark for 2 days before transferring to 1.0ZR screening medium (MS + 1.0 mg / L ZR (zeatin riboside) + 400 mg / L Cef (cephalosporin) + 100 mg / L Km (karatomycin)) for resistance screening. Subculture every two weeks. When resistant shoots emerge, transfer the explants to 0.2ZR + 200 mg / L Cef (cephalosporin) + 100 mg / L Km (karatomycin). After 20-30 days, explants are excised and inserted into rooting medium (RM) to induce rooting. Plants with well-developed root systems are transplanted into pots. See Table 3 for the specific medium formula.

[0043] Table 3 Tomato genetic transformation medium formula

[0044] Example 4 This embodiment provides the above-mentioned detection of the expression level of the overexpressing plant, and the detection method is as follows: Genetic transformation ​ The genomic DNA of leaves of the T0 generation overexpressing plants was extracted by CTAB method. The detection primers were 35S and 35S- ​ -OE-R, PCR detection was performed using gDNA from T0 plants as template, overexpression plasmid as positive control, and wild-type background material as negative control.

[0045] The primers were designed for the T0 generation plants that were positive in PCR test and real-time fluorescence quantitative test was performed. ​ The primer sequences for the gene overexpression level are: ​ -Qpcr-FW:ACATCTTATTTTTGTGGGGCAC; ​ -Qpcr-RV:TCCATCCCAAACTAGTGTACAC.

[0046] Real-time fluorescence quantitative experimental method: RNA was extracted from T0 plants that tested positive for PCR and reverse transcribed into cDNA. The reaction system was: 5µL SYBR Mix, 0.5µL each of forward and reverse primers (10nmol / L), and 4µL sample cDNA. The reaction program was: 95°C for 30s, 95°C for 5s, 55°C for 10s, and 72°C for 15s, for a total of 40 cycles (if the melting curve peak time is later than 30 cycles, add 10 cycles), cool at 40°C for 10s, and then collect and analyze the melting curve. Each sample was technically replicated three times, and the tomato endogenous actin gene (GenBank accession no. BT013524) was used as an internal reference. The data obtained were analyzed using the ∆∆Ct method, and the results are shown in the table. ​ Finally, we screened out SlCT1-OE-14 and SlCT1-OE-16, which overexpressed SlCT1 more than 100-fold compared with the control AC, for subsequent material verification.

[0047] Example 5 This embodiment provides the above acquisition ​ and ​ Knockout plants were tested using the following method: ​ and ​ Genomic DNA from leaves of the double knockout plants of generation T0 was extracted using the CTAB method. A PCR reaction was performed using the forward and reverse primers of the vector to detect whether the T0 plants had the vector pTX041 inserted. The primer sequences were the same as in Example 3, i.e., PTX-FW and PTX-RV.

[0048] PCR amplification using gDNA of CRISPR / Cas9 gene-edited plants with vector pTX041 inserted as template ​ and ​ .

[0049] Among them, the primer sequence for amplifying SlCT1 is: ​ -DKO-jc-F:TGGGATTTTGGGGAAAATCAAAG; ​ -DKO-jc-R:CCAAGGCATCTACTATCTT; The primer sequences for amplifying the SlCT2 gene are: ​ -DKO-jc-F:GGCACATTCATTACTTGCGT; ​ -DKO-jc-R:GGAATGCTTAGGGATAGC.

[0050] After sequencing, the sequencing results were compared, and the peak diagram was as follows ​ Finally, we screened out the ​ and ​ Double mutant plants with mutations: DKO-43 and DKO-47 were used as materials for subsequent verification.

[0051] Example 6 This embodiment provides ​ Overexpression materials and ​ The resistance of double mutant plants to Botrytis cinerea was identified as follows: The inoculation experiment of Botrytis cinerea was as follows: the back of mature leaflets of compound leaves of healthy plants with the same growth potential were inoculated with Botrytis cinerea spores. The concentration of Botrytis cinerea spore suspension was 10 5 / ml, constant temperature of 22℃, 16h light / 8h dark, relative humidity of 75% for 72h. The results of multiple inoculations showed that compared with the control material ​ The gray mold lesions on the leaves of the overexpressing transgenic plants were smaller, while the lesions on the leaves of the double mutant materials were significantly larger than those of the control. ​ , the statistical graph of lesion area is shown in ​ .

[0052] Example 7 This example studies materials resistant to gray mold and materials susceptible to gray mold to obtain ​ The promoter difference sequence is as follows: Tomato GWAS population validation of the more resistant gray mold materials: TS6, TS13, TS29, TS42, TS57, TS61, TS62, TS63, TS74, TS75, TS86, TS87, TS91, TS105, TS107, TS109, TS118, TS125, TS129, TS131, TS148, TS149, TS151, TS154, TS179, TS185, TS203, TS204, TS213, TS234, TS238, TS239, TS242, TS284, TS286, TS295, TS297; Materials more susceptible to gray mold: TS1, TS2, TS3, TS41, TS46, TS47, TS54, TS58, TS60, TS64, TS71, TS82, TS95, TS98, TS100, TS112, TS114, TS115, TS132, TS138, TS139, TS142, TS159, TS171, TS180, TS192, TS200, TS245, TS249, TS266, TS268, TS274, TS276, TS292, TS254.

[0053] The gDNA in the above materials were extracted by CTAB method. ​ Design primers for the promoter region: ​ -290-jc-F:AGGCTTTTAAGTAATGCTTG; ​ -290-jc-R:GTGGGTGGTCCATCATCCTC.

[0054] PCR was performed to detect the size of the fragment in the DNA of the above materials. The gel electrophoresis results were as follows: ​ As shown in the following statistics, ​ shown.

[0055] Depend on ​ 、 6 As shown: The larger bands in lipose gel electrophoresis (about 713bp) are materials containing 290bp in the promoter, and the smaller bands (about 423bp) are materials that do not contain 290bp in the promoter.

[0056] Example 8 This embodiment provides a creative destruction ​ The mutants of promoter 290 bp are as follows: 8.1 Target determination and primer design Search on the SGN website (https: / / www.sgn.cornell.edu / search / locus) ​ The promoter sequence of the gene is the same as described in Example 2.1. Enter the promoter sequence into the CRISPR design website and select two suitable targets based on the off-target rate and other factors: ​ Promoter target site 1 (GAGCGGTGAACGAGATGGG), target site 2 (AGATTAGTAAAATTTATGT), artificially synthesized primers containing two target sites: PTX-p290KO-F: GAATCTAACAGTGTAGTTTGGAGCGGTGAACGAGATGGGGTTTTAGAGCTAGAAATAGC; PTX-p290KO-R: GCTATTTCTAGCTCTAAAACTCACTTAGACAAAGTGTGTCCAAACTACACTGTTAGATT.

[0057] Using the 043 plasmid as a template (from Cui Xia's group at the Institute of Vegetable and Floriculture, Chinese Academy of Agricultural Sciences, Song J, Zhang S, Wang X, et al. Variations in BothFTL1 and SP5G, Two Tomato FT Paralogs, Control Day-Neutral Flowering [J]. Molecular Plant, 2020, 13(7)), a PCR fragment containing two target sites was amplified.

[0058] 8.2 Amplification and transformation into Escherichia coli The PTX plasmid was digested with BsaI (NEB) at 37°C for >3 hours. The digestion product was examined on a 1.0% agarose gel and the large PTX fragment (approximately 18K) was recovered using a gel recovery kit. The amplified fragment containing both target sites and the vector were mixed at a 1:1 ratio. 1 μL of Exnase II, 2 μL of 5X CE II Buffer, 25-100 ng of the linearized cloning vector, and 10-100 ng of the insert amplified product were added, and the volume was brought up to 10 μL with sterile water. Ligation was performed at 37°C for 30 minutes. All ligation products were heat-shocked and transformed into Escherichia coli TransT1. Positive colonies were selected on Kan-resistant LB plates, picked, and the culture suspension was assayed by PCR. PCR products were examined on a 1.0% agarose gel for PTX-FW and PTX-RV. The empty vector fragment size was 1820 bp, while the vector containing both target sites was 1169 bp. Correctly identified culture suspensions were sent for sequencing (Tianyi Huiyuan). Perform sequence alignment, shake the correct bacterial solution, and extract the plasmid using the small-volume method.

[0059] 8.3 Creation of transgenic plants According to the method in step 3.2 of Example 3, the vector with the 290 bp promoter edited was transferred into Agrobacterium, and then Agrobacterium-mediated tomato transformation was performed to create transgenic plants.

[0060] 8.4 Editing Detection in Transgenic Plants Design detection primers in the promoter region (the region containing the two editing target sites): ​-290-jc-F:AGGCTTTTAAGTAATGCTTG; ​ -290-jc-R:GTGGGTGGTCCATCATCCTC.

[0061] Amplify the fragment, send the PCR stock solution to Tianyi Huiyuan sequencing, and finally screen out the ​ The strain p290-KO-19 with a homozygous mutation in the 290 bp region of the promoter was used as the material for subsequent research, and its sequencing peak alignment is shown in Figure 7. ​ The results showed that the 290-KO-19 promoter sequence was missing 37bp near the target site.

[0062] Example 9 This embodiment aims at the above-mentioned ​ The resistance of mutants in the 290 bp promoter region was tested as follows: Refer to the method of implementation case 4, use primer ​ -qPCR-FW and ​ -QPCR-RV, real-time fluorescence quantitative detection of control materials AC and p290-KO-19 materials ​ The test results are as follows ​ As shown, ​ Promoter 290bp mutant material p290-KO-19 ​ The expression level of AC was significantly higher than that of AC.

[0063] Further, according to the method in Example 6, the resistance of AC and p290-KO-19 to Botrytis cinerea was tested. ​ As shown, after 72 hours of inoculation with Botrytis cinerea, the lesion area of ​​p290-KO-19 was significantly smaller than that of the control AC, indicating that ​ Mutations in the 290bp promoter region can enhance resistance to Botrytis cinerea.

[0064] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0065] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. SlCT1 The application of the gene in regulating resistance to tomato gray mold is characterized in that: described SlCT1 The nucleotide sequence of the gene is shown in a or b: a: the sequence shown in SEQ ID NO.1; b: A nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added to the sequence shown in SEQ ID NO.1 and which can encode the amino acid sequence shown in SEQ ID NO.

2.

2. A method for regulating tomato gray mold resistance, characterized in that: include: By overexpressing tomato SlCT1 Genes to improve gray mold resistance in tomatoes; or, by knocking out SlCT1 Genes and their homologous genes SlCT2 , reducing the gray mold resistance of tomatoes, the SlCT2 The gene nucleotide sequence is shown in SEQ ID NO.3; or, by editing SlCT1 Gene promoter sequence, so SlCT1 Gene overexpression improves gray mold resistance in tomatoes, the SlCT1 The gene promoter sequence is shown in SEQ ID NO.

4.

3. The method according to claim 2, characterized in that By overexpressing tomato SlCT1 The specific steps for genetically improving tomato gray mold resistance are as follows: construct an overexpression vector and transfect tomatoes with Agrobacterium to obtain overexpression plants, thereby improving tomato gray mold resistance. The primers used to construct the overexpression vector are as follows: 35S- SlCT1 -OE-F:CATTTGGAGAGGACACGCTCGAG ATGCAAGGCTCAATTCATTAC; 35S- SlCT1 -OE-R:TCTCATTAAAGCAGGACTCTAGACACAAGGCCAGGCATGGGGC。 4. The method according to claim 2, characterized in that By simultaneous knockout SlCT1 Genes and their homologous genes SlCT2 The specific steps to reduce the gray mold resistance of tomatoes are as follows: construct a CRISPR / Cas9 multi-target knockout vector and transfect tomatoes with Agrobacterium to obtain SlCT1 / 2 Double mutant plants can reduce the gray mold resistance of tomatoes. The target fragments for constructing the CRISPR / Cas9 multi-target knockout vector are as follows: SlCT1 Gene target 1: TTGTGTACACTAGTTTGGGATGG; SlCT1 Gene target 2: GGGGTTGAATTTTGATTTCG; SlCT2 Gene target 3: TTCTTGGAATTCCGTTCCAC; SlCT2 Gene target 4: ATGATGTGGCTTTCTCTACT.

5. The method according to claim 2, characterized in that By editing SlCT1 The specific steps of improving the gray mold resistance of tomatoes by using gene promoter sequence are as follows: constructing CRISPR / Cas9 editing system and transfecting tomatoes with Agrobacterium to create a SlCT1 The mutant plants of the promoter can improve the gray mold resistance of tomatoes; the target fragments of CRISPR / Cas9 are constructed as follows: SlCT1 Promoter target 1: GAGCGGTGAACGAGATGGG; SlCT1 Promoter target 2: AGATTAGTAAAATTTATGT.

6. A method for identifying tomato plants SlCT1 A molecular marker of gene expression, characterized in that The molecular marker nucleotide sequence is shown in SEQ ID NO.

5.

7. A primer set for detecting the molecular marker according to claim 6, characterized in that: The primer set is specifically as follows: SlCT1-290 -jc-F:AGGCTTTTAAGTAATGCTTG; SlCT1-290 -jc-R:GTGGTGGTCCATCATCCTC。 8. A kit comprising the primer set according to claim 7.

9. Use of the primer set according to claim 7 or the kit according to claim 8 in breeding tomato varieties resistant to gray mold.

10. Containing the composition according to claim 1 SlCT1 The application of gene vectors and engineered bacteria in breeding tomato varieties resistant to gray mold.

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