Application of SlRIK1 gene and its protein in immune regulation of tomato bacterial wilt

Abstract

The present invention belongs to the field of plant genetic engineering, and specifically relates to the application of the SlRIK1 gene and its protein in immune regulation of tomato bacterial wilt. The present invention provides the SlRIK1 gene and the SlRIK1 protein encoded by the SlRIK1 gene, which are closely related to tomato bacterial wilt; further, the present invention provides a biological material containing the SlRIK1 gene, which can be used to regulate the disease resistance of tomatoes and cultivate disease-resistant tomatoes. Experiments have shown that by inhibiting the expression of the SlRIK1 gene in target tomatoes, tomatoes that are sensitive to bacterial wilt can be obtained; by promoting the expression of the SlRIK1 gene in target tomatoes, tomatoes that are resistant to bacterial wilt can be obtained. The present invention provides a basis for cultivating disease-resistant tomatoes, which is beneficial to reducing agricultural losses and improving economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of SlRIK1 gene and protein thereof in immune regulation of tomato bacterial wilt. Background Art

[0002] Tomato bacterial wilt is a devastating bacterial disease caused by Ralstonia solanacearum that can cause huge economic losses. The pathogen usually invades the plant through wounds and natural openings in the root system, first colonizing the intercellular spaces of the root system, then invading the xylem vessels and proliferating in the vascular bundles, eventually blocking the vascular bundles and causing the plant to wilt and die, causing serious losses to agricultural production.

[0003] The bacterial wilt pathogen can lurk in the soil for a long time and spread through running water, so its prevention and control process is difficult. At present, the prevention and control of bacterial wilt in production mainly includes chemical control, agricultural control and biological control. However, chemical control such as long-term application of pesticides will enhance the resistance of pathogens; agricultural control such as grafting and soil improvement cannot fundamentally prevent the spread of the disease; although biological control is relatively friendly to the environment, it is easily affected by factors such as temperature, humidity and soil microbial diversity during the biological control process, and the prevention effect is not ideal. At present, there is no research or report on the use of SlRIK1 gene to control tomato bacterial wilt. Summary of the invention

[0004] The purpose of the present invention is to provide the application of SlRIK1 gene and its protein in immune regulation of tomato bacterial wilt. The gene is closely related to tomato bacterial wilt, so it can not only be used to regulate the disease resistance of tomatoes but also to cultivate disease-resistant tomatoes.

[0005] The present invention provides the use of SlRIK1 gene, SlRIK1 protein encoded by SlRIK1 gene or biological material containing SlRIK1 gene in regulating tomato disease resistance and / or cultivating disease-resistant tomato; the disease resistance includes resistance to diseases caused by Ralstonia solanacearum.

[0006] Preferably, the disease includes bacterial wilt.

[0007] Preferably, the regulation includes: promoting the expression of the SlRIK1 gene or increasing the content of the SlRIK1 protein to enhance the resistance of tomatoes to diseases; or inhibiting the expression of the SlRIK1 gene or reducing the content of the SlRIK1 protein to reduce the resistance of tomatoes to diseases.

[0008] Preferably, the biological material containing the S1RIK1 gene includes a biological material that inhibits the expression of the S1RIK1 gene and / or a biological material that overexpresses the S1RIK1 gene.

[0009] Preferably, the biological material for inhibiting the expression of the SlRIK1 gene includes: sgRNA, an expression vector containing the sgRNA, or an engineered bacterium containing the sgRNA; the nucleotide sequence of the sgRNA is shown in SEQ ID NO.7.

[0010] Preferably, the biological material overexpressing the SlRIK1 gene includes: a recombinant expression vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the SlRIK1 gene.

[0011] Preferably, the basic vector of the recombinant expression vector includes pCAMBIA2300.

[0012] The present invention provides a method for cultivating tomatoes, comprising the following steps: obtaining bacterial wilt-sensitive tomatoes by inhibiting the expression of SlRIK1 gene in target tomatoes; or obtaining bacterial wilt-resistant tomatoes by promoting the expression of SlRIK1 gene in target tomatoes.

[0013] Preferably, the SlRIK1 gene in the target tomato is knocked out by sgRNA to inhibit the expression of the SlRIK1 gene in the target tomato; or the SlRIK1 gene is introduced into the target tomato to promote the expression of the SlRIK1 gene in the target tomato.

[0014] Preferably, the primers for amplifying the SlRIK1 gene include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.8; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.9.

[0015] Beneficial effects: The present invention provides the use of the SlRIK1 gene, the SlRIK1 protein encoded by the SlRIK1 gene or the biological material containing the SlRIK1 gene in regulating the disease resistance of tomatoes and / or cultivating disease-resistant tomatoes; the disease resistance includes resistance to diseases caused by Ralstonia solanacearum, and the SlRIK1 gene is closely related to tomato bacterial wilt, so the protein encoding the SlRIK1 gene and the biological material containing the gene can not only be used to regulate the disease resistance of tomatoes but also cultivate disease-resistant tomatoes.

[0016] Based on the above technical advantages, the present invention also provides a method for cultivating tomatoes. Experiments have shown that by inhibiting the expression of the SlRIK1 gene in the target tomatoes, tomatoes sensitive to bacterial wilt can be obtained; or by promoting the expression of the SlRIK1 gene in the target tomatoes, tomatoes resistant to bacterial wilt can be obtained, which provides a basis for cultivating disease-resistant tomatoes, is conducive to reducing agricultural losses and improving economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 The results of screening kinase genes involved in positive regulation of bacterial wilt resistance in Example 1, wherein A: expression dynamic diagram of SlRIKs after infection with Ralstonia solanacearum, B: disease photos of tomatoes after inoculation with Ralstonia solanacearum after silencing SlRIKs;

[0019] Figure 2 The figure is a diagram showing the change of tomato resistance to bacterial wilt after the hairy roots were transformed to overexpress SlRIK1 in Example 1, wherein A: western-blot identification of positive hairy roots, B: tomato bacterial wilt disease photos, C: tomato bacterial wilt disease index line graph, D: tomato survival rate line graph;

[0020] Figure 3 Schematic diagram of the construction of the knockout vector and the construction method of the gene knockout mutant in Example 2, wherein A: gDNA design map, B: sequencing verification map of the gene knockout mutant;

[0021] Figure 4 The growth phenotype diagram of the knockout mutant in Example 2, wherein A: a photo of a tomato at five weeks of growth, B: a bar graph of plant height of a tomato at five weeks of growth;

[0022] Figure 5 The results of the bacterial wilt resistance test of the knockout mutants in Example 3 are shown, wherein A: a line graph of the disease index of tomato root wounds inoculated with the bacterial wilt strain UW551, B: a line graph of the survival rate of tomato root wounds inoculated with the bacterial wilt strain UW551, and C: a statistical bar graph of the growth of bacterial wilt in the xylem of tomato root wounds inoculated with the bacterial wilt strain UW551 for 3 days and 5 days;

[0023] Figure 6 These are the results of the knockout mutants immunized against the csp22 polypeptide in Example 4, wherein A: a line graph of ROS burst in tomato roots triggered by the csp22 polypeptide, B: a line graph of ROS burst in tomato leaves triggered by the csp22 polypeptide, C: a graph of MAPK phosphorylation in tomato roots triggered by the csp22 polypeptide, and D: a graph of MAPK phosphorylation in tomato leaves triggered by the csp22 polypeptide;

[0024] Figure 7 The expression results of tomato root immunity-related genes triggered by csp22 polypeptide in Example 4 are shown in Figure A, wherein A: calorimetric diagram of tomato root salicylic acid-related immunity gene expression, B: calorimetric diagram of tomato root ethylene-related immunity gene expression, C: calorimetric diagram of tomato root systemic acquired resistance-related immunity gene expression, and D: verification bar graph of tomato root immunity-related gene expression;

[0025] Figure 8 The identification diagram of the transgenic tomato overexpressing SlRIK1 in Example 5, wherein A: the map of the binary vector pCAMBIA2300-35S:MCS-HA used for transgenic, B: the result diagram of western-blot identification of T1 generation positive plants, C: the bar graph of qPCR detection of the expression level of SlRIK1 in T2 generation positive plants;

[0026] Fig. 9 The results of the bacterial wilt resistance test of the S1RIK1-overexpressing transgenic tomatoes in Example 5 are shown in Figure A, wherein A: a photo of tomato bacterial wilt disease, B: a statistical graph of the growth of bacterial wilt fungi on tomato stems, C: a line graph of tomato bacterial wilt disease index, and D: a line graph of tomato survival rate;

[0027] Fig.10 The figures are the results of the development-related trait detection of the transgenic tomatoes overexpressing SlRIK1 in Example 5, wherein A: photos of 5-week-old tomato plants, immature tomatoes and mature tomatoes, B: a bar graph of statistical data on plant height of 5-week-old tomato plants, C: a bar graph of the number of fruits produced by a single tomato plant, and D: a bar graph of the weight of a single tomato fruit. DETAILED DESCRIPTION

[0028] The present invention provides the use of SlRIK1 gene, SlRIK1 protein encoded by SlRIK1 gene or biological material containing SlRIK1 gene in regulating tomato disease resistance and / or cultivating disease-resistant tomatoes; the disease resistance includes resistance to diseases caused by Ralstonia solanacearum; the disease preferably includes bacterial wilt.

[0029]

[0030] The amino acid sequence of the SlRIK1 protein encoded by the SlRIK1 gene of the present invention is shown in SEQ ID NO.2, specifically: MGCFSCFSS QEKKAFKRIHSKSKETTDVVRQRETLLNQQQQQRPHSLSRPKPQQDHRSHSQPLSENTRKVPAETPHQKSSNKGKGQNIAAQTFTFRELATATKNFRSECLIGEGGFGRVYKGHLNKTGQTVAVKQLDRNGLQGNREFLVEVLMLSLLHHSNLVNLIGYCADGDQRLLVYEYMSLGSLEDHLLDLERDKLPLDWFTRMKIALHAAKGLEYLHHRANPPVIYRDLKSSNILLDKEHNAK LSDFGLAKVGPTGENTHVSSRVMGTYGYCAPEYQQTGKLTIKSDVYSFGVVLLELITGRRAVDITRFGHQQNLVTWAEPIFKDTKRHIELADPLLGANFPRKSFSQAVAIASMCLQDDPVVRPLISDVVTALSTLSTPETELVSPFASPTRAPSPMHMSTEENIISTRECQDEVAEAIEWGSNSRKQNARSLASCGSSV*, this protein is closely related to tomato bacterial wilt disease, and can therefore be used to regulate tomato disease resistance and cultivate disease-resistant tomatoes.

[0031] The regulation of the present invention preferably includes: promoting the expression of the SlRIK1 gene in tomatoes or increasing the content of the SlRIK1 protein in tomatoes, thereby improving the resistance of tomatoes to bacterial wilt; or inhibiting the expression of the SlRIK1 gene in tomatoes or reducing the content of the SlRIK1 protein in tomatoes, thereby reducing the resistance of tomatoes to bacterial wilt.

[0032] The biomaterial containing the SlRIK1 gene of the present invention preferably includes a biomaterial that inhibits the expression of the SlRIK1 gene and / or a biomaterial that overexpresses the SlRIK1 gene; the biomaterial that inhibits the expression of the SlRIK1 gene preferably includes: sgRNA, an expression vector containing the sgRNA or an engineered bacterium containing the sgRNA; the nucleotide sequence of the sgRNA is preferably as shown in SEQ ID NO.3, specifically 5'-GAGGTTGTGAATGAGAACGG-3'; the initial vector for constructing the expression vector is preferably PTX; there is no special requirement for the method of constructing the expression vector, and the technology well known in the art can be used.

[0033] The biological material for overexpressing the SlRIK1 gene of the present invention preferably includes: a recombinant expression vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the SlRIK1 gene; the basic vector of the recombinant expression vector preferably includes pCAMBIA2300; the recombinant expression vector preferably includes pCAMBIA2300-35S:SlRIK1-HA; the construction method of the recombinant expression vector preferably includes: pCAMBIA2300 is double-digested with SalⅠ and EcoRI, and then connected with CaMV 35S promoter:MCS-HA-NOS terminator to obtain a pCAMBIA2300-35S:MCS-HA vector; the pCAMBIA2300-35S:MCS-HA vector is digested with BamHI and StuⅠ, and then connected with the SlRIK1 gene to obtain the recombinant expression vector. The initial microorganism used to construct the recombinant microorganism of the present invention is preferably Agrobacterium tumefaciens; the recombinant microorganism is preferably obtained by transferring the recombinant expression vector into the initial microorganism; there is no special requirement for the transfer method, and the technology well known in the art can be used.

[0034] The present invention provides a method for cultivating tomatoes, comprising the following steps: obtaining a tomato susceptible to bacterial wilt by inhibiting the expression of the SlRIK1 gene in the target tomato. The present invention preferably knocks out the SlRIK1 gene in the target tomato by sgRNA to inhibit the expression of the SlRIK1 gene in the target tomato; the sgRNA nucleotide sequence is preferably as shown in SEQ ID NO.3; the sequence of the SEQ ID NO.3 has been described in detail above and will not be repeated here; when knocking out the SlRIK1 gene, it is preferably achieved by introducing a recombinant vector pTX-SlRIK1gDNA into the tomato; the method of introduction has no special requirements and can be used in a manner well known in the art.

[0035] The present invention also provides a method for cultivating tomatoes, comprising the following steps: obtaining a tomato resistant to bacterial wilt by promoting the expression of the SlRIK1 gene in the target tomato. That is, by introducing the SlRIK1 gene into the target tomato, the expression of the SlRIK1 gene in the target tomato is promoted; there is no special requirement for the method of introduction, and a method well known in the art can be used; the primers for amplifying the SlRIK1 gene in the present invention preferably include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is preferably as shown in SEQ ID NO.4, specifically 5'-GGATCCATGGGTTGTTTTTCATGTTTTTCA-3'; the nucleotide sequence of the downstream primer is preferably as shown in SEQ ID NO.5, specifically 5'-AGGCCTCACTGAAGAACCACAAGATGC-3'.

[0036] Experiments have shown that by adopting the technical solution provided by the present invention, tomatoes resistant to bacterial wilt can be obtained. The present invention provides a scientific basis for cultivating disease-resistant tomatoes, which is not only beneficial to reducing agricultural losses, but also beneficial to improving agricultural economic benefits.

[0037] To further illustrate the present invention, the application of the SlRIK1 gene and its protein provided by the present invention in immune regulation of tomato bacterial wilt is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Preparation before the experiment:

[0039] 1. Plant Materials

[0040] Tomato (Solanum lycopersicum), Moneymaker, a susceptible variety to bacterial wilt (from Wang Taotao's laboratory at Huazhong Agricultural University), Hawaii 7996, a resistant variety (from Wang Taotao's laboratory at Huazhong Agricultural University), the characteristics of Hawaii 7996 are disclosed in the literature: Kwak MJ, Kong HG, Choi K, Kwon SK, Song JY, Lee J, Lee PA, Choi SY, Seo M, Lee HJ, Jung EJ, Park H, Roy N, Kim H, Lee MM, Rubin EM, Lee SW, Kim JF. Rhizosphere microbiome structure alters to enable wilt resistance in tomato. Nat Biotechnol. 2018 Oct 8. doi: 10.1038 / nbt.4232.

[0041] 2. Test strains and vectors

[0042] The plant expression vector pCAMBIA2300 (purchased from Abcam) used for constructing tomato transgenic plants; the gene editing vector pTX (from Wang Taotao's laboratory at Huazhong Agricultural University); the gene editing vector pTX has been disclosed in Luo D, Sun W, Cai J, Hu G, Zhang D, Zhang X, Larkin RM, Zhang J, Yang C, Ye Z, Wang T. SlBBX20 attenuates JA signalling and regulates resistance to Botrytis cinerea by inhibiting SlMED25 in tomato. Plant Biotechnol J. 2023Apr; 21(4): 792-805. doi: 10.1111 / pbi.13997.

[0043] Competent cells: Escherichia coli MC1061 (Escherichia coli) used for vector construction and Agrobacterium tumefaciens GV3101 (Agrobacterium tumefaciens) used for gene expression were purchased from Wuhan Bovino & Green Day Biotechnology Co., Ltd., and MSU440 (Agrobacterium tumefaciens) used for hairy root transformation was from Alberto Macho's laboratory, Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences.

[0044] Ralstonia solanacearum GMI1000, also known as Ralstonia solanacearum GMI1000, was used for pathogen inoculation.

[0045] 3. Main Reagents

[0046] Taq, Pfu DNA Polymerase, Pfu buffer, and dNTPs were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Gold Mix TSE101 was purchased from Qingke Biotechnology Co., Ltd.; reverse transcription kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; restriction endonucleases were purchased from Baoriyi Biological Co., Ltd.; SYBR was purchased from Mona Biotechnology Co., Ltd.; Trizol was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; column DNA recovery kit was purchased from Shanghai Shenggong Bioengineering Co., Ltd.; other conventional reagents were domestic analytical or chemical pure reagents; primers were synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd.; sequencing was completed by Qingke Sequencing Co., Ltd. HA Western Blot antibody was purchased from Roche, catalog number 12013819001, and was diluted 2000 times with 1×PBST+5% Milk.

[0047] 4. Main instruments

[0048] Electroporator (EppendorfEporator, Eppendorf, Germany), PCR instrument (Mastercycler nexus, Eppendorf, Germany), high-speed centrifuge (5810R, Eppendorf, Germany), constant temperature shaking incubator (MQL-61R, Minquan), plant growth chamber (HP1500GS-B, Ruihua), electrophoresis instrument (EPS 600, Shanghai Tianneng), one-tenth balance (JY5002, Shanghai Sunny Hengping), clean bench (DL-CJ-2NDI, Beijing Donglianhal), water bath (XMTD6000, Beijing Changfeng), gel imager (ChemiDoc XRS+, BIO-RAD, USA), SLR camera (B700, Nikon, Japan), ultra-micro spectrophotometer (DeNovix DS11, Beijing Beihui), microplate reader (SPARK, Switzerland TECAN), UV-visible spectrophotometer (UV1300, Shanghai Meixi Instrument), laser confocal microscope (N-STORM, Japan Nikon).

[0049] 5. Main culture medium

[0050] CPG medium: 10 g of bacterial peptone No. 2, 1 g of acid hydrolyzed casein, 5 g of glucose, dissolved in 1 L of ddH2O, sterilized at 121°C for 20 min. For solid medium, 15 g of agar powder should be added.

[0051] 1 / 2MS medium: Murashige and Skoog medium 2.15 g, morpholineethanesulfonic acid (MES) 0.5 g, dissolved in 800 mL ddH2O, adjusted to pH 5.8 with 5 M KOH, sucrose 5.0 g, dilute to 1 L. For solid medium, 8 g agar powder should be added.

[0052] KCMS medium (preculture, co-culture medium): Murashige and Skoog medium 4.3 g, inositol (20 mg / mL) 5 mL, vitamin B1 (1 mg / mL) 1.3 mL, 2,4-D (1 mg / mL) 200 μL, KH2PO4 (100 mg / mL) 2 mL, kinetin (1 mg / mL) 100 μL, sucrose 30.0 g, adjusted to pH 5.8, made up to 1 L with ddH2O, agar 7.4 g, autoclaved at 121°C for 30 min.

[0053] 2Z medium (screening medium): Murashige and Skoog medium 4.3 g, organic matter (200×) 5 mL, sucrose 30.0 g, adjusted to pH 5.8, ddH2O to 1 L, agar 7.4 g, autoclave at 121°C for 30 min, add IAA (1 mg / mL) 100 μL, zeatin (1 mg / mL) 2 mL, kanamycin (100 mg / mL) 1 mL, and cephalosporin (300 mg / mL) 1.2 mL when the temperature is cooled to 60°C.

[0054] 0.2Z medium (subculture medium): Murashige and Skoog medium 4.3 g, organic matter (200×) 5 mL, sucrose 30.0 g, adjusted to pH 5.8, ddH2O to 1 L, agar 7.4 g, autoclave at 121°C for 30 min, add zeatin (1 mg / mL) 200 μL, kanamycin (100 mg / mL) 1 mL, and cephalosporin (300 mg / mL) 1.2 mL when the temperature is cooled to 60°C.

[0055] Rooting medium: MS Salt 4.3 g, organic matter (200×) 5 mL, IBA (1 mg / mL) 2 mL, sucrose 30.0 g, agar 7.4 g, adjusted to pH 5.8, made up to 1 L with ddH2O, autoclaved at 121°C for 30 min, added kanamycin (100 mg / mL) 500 μL, cephalexin (300 mg / mL) 1.2 mL when cooled to 60°C.

[0056] Example 1 Screening for cytoplasmic receptor kinases that positively regulate bacterial wilt resistance in tomatoes

[0057] 1) Transcriptome data of tomato variety Hawaii 7996 before and after infection with Ralstonia solanacearum (data document: French E, Kim BS, Rivera-Zuluaga K, Iyer-Pascuzzi AS. Whole Root Transcriptomic Analysis Suggests a Role for Auxin Pathways in Resistance to Ralstonia solanacearum in Tomato. Mol Plant Microbe Interact.2018Apr;31(4):432-444.doi:10.1094 / MPMI-08-17-0209-R.) were used for comparative analysis to obtain the expression of tomato cytoplasmic receptor kinase encoding genes after 24 and 48 h of R. solanacearum treatment. Five genes with the highest induced expression folds were selected and named SlRIK1 (Solyc09g061330), SlRIK2 (Solyc06g005520), SlRIK3 (Solyc01g111950), SlRIK4 (Solyc09g008010) and SlRIK5 (Solyc07g064340). The content in brackets indicates the gene number of the corresponding gene in the tomato genome database (https: / / www.solgenomics.net / ); the induction folds of SlRIK1 to SlRIK5 in the transcriptome are shown in Figure 1 A( Figure 1 In A, RIK1~RIK5 represent the expression levels of SlRIK1~SlRIK5 respectively; mock represents the expression of each gene without inoculation of Ralstonia solanacearum, 24hpi represents the expression of each gene after 24h; 48hpi represents the expression of each gene after 48h).

[0058] Depend on Figure 1 As can be seen from A, after inoculation with R. solanacearum, the expression levels of SlRIK1-5 in tomato roots increased, which may be potential genes related to resistance to R. solanacearum.

[0059] Among them, the amino acid sequence of the SlRIK2 protein encoded by SlRIK2 (Solyc06g005520) is as shown in SEQ ID NO. 32, specifically: MKC FYFTKDEEEEGGTLSSRESKVSWVRSLSVASSSVDTRKSRSDLDSESRDFTETFEFYEFLTQRRANDLRVFSFSELKMATKGFSRGLMIGEGGFGCVYRGIVSAPCSDLKMEVAVKRLNRHGFQGHKEWINEVNFLGVVKHPNLVKLIGYCAEDDERGMQRLLVYELMRNKSLEDHLLARAAAPLSWTLRLKIAQDAARGLAYLHEEMDFQLIFRDFKPSNILLDEDFIAKLSDFGLARQGPTAGLTHVSTSVVGTVGYAAPEYVQTGRLTAKSDVWSFGVVLYELITGRRVLERNLPRAEQKLLEWVRPYVSDTKKFHFILDPRLEGHDCIKSAQRLASLANKCLAKNARSRPRMSEVVDMLENIIIDAEAVPETLKETENVNEEVDDTKEEGREEVELGKPESNSQKWGFDFKEMVSFRNKSIGKLDWRNWTAGLVKTS;

[0060] The amino acid sequence of the SlRIK3 protein encoded by SlRIK3 (Solyc01g111950) is shown in SEQ ID NO. 33, specifically: MFGSSSGK GGISSLLRCRVVPKAKRRTIVVGLKSDNSSREMLLRLLNLVVVPGDYVLSVHVQQPNDTFDPNTFHIHEDLCKSKQVDFQIKVCVADTYITELSNQVRINFATMLAVGCSSSRPTDQIAGKILKELPPTCSLLVMDNGGKILLQRPGTSQEGAPIKVFQSPQSSLSVSSSSTQSGDKYQIHKSLSMTCSSTTTTSSQPTRNATFPRIKKLNNAAAKSLFERIACLESMGCARRFTTDELSCATDNFSPSLLTGVGGHSQVYRANLENGQLAAVKVLKNTRYAEDDLFQEVEILSNLKHENLIQLVGYSYSKDMQAIVYNLQKDSLKQRLKQLNWNTRMQVAIGVARGLEYLHSQTPPIVHRDVKSSNILLSDDCHPQLSDFGSAAVHQETQQDSACVKPIHVVGTFGYLAPEYIMYGKVDEKVDVYSYGVVLLELITGKRAIEKDLEAHHESLVLWARSLLSCGLSDRLVDPDINENYNKDEMKTMMFAARLCLLHSSSRRPKMKTILRLFEEPEHWLELQRKREELLDGIGSEDETCLCRYYGSDSEEGMFIEDS;

[0061] The amino acid sequence of the SlRIK4 protein encoded by SlRIK4 (Solyc09g008010) is shown in SEQ ID NO. 34, specifically: MGGYFSSI LKDMKCFPFYNAEKKEEPKTTKSNPVLSSSFGLFDSEFRHSHRESSSQNVSDTSSESRGRSQIPSLSDRPSDLRAFTFSELKAATKNFNRTTKIGEGGFGCVYKATVKTGEDSSKKIDVAIKQLGRRGLQGHKEWVTEVNVLGVAEHKNLVKLVGYCAEDDERGIQRLLVYEYMPNRSVENHLSARSETPLSWAMRLKIAQDAARGLAYLHEEMDVQIIFRDFKSSNILLDEQWNAKLSDFGLARLGPPEGLTHVSTAVVGTMGYAAPEYVQTGRLTSKSDVWSYGVFLYELITGRRPLDRNRPRSEQKLLEWVKPYISDSKKFQQIIDPRLDGKISRSAQKLSIVANRCLVRHAKTRPKMSEVLEMVNKVVETSTGIGNPGPPVRIAEPTSPESTRKGKRKVDTKLGDGSRLVRIWSTKLTNTC;

[0062] The amino acid sequence of the SlRIK5 protein encoded by SlRIK5 (Solyc07g064340) is shown in SEQ ID NO. 35, specifically: MILGRLVK RDWLIWFPCCMTMDDNNQISSLEDSIIHQYKSANSQAQFANISLKTDSSRRRYIASEIEKFGKGNISAQAFTFRELCLATENFDSECLLGSGGFGKVYKGHIKSKNMAVAVKQLDRNGFQGTKEFLVEVLLLSLLRHSNLVNLIGYCSDGDQRILVYELMSNDSLEVHLLELGPDQKPLDWYTRMKIATGAARGLAYLHETANPPVIYRDFKSSNILLDENFDPKLSDFGLAKLGPTGDSSHVSTRVMGTYGYCAPDYACTGKLTIKSDVYSFGVVLLEIISGRRVIDSSRPSEEKNLIKWARPLISDKKLHLIADPLLRGNYPRRGLDKALTVATMCLQEDASTRPLMSEVVTALDYILNIKKHDDDHKEETADDTYKSPPALQTITSHVDRIASNKPNCVRERY。

[0063] 2) Gene silencing of the above genes SlRIK1, SlRIK2, SlRIK3, SlRIK4, and SlRIK5 was performed to verify the regulatory effect of SlRIK1 to SlRIK5 genes on tomato bacterial wilt resistance. The process is as follows:

[0064] The VIGS target sites of SlRIK1 to SlRIK5 were designed for PCR amplification. The length of each PCR product was about 500 bp. The primers used for PCR amplification are shown in Table 1.

[0065] Table 1 Primer sequence information

[0066]

[0067]

[0068] The PCR reaction system, with a total volume of 50 μL, consisted of 1 μL Hawaii 7996 cDNA, 45 μL Gold Mix, 2 μL upstream primer and 2 μL downstream primer; PCR reaction program: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; final extension at 72°C for 5 min; and storage at 12°C for 10 min.

[0069] After obtaining each PCR product, each PCR product was digested with EcoRⅠ and KpnⅠ restriction endonucleases to recover the target fragment; at the same time, the pYL156 vector was digested with EcoRⅠ and KpnⅠ and the vector was recovered; the vector and the target fragment were connected at 16°C for 10 hours using T4 DNA ligase to obtain a connection product; the connection product was transformed into MC1061 competent state, spread on an LB plate with 50μg / mL kanamycin resistance, cultured at 37°C for 12 hours, and a single colony was picked and cultured in LB liquid culture medium with 50μg / mL kanamycin resistance at 37°C, 190r / min for 8 hours, the plasmid was extracted, and double enzyme digestion verification was performed using EcoRⅠ and KpnⅠ. After cutting the correct fragment, the VIGS gene silencing vector of SlRIK1~SlRIK5 was successfully constructed.

[0070] The SlRIK1-SlRIK5 genes were silenced in the Hawaii 7996 background using VIGS and inoculated with Ralstonia solanacearum. Specifically, the concentration of the Agrobacterium solution containing the VIGS gene silencing vectors of each of the SlRIK1-SlRIK5 genes was adjusted to OD 600 =1.5, and the bacterial solution was injected into the cotyledons of tomato Hawaii7996, and inoculated with Ralstonia solanacearum GMI1000 when the tomato grew to 3 weeks old, N=3, the inoculation method was root inoculation, and each tomato was inoculated with 10mL of the concentration of OD600 = 0.1 GMI1000 solution, after inoculation, the tomatoes were placed in an incubator at 28°C and 65% humidity, and the disease condition of the tomatoes was observed 7 days after inoculation. The results are shown in Figure 1 B( Figure 1 In B, TRV-GFP represents the negative control plant without gene silencing; TRV-RIK1 to TRV-RIK5 represent the plants with SlRIK1 to SlRIK5 gene silencing, respectively)

[0071] Depend on Figure 1 As can be seen in B, Hawaii 7996 became more susceptible to the disease after SlRIK1 silencing.

[0072] 3) Primers for amplifying SlRIK1 were designed according to SEQ ID NO.1 for PCR amplification, wherein the upstream primer SlRIK1_FLFP_BamHI is: 5'-GGATCCATGGGTTGTTTTTCATGTTTTTCA-3' (SEQ ID NO.4), and the downstream primer SlRIK1_FLRP_StuI is: 5'-AGG CCTCACTGAAGAACCACAAGATGC-3' (SEQ ID NO.5); the PCR reaction system, with a total volume of 50 μL, consists of: 1 μL Hawaii 7996 cDNA, 45 μL gold medal Mix, 2 μL upstream primer and 2 μL downstream primer; PCR reaction procedure: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min 30 s, for a total of 35 cycles; final extension at 72°C for 10 min; storage at 12°C for 10 min.

[0073] After obtaining the PCR product, the PCR product was digested with BamHⅠ and StuⅠ restriction endonucleases to recover the target fragment; at the same time, the pCAMBIA2300-35S:MCS-HA vector was digested with BamHⅠ and StuⅠ and recovered (in which, the CaMV35S promoter:MCS-HA-NOSterminator reading frame was connected to the original plasmid pCAMBIA2300 by double digestion with SalⅠ and EcoRⅠ, thereby obtaining the pCAMBIA2300-35S:MCS-HA vector); T4 DNA ligase was used to connect the vector and the target fragment at 16°C for 10 h to obtain a ligation product. The ligation product was then transformed into MC1061 competent cells, spread on an LB plate with 50 μg / mL kanamycin resistance, and cultured at 37°C for 12 h. A single colony was picked and cultured in LB liquid culture medium with 50 μg / mL kanamycin resistance at 37°C, 190 r / min, for 8 h. The plasmid was extracted and double enzyme digestion was performed using BamHⅠ and StuⅠ to verify that the correct fragment was cut out, and the pCAMBIA2300-35S:SlRIK1-HA binary expression vector was successfully constructed.

[0074] The pCAMBIA2300-35S:SlRIK1-HA binary expression vector was overexpressed in the Moneymaker background and inoculated with Ralstonia solanacearum GMI1000 by hairy root transformation, that is, the roots of seedlings grown on 1 / 2MS medium for 1 week were excised, and a single colony of Agrobacterium rhizogenes containing the pCAMBIA2300-35S:SlRIK1-HA binary expression vector was dipped into the wound. After positive hairy roots grew out, samples were taken to detect protein expression, and the tomato plants were transplanted to nutrient soil for further cultivation for 3 weeks, and inoculated with GMI1000 bacterial solution. The inoculation method was root irrigation inoculation, and the inoculation concentration was OD 600 =0.1, the inoculation volume was 10 mL per tomato, and then the tomatoes were placed in an incubator at 28°C and 65% humidity to grow. The hairy roots of 7 tomatoes were randomly selected to detect the expression of SlRIK1-HA in the hairy roots of tomatoes. The hairy roots of each tomato were positive. The results are shown in Figure 2 A in the figure; after 7 days, the growth of tomato plants was observed. Figure 2 and the disease index of tomato plants (the same as the judgment criteria in Example 3) and the survival rate were counted every day within 1 to 6 days after inoculation. The results are shown in Figure 2 C and D in Figure 2 In the figure, A represents the verification of the expression of SlRIK1-HA in tomato hairy roots; B represents the disease photos after inoculation with Ralstonia solanacearum; C represents the disease index line graph after inoculation with Ralstonia solanacearum (N=7); D represents the tomato survival rate line graph after inoculation with Ralstonia solanacearum (N=7).

[0075] Table 2 Disease index and survival rate of each treatment

[0076]

[0077]

[0078] From Table 2 and Figure 2 It can be seen that SlRIK1 may be involved in the positive regulation of tomato resistance to Ralstonia solanacearum.

[0079] Example 2 Knockout of target gene S1RIK1

[0080] gDNA primers for SlRIK1 (SEQ ID NO.1, gene accession number 104649311) were designed, and the length of gDNA (SEQ ID NO.3) was 20 bp; PCR amplification was performed using the p043 vector (published in Luo D, Sun W, Cai J, Hu G, Zhang D, Zhang X, Larkin RM, Zhang J, Yang C, Ye Z, Wang T. SlBBX20 attenuates JA signalling and regulates resistance to Botrytis cinerea by inhibiting SlMED25 in tomato. Plant Biotechnol J. 2023Apr; 21(4): 792-805. doi: 10.1111 / pbi.13997) as a template, and the forward and reverse primers containing the above gDNA sequence included: forward primer pTX-SlRIK1gDNA_FP: 5'-GAATCTAACAGTGTAGTTTGGA GGTTGTGAATGAGAACGGGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO.16), reverse primer pTX-SlRIK1gDNA_RP: 5'-GCTATTTCTAGCTCTAAAACCACCTATTAGACATTCTTGGCAAACTACACTGTTAGATTC-3'(SEQ ID NO.17); PCR reaction system: 043 vector 1μL, Taq mix 25μL, forward primer 1μL, reverse primer 1μL, ddH2O supplemented to 50μL; PCR reaction program: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, a total of 35 cycles; 72℃ final extension for 5min; 12℃ storage for 10min.

[0081] After the above PCR products were recovered, the target fragment was obtained; the target fragment was homologously recombined with the pTX vector after BsaⅠ digestion, and the recombinant product was heat-shock transformed into MC1061. After the bacterial solution PCR was verified to be correct, the correct single clone was taken for micro-plasmid extraction, and then the plasmid was electroporated into GV 3101. The pTX-SlRIK1gDNA vector was transferred into tomato explants using Agrobacterium-mediated genetic transformation technology, and the T1 generation gene-edited plants were obtained after screening with 2Z medium and rooting medium, and the DNA of the T1 generation plants was extracted.

[0082] The primers used to identify positive T1 plants were: slrik1 ID_FP: 5'-AGGCATTCAAAAGAATACATAGCAA-3' (SEQ ID NO.18), slrik1 ID_RP: 5'-TTTTATGTCTACAACCGTCTCCC-3' (SEQ ID NO.19). PCR amplification was performed using DNA of T1 plants as a template. The PCR amplification system was 2 μL of template DNA, 1 μL of slrik1 ID_FP, 1 μL of slrik1 ID_RP, 25 μL of Taq mix, and 50 μL made up with ddH2O. The PCR reaction procedure was: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles; final extension at 72°C for 5 min; and storage at 12°C for 10 min.

[0083] The above PCR products were sent to Qingke Biotechnology Co., Ltd. for sequencing. Two homozygous mutant lines, slrik1-6 and slrik1-16, were identified from 20 T1 plants. The sequencing results are shown in Figure 3 (exist Figure 3 In the figure, A represents the gene map of SlRIK1; B represents the comparison results of the measured sequences of slrik1-6 and slrik1-16 with the reference genome sequence).

[0084] slrik1-6, slrik1-16 and wild-type Hawaii 7996 were cultured in a culture room at 22°C, 75% humidity, 12h light and 12h dark photoperiod. One strain was treated, and each treatment was replicated 10 times. After 5 weeks, the growth of each plant was observed and the height of the plant was counted. The results are shown in Figure 4 (exist Figure 4 In the figure, A shows photos of wild-type and mutant tomatoes at 5 weeks old; B shows a bar graph of plant height data of wild-type and mutant tomatoes at 5 weeks old, N=10, pvalues ​​are 0.5964 and 0.8290, respectively, using one-way ANOVA statistical analysis).

[0085] Depend on Figure 4It can be seen that the growth and development of the slrik1-6 and slrik1-16 mutant lines were not significantly affected, and the plant height was not significantly different from that of wild-type tomatoes when they grew to 5 weeks old.

[0086] Example 3 Detection of resistance of slrik1 mutant to bacterial wilt

[0087] Hawaii 7996, slrik1-6 and slrik1-16 were cultured in 32-hole plug trays. When the tomatoes were grown to 4 weeks old, they were inoculated with Ralstonia solanacearum. The Ralstonia solanacearum was inoculated by root irrigation, that is, Ralstonia solanacearum was cultured in CPG liquid medium to the logarithmic growth phase, and the cells were collected after centrifugation at 4000r / min for 15 minutes. The Ralstonia solanacearum cells were resuspended with distilled water and the OD600 of the cell solution was adjusted to 0.1. The tomato roots were wounded with a scalpel, and then 10mL of the Ralstonia solanacearum cell solution was inoculated on each tomato. Each genotype was inoculated with 15 replicates. The inoculated tomatoes were placed in a light incubator at 28°C, RH 75%, 12h light / 12h dark for growth, and the disease index within 14 days after inoculation was counted.

[0088] The degree of leaf wilting of the plants was divided into 5 disease levels: level 0, no wilting symptoms; level 1, 1% to 25% leaf wilting; level 2, 26% to 50% leaf wilting; level 3, 51% to 75% leaf wilting; level 4, 76% to 100% leaf wilting. Disease index greater than or equal to 2 was considered death. The incidence rate and disease index were calculated and recorded (refer to Morcillo RJL, Zhao A, Tamayo-Navarrete MI, García-Garrido JM, Macho AP. Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease. J Vis Exp. 2020 Mar 11; (157). doi: 10.3791 / 60302.). The results are shown in Figure 5 A and B in Figure 5In the figure, A represents the line graph of disease index after inoculation with Ralstonia solanacearum, N=15; B represents the line graph of survival rate after inoculation with Ralstonia solanacearum, N=15, pvalues ​​are 0.0124 and 0.0031, respectively, and log-ranktest statistical analysis is used); 3d or 5d after inoculation, stem samples were taken to count the growth of Ralstonia solanacearum (reference: Xian L, Yu G, Wei Y, Rufian JS, Li Y, Zhuang H, Xue H, Morcillo RJL, Macho AP. A Bacterial Effector Protein Hijacks Plant Metabolism to Support Pathogen Nutrition. Cell Host Microbe. 2020 Oct 7; 28(4): 548-557.e7.doi:10.1016 / j.chom.2020.07.003.), the results are shown in Figure 5 C( Figure 5 C in the figure represents the bar graph of the growth of Ralstonia solanacearum on tomato stems 3 or 5 days after inoculation, N=8, pvalues ​​are 0.0037, 0.0032, 0.0014, and 0.0001, respectively, using two-way ANOWA statistical analysis).

[0089] Depend on Figure 5 It can be seen that the growth of Ralstonia solanacearum in the stems of slrik1-6 and slrik1-16 was significantly higher than that of the wild type Hawaii 7996, and the resistance of slrik1-6 and slrik1-16 to bacterial wilt was weaker than that of the wild type Hawaii 7996. This shows that SlRIK1 is involved in the positive regulation of tomato resistance to bacterial wilt.

[0090] Example 4 Detection of the immune response of knockout mutants to csp22 polypeptide

[0091] 1) Detect the production of reactive oxygen species in roots and leaves of wild-type Hawaii 7996 and mutants (slrik1-6 and slrik1-16) tomatoes after treatment with csp22 polypeptide. The specific method is as follows: slrik1-6, slrik1-16 and wild-type Hawaii 7996 were cultured in a culture room at 22°C, 75% humidity, and a 12-h light and 12-h dark photoperiod. When the tomatoes were 4 weeks old, leaf discs were taken for 9 hours of recovery, and then the reaction solution: 5 μM L-012, 10 μg / mL peroxidase, 500 nM csp22 were added, and the active oxygen burst was detected in an ELISA instrument; slrik1-6, slrik1-16 and wild-type Hawaii The seeds of 7996 were disinfected with 75% ethanol and 50% 84 disinfectant for 5 minutes respectively, planted on 1 / 2MS medium, and grown in a culture room at 22°C, 75% humidity, and a photoperiod of 12 h light and 12 h dark. When the tomatoes were 10 days old, the roots were taken for 9 hours of recovery, and then the reaction solution was added: 5 μM L-012, 10 μg / mL peroxidase, 500 nM csp22, and the active oxygen burst was detected by an enzyme reader. Reference for detecting reactive oxygen species: Wei Y, Caceres-Moreno C, Jimenez-Gongora T, Wang K, Sang Y, Lozano-Duran R, Macho AP. The Ralstonia solanacearum csp22 peptide, but not flagellin-derived peptides, is perceived by plants from the Solanaceae family. Plant Biotechnol J. 2018 Jul; 16(7): 1349-1362. doi: 10.1111 / pbi.12874. Results are shown in Figure 6 A and B in Figure 6 In the figure, A is the line graph of the amount of active oxygen produced in tomato leaves, N=12; B is the line graph of the amount of active oxygen produced in tomato roots, N=10)

[0092] Depend on Figure 6 As can be seen from A and B, compared with the roots of wild-type tomatoes, the production of reactive oxygen species in the roots of slrik1 mutant tomatoes decreased by 50%; the production of reactive oxygen species in the leaves of slrik1 mutant tomatoes decreased by 70% to 90%.

[0093] 2) Detecting the MAPK activation in roots and leaves of wild-type and mutant tomatoes after treatment with csp22 polypeptide, specifically, culturing slrik1-6, slrik1-16 and wild-type Hawaii 7996 in a culture room at 22°C, 75% humidity, 12h light and 12h dark photoperiod, taking leaf discs when the tomatoes were 4 weeks old, resuscitating them in sterile water for 9h, then adding 500nM csp22 for 0-15min, collecting the samples and freezing them in liquid nitrogen, grinding them, adding 50μL 2×SDS buffer, running western blot on the samples, and detecting MAPK activation with pERK antibody; culturing slrik1-6, slrik1-16 and wild-type Hawaii The seeds of 7996 were disinfected with 75% ethanol and 50% 84 disinfectant for 5 minutes, planted on 1 / 2MS medium, and grown in a culture room at 22°C, 75% humidity, and a 12-h light and 12-h dark photoperiod. When the tomatoes were 10 days old, they were resuscitated in sterile water for 9 hours, and then 500nM csp22 was added for treatment for 0-15 minutes, and the roots were quickly frozen in liquid nitrogen. After grinding, 50μL 2×SDS buffer was added, and the samples were run for western blot electrophoresis, and MAPK activation was detected with pERK antibody. References for detecting MAPK activation: Qi P, Huang M, Hu X, Zhang Y, Wang Y, Li P, Chen S, Zhang D, Cao S, Zhu W, Xie J, Cheng J, Fu Y, Jiang D, Yu X, Li BA Ralstonia solanacearum effector targets TGA transcription factors to subvert salicylic acid signaling. Plant Cell. 2022Apr 26; 34(5): 1666-1683. doi: 10.1093 / plcell / koac015. Results are shown in Figure 6 C and D in Figure 6 (C in the middle represents the MAPK activation detection picture of tomato roots; D represents the MAPK activation detection picture of tomato leaves).

[0094] Depend on Figure 6 As can be seen from C and D, compared with the roots and leaves of wild-type tomatoes, the MAPK activation in the roots and leaves of the slrik1 mutant tomatoes was significantly weakened.

[0095] 3) Detect the changes in the transcriptome of wild-type and mutant tomato roots after treatment with csp22 polypeptide. The specific method is as follows: the seeds of slrik1-16 and wild-type Hawaii 7996 were disinfected with 75% ethanol and 50% 84 disinfectant for 5 minutes, planted on 1 / 2MS culture medium, and grown in a culture room at 22°C, 75% humidity, 12h light and 12h dark photoperiod. When the tomatoes were 10 days old, they were revived in sterile water for 9 hours, then 500nM csp22 was added for treatment for 0-15 minutes, and the roots were quickly frozen in liquid nitrogen. The samples were sent to Paisono for transcriptome detection. The results are shown in Figure 7 A~C (in Figure 7 In the figure, A represents the heat map of salicylic acid-related immune gene expression; B represents the heat map of ethylene-related immune gene expression; C represents the heat map of systemic acquired resistance-related immune gene expression. In C, the expression verification bar graph shows that the pvalues ​​of SlETR after 1 h of csp22 treatment (1 hpi) were <0.0001 and <0.0001, respectively; the pvalues ​​of SlPR3 after 3 h of csp22 treatment (3 hpi) were 0.0078 and 0.0276, respectively; the pvalues ​​of SlPR3 at 1 hpi were The pvalues ​​of SlSARD1 at 1 hpi were <0.0001 and <0.0001, and the pvalues ​​at 3 hpi were 0.0002 and 0.0003, respectively; the pvalues ​​of SlPR2 at 1 hpi were <0.0001 and <0.0001, and the pvalues ​​at 3 hpi were <0.0001 and <0.0001, respectively).

[0096] Depend on Figure 7 As can be seen from Figures A to C, compared with the roots of wild-type tomatoes, the activation of salicylic acid-related (SApathway), ethylene-related (ETpathway) and systemic acquired resistance-related (SAR) immune genes in the roots of slrik1 mutant tomatoes was significantly weakened.

[0097] 4) Select the representative immune genes SlETR (Solyc11g006160), SlPR3 (Solyc07g009500), SlSARD1 (Solyc03g119250) and SlPR2 (Solyc10g079860) in step 3) for qPCR to verify the expression level of each gene. The results are shown in Figure 7 D (in Figure 7 In the figure, D represents the expression level of different immune genes). The primer information used for qPCR verification of SlETR, SlPR3, SlSARD1 and SlPR2 is as follows:

[0098] SlETR_qPCR_FP:5'-GCTGCAGCAGATGAGGCG-3' (SEQ ID NO.20),

[0099] SlETR_qPCR_RP:5'-CCCATCACCTGCCCTGTAA-3' (SEQ ID NO. 21);

[0100] SlSARD1_qPCR_FP:5'-CGTCGAGTGGTTGTTGAGGA-3' (SEQ ID NO.22),

[0101] SlSARD1_qPCR_RP:5'-CGACGACTTTGCTGCTGGTA-3'(SEQ ID NO.23);

[0102] SlPR2_qPCR_FP:5'-CACAGGTGCACAAACAGGAGT-3' (SEQ ID NO.24),

[0103] SlPR2_qPCR_RP:5'-GCTTGGAGAGTTGGTTGATGAGG-3' (SEQ ID NO. 25);

[0104] SlPR3_qPCR_FP:5'-GTAAGCGTAAGTGTCCCAACAAG-3' (SEQ ID NO.26),

[0105] SlPR3_qPCR_RP:5'-CAGTTGAAGTCACCATTGTCCAA-3' (SEQ ID NO. 27);

[0106] SlACTIN_qPCR_FP:5'-GTATGTTGCTATTCAGGCTGTG-3' (SEQ ID NO.28),

[0107] SlACTIN_qPCR_RP: 5'-GCAAAGCATAACCTCCGTAAAT-3' (SEQ ID NO. 29).

[0108] Depend on Figure 7 As can be seen from Figure 3, the induction levels of SlETR, SlPR3, SlSARD1 and SlPR2 genes in the slrik1 mutant were significantly reduced or even not induced.

[0109] Example 5 Detection of resistance of S1RIK1 overexpressing transgenic plants to bacterial wilt

[0110] 1) Transform pCAMBIA2300-35S:SlRIK1-HA into Moneymaker explants using Agrobacterium-mediated genetic transformation (see pCAMBIA2300 vector map for details). Figure 8 A), explants were induced to produce callus tissue, callus tissue was induced to produce regenerated buds, and regenerated buds were transferred to rooting medium to obtain 37 T1 transgenic plants, which were taken out and transferred to jiffypot for further culture. The roots were quickly frozen in liquid nitrogen and then fully ground. After adding 50 μL 2×SDS buffer, they were denatured at 95°C for 10 min. The expression of SlRIK1-HA in the roots of transgenic plants was detected by western blot. The results are shown in Figure 8 B in Figure 8 In the figure, OX-5 indicates OXS1RIK1-5; OX-6 indicates OXS1RIK1-6; B indicates a western blot image of positive transgenic tomatoes in the T1 generation)

[0111] Depend on Figure 8 As can be seen in B, chemiluminescent signals can be detected in the root samples of OXSlRIK1-5 and OXSlRIK1-6, indicating that these two plants are positive transgenic lines.

[0112] 2) After obtaining the T1 generation seeds of OXSlRIK1-5 and OXSlRIK1-6, the seeds of OXSlRIK1-5, OXSlRIK1-6 and Moneymaker were planted on 1 / 2MS medium, each strain was a treatment, and each treatment was repeated 3 times. After 10 days, the roots of the seedlings were taken to extract RNA and reverse transcribed to obtain cDNA. The expression level of SlRIK1 was detected by qPCR. The expression results are shown in Tables 3 and Figure 8 C in Figure 8 In the figure, C represents the bar graph of the expression level of SlRIK1 in T2 generation positive transgenic tomatoes); the primers used for qPCR detection are SlRIK1_qPCR_FP: 5'-CATCTGCTTGATCTTG AACGAG-3' (SEQ ID NO.30), SlRIK1_qPCR_RP: 5'-GCTAACCCGAAATCTGACAGTT-3' (SEQ ID NO.31).

[0113] Table 3 Expression of SlRIK1 in different treatments

[0114] - OXS1RIK1-5 OXS1RIK1-6 Moneymaker Expression level (repeat 1) 6.86 12.01 1 Expression (repeat 2) 6.06 10.53 1 Expression level (repeat 3) 10.70 8.26 1

[0115] From Table 3 and Figure 8 As can be seen from C, the expression levels of OXSlRIK1-5 and OXSlRIK1-6 increased by 7 to 10 times compared with Moneymaker.

[0116] 3) T1 seeds of Moneymaker, OXS1RIK1-5 and OXS1RIK1-6 were planted in nutrient soil. When the tomatoes were 4 weeks old, 10 mL of Ralstonia solanacearum solution with OD600 = 0.1 was inoculated into the roots. The growth of Ralstonia solanacearum on the stems was counted 5 days after inoculation, and the disease index and incidence rate within 14 days after inoculation were counted. The statistical method was the same as in Example 3. The results are shown in Fig. 9 (exist Fig. 9 In the figure, A shows the photos of tomato disease after inoculation with Ralstonia solanacearum; B shows the bar graph of the growth of Ralstonia solanacearum on tomato stems 5 days after inoculation with Ralstonia solanacearum, N=8, pvalues ​​are 0.0004 and 0.0002, respectively, using one-way ANOVA statistical analysis; C shows the line graph of tomato disease index after inoculation with Ralstonia solanacearum, N=15; D shows the line graph of tomato survival rate after inoculation with Ralstonia solanacearum, N=15, pvalues ​​are <0.0001 and 0.0003, respectively, using log-rank test statistical analysis).

[0117] Depend on Fig. 9 It can be seen that the growth of R. solanacearum in the stems of OXSlRIK1-5 and OXSlRIK1-6 was significantly smaller than that of Moneymaker, the disease index was lower than that of Moneymaker, and the survival rate was higher than that of Moneymaker.

[0118] 4) The T1 seeds of Moneymaker, OXS1RIK1-5 and OXS1RIK1-6 were planted in nutrient soil to observe the developmental characteristics of tomatoes throughout the growth period. The results are shown in Fig.10 A( Fig.10 A represents the photos of tomato plants, immature fruits and mature fruits at 5 weeks of growth); the plant height of tomatoes was measured when they were 5 weeks old, and the results are shown in Fig.10 B( Fig.10 B represents the plant height data bar graph, N = 21, pvalues ​​are 0.9104 and 0.5207 respectively, using one-way ANOWA statistical analysis); the number of fruits per tomato plant is counted, and the results are shown in Fig.10 C( Fig.10 C represents the number of fruits, N = 11, pvalues ​​are 0.2202 and 0.9381, using one-way ANOWA statistical analysis); the weight of tomato fruits is counted, and the results are shown in Fig.10 D( Fig.10 D represents the fruit weight bar graph, N = 16, pvalues ​​are 0.8141 and 0.9996, respectively, using one-way ANOWA statistical analysis).

[0119] Depend on Fig.10It can be seen that there is no significant difference in plant height, fruit number, or fruit weight between OXSlRIK1-5, OXSlRIK1-6 and the wild-type Moneymaker.

[0120] In summary, the technical solution provided by the present invention can not only be used to regulate the disease resistance of tomatoes but also to cultivate disease-resistant tomatoes, that is, by inhibiting the expression of the SlRIK1 gene in the target tomatoes, tomatoes sensitive to bacterial wilt can be obtained; or by promoting the expression of the SlRIK1 gene in the target tomatoes, tomatoes resistant to bacterial wilt can be obtained, which provides a basis for cultivating disease-resistant tomatoes, is beneficial to reducing agricultural losses and improving economic benefits.

[0121] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of the SlRIK1 gene, the SlRIK1 protein encoded by the SlRIK1 gene or a biological material containing the SlRIK1 gene in regulating tomato disease resistance and / or cultivating disease-resistant tomatoes; the nucleotide sequence of the SlRIK1 gene is shown in SEQ ID NO.1; The disease resistance includes resistance to Ralstonia solanacearum ( Ralstonia solanacearum ) caused by diseases.

2. The use according to claim 1, characterized in that: The diseases include bacterial wilt.

3. The use according to claim 1, characterized in that: The regulation includes: promoting the expression of SlRIK1 gene or increasing the content of SlRIK1 protein to enhance the resistance of tomatoes to diseases; Either inhibit the expression of SlRIK1 gene or reduce the content of SlRIK1 protein to reduce the resistance of tomatoes to diseases.

4. The use according to claim 1, characterized in that: The biological material containing the S1RIK1 gene includes a biological material in which the expression of the S1RIK1 gene is suppressed and / or a biological material in which the S1RIK1 gene is overexpressed.

5. The use according to claim 4, characterized in that: The biological material for inhibiting the expression of the SlRIK1 gene includes: sgRNA, an expression vector containing the sgRNA, or an engineered bacterium containing the sgRNA; The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

3.

6. The use according to claim 4, characterized in that: The biological materials for overexpressing the SlRIK1 gene include: a recombinant expression vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the SlRIK1 gene.

7. The use according to claim 6, characterized in that: The basic vector of the recombinant expression vector includes pCAMBIA2300.

8. A method for growing tomatoes, characterized in that: The steps include: By inhibiting the expression of SlRIK1 gene in target tomatoes, a tomato susceptible to bacterial wilt was obtained; Or by promoting the expression of SlRIK1 gene in target tomatoes, to obtain tomatoes resistant to bacterial wilt; The nucleotide sequence of the S1RIK1 gene is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that The SlRIK1 gene in the target tomato is knocked out by sgRNA to inhibit the expression of the SlRIK1 gene in the target tomato; Alternatively, the SlRIK1 gene may be introduced into the target tomato to promote the expression of the SlRIK1 gene in the target tomato.

10. The method according to claim 8 or 9, characterized in that: The primers for amplifying the S1RIK1 gene include an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

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