Phenolamine and salicylic acid biosynthesized bifunctional gene cluster and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a difunctional gene cluster biosynthesized by phenol amine and salicylic acid and application of the difunctional gene cluster. The gene cluster comprises five genes, the five genes are S1EPS1, S1PBS3, S1CoAOMT2, S1CoOOMT3 and S1CoAOMT4, the nucleotide sequence of the S1EPS1 gene is as shown in SEQ ID No.1, the nucleotide sequence of the S1PBS3 gene is as shown in SEQ ID No.3, the nucleotide sequence of the S1CoAOMT2 gene is as shown in SEQ ID No.5, the nucleotide sequence of the S1CoOOMT3 gene is as shown in SEQ ID No.7, and the nucleotide sequence of the S1CoAOMT4 gene is as shown in SEQ ID No.9. The invention further discloses a preparation method of the gene cluster. The overexpressed gene cluster component SlEPS1 gene can significantly enhance accumulation of spermidine derivatives and salicylic acid of tomato plants so as to enhance the resistance to Pst DC3000, lays a foundation for genetic improvement in the aspect of tomato disease resistance by using the gene cluster, and also provides technical reference for excavation of related metabolic gene clusters in other crops and in the aspect of resistance breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a bifunctional gene cluster for biosynthesis of phenolamine and salicylic acid and application thereof. Background Art

[0002] Plants grow in a fixed position and cannot move. In order to adapt to the ever-changing environment around them, such as abiotic stresses such as drought, salinity, high temperature, ultraviolet radiation and cold damage, biotic stresses such as pests and diseases, animal bites, and human activities, plants produce a variety of metabolites. These metabolites are often not necessary for normal cell activities, but they are very important for plants to adapt to the environment. This process is called plant secondary metabolism, and these metabolites are collectively called plant secondary metabolites.

[0003] tomato( Solanum lycopersicum L. ) is a widely consumed vegetable crop worldwide and a key model organism for scientific research due to its agricultural significance. During the process of tomato domestication and improvement, the excessive emphasis on high yield has led to a significant increase in the susceptibility of modern cultivated tomatoes to pathogens including Pseudomonas syringae, which may result in reduced yield and quality. The ancestors of wild tomatoes (Solanum pimpinellifolium, PIM), cerasiforme (CER), and S. lycopersicum (BIG) are all tomato varieties and show significant genetic diversity. They have evolved complex chemical defense mechanisms involving multiple metabolites, including phenolamines and the phytohormone salicylic acid (SA), which play a key role in the plant's immune response against pathogens.

[0004] Phenolamines are conjugates of hydroxycinnamic acid derivatives and polyamines (PAs), which are biosynthesized by hydroxycinnamoyltransferases (HCTs), key enzymes in the initial stage of the phenolamine biosynthetic pathway. In addition, 159 phenolamines have been reported to have beneficial effects in plant defense against biotic stresses. However, research on the effects of phenolamines on tomato biotic stress resistance, especially tolerance to bacterial diseases, is still limited.

[0005] Salicylic acid (SA) is a key phytohormone in plant immunity and is essential for resistance to biopathogens such as P. syringae. Mutations in key genes involved in SA biosynthesis, including ICS1, PBS3, and EPS1, lead to increased susceptibility to P. syringae and characteristic impairments in SA synthesis. The benzoate pathway contributes relatively little to SA biosynthesis, accounting for only 10% of the total, and has been identified as independent of the phenylalanine pathway. In addition, SA not only activates local defense responses but also initiates systemic defense. SA plays an important role in the synthesis and metabolism of PAs, and pathogen infection can also activate PAs, leading to local SA accumulation and triggering systemic defense responses. Although these findings reveal the interaction between SA and PA in plant defense mechanisms, the synergistic regulatory mechanism with SA during biosynthesis needs to be further explored.

[0006] Therefore, it is of great significance to analyze the bifunctional gene cluster of phenolamine and salicylic acid biosynthesis and further explore its disease resistance mechanism. It can not only lay the foundation for subsequent research on the synthesis mechanism of tomato phenolamines, but also play a very important role in understanding the mechanism of plant adaptation to the environment and improving the yield and quality of crops under adverse conditions. Summary of the invention

[0007] The purpose of the present invention is to enrich the types of current plant disease resistance genes and substances, isolate and clone a bifunctional gene cluster BGC2 for the biosynthesis of spermidine derivatives and salicylic acid in tomatoes, and verify that the gene cluster is involved in the biosynthesis of phenolamines and salicylic acid by in vitro enzyme activity assay, tobacco transient expression and overexpression transgenic tomatoes, and that its products confer disease resistance to tomatoes. The present invention lays a foundation for genetic improvement of tomato disease resistance using the gene cluster.

[0008] In order to achieve the above object, the present invention provides the following technical solutions: The first aspect of the present invention provides a bifunctional gene cluster for the biosynthesis of phenolamines and salicylic acid, the gene cluster comprising five genes, the five genes being SlEPS1 , SlPBS3 , SlCoAOMT2 , SlCoOOMT3 and SlCoAOMT4 ,in, SlEPS1 The nucleotide sequence of the gene is shown in SEQ ID No. 1. SlPBS3 The nucleotide sequence of the gene is shown in SEQ ID No. 3. SlCoAOMT2 The nucleotide sequence of the gene is shown in SEQ ID No. 5. SlCoOOMT3 The nucleotide sequence of the gene is shown in SEQ ID No. 7, SlCoAOMT4 The nucleotide sequence of the gene is shown in SEQ ID No.9.

[0009] Furthermore, the SlEPS1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2. SlPBS3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.4. SlCoAOMT2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.6. SlCoOOMT3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.8. SlCoAOMT4 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.10.

[0010] The second aspect of the present invention provides the above gene cluster in improving tomato resistance PstDC3000 Application on pathogenic bacteria, wherein the application method is to overexpress gene cluster components SlEPS1 Gene, enhancer Pst DC3000 Resistance to pathogens.

[0011] Furthermore, the overexpressed gene cluster components SlEPS1 Gene, enhancer Pst DC3000 The ability to resist pathogens includes the following steps: (1) Extract total RNA from tomato flowers, reverse transcribe to obtain cDNA, use cDNA as template, and use primers SlEPS1 -F and SlEPS1 -R for PCR amplification; (2) The PCR product obtained in step (1) was connected to the pDonr207 entry vector through the BP reaction of the GATEWAY cloning technology, and the positive clones were screened and sequenced to obtain SlEPS1 The full-length cDNA of the gene was then linked into the overexpression vector pBI121 through the LR reaction of GATEWAY cloning technology to construct pBI- SlEPS1 Carrier; (3) The pBI- SlEPS1 The vector was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

[0012] Furthermore, the primer SlEPS1 -F and primers SlEPS1 The primer sequences for -R are as follows: SlEPS1 -F: 5'-AAAAAGCAGGCTTAATGACAAAACTTCGAGTAGTATCAG-3'; SlEPS1 -R: 5'-AGAAAGCTGGGTATTAAGCCGATTTCAATTTTTACTGAC-3'.

[0013] The third aspect of the present invention provides the components of the above gene cluster SlEPS1 Application of genes in enhancing the accumulation of spermidine derivatives and salicylic acid in tomato plants, wherein the application is to overexpress the genes in tomatoes SlEPS1 Gene.

[0014] Beneficial effects of the present invention: The present invention cloned a metabolic gene cluster controlling the biosynthesis of phenolamines and salicylic acid in tomatoes for the first time, and verified that the gene cluster was responsible for the biosynthesis of phenolamines and salicylic acid by means of in vitro enzyme activity assay, tobacco transient expression and tomato overexpression transgenic technology. The final products, spermidine derivatives and salicylic acid, can enhance the disease resistance of plants, laying a foundation for the genetic improvement of tomato resistance using the gene cluster, and also providing technical reference for the exploration of related metabolic gene clusters in other crops and resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Identification of the phenolamine and salicylic acid biosynthetic gene cluster BGC2: Figure 1 A in the figure shows the Manhattan plot of the GWAS results of diFer-Spd (n=401), the horizontal dashed line indicates the genome-wide significance threshold, and the upper part of the panel shows the genomic structure of the candidate metabolic gene cluster; Figure 1 Panel B shows the phylogenetic tree analysis of SlEPS1; Figure 1 Panel C shows tissue expression profile analysis of SlEPS1.

[0016] Figure 2 Preliminary verification of the components of the phenolamine and salicylic acid biosynthesis gene cluster BGC2: Figure 2 A in the figure shows the SDS-PAGE gel electrophoresis of prokaryotic protein expression SlEPS1 (76 kDa), SlCoAOMT2 (53 kDa), SlCoAOMT3 (53 kDa) and SlCoAOMT4 (53 kDa) and GST fusion protein after purification; Figure 2 Figure B shows the chromatogram showing the enzyme activity results of the reaction of the recombinant proteins SlEPS1, SlEPS1-mut and AtEPS1 with the donor Fer-CoA and the substrate Spd; Figure 2 Figure C shows that the chromatograms sequentially display the enzyme activity results of the reactions of the recombinant proteins SlCoAOMT2, SlCoAOMT3 and SlCoAOMT4 with the substrate Caf-CoA and the donor SAM.

[0017] Figure 3 Dual-function validation of the gene cluster component SlEPS1: Figure 3A in the figure shows the results of tobacco transiently expressing SlEPS1, SlPBS3, SlICS1, AtEPS1, AtPBS3 and AtICS1 to synthesize SA; Figure 3 B shows the results of tobacco transiently expressing SlEPS1 and AtEPS1 to synthesize Fer-Spd and diFer-Spd; Figure 3 C in the figure shows the metabolic pathway for the synthesis of phenolamines and SA.

[0018] Figure 4 The component genes of gene cluster BGC2 are expressed in different tissues and in Pst DC3000 Expression pattern after treatment: Figure 4 A in the figure shows the heat map of the expression distribution of each component gene of gene cluster BGC2 in different tissues; Figure 4 BGC2 component genes in the B display gene cluster SlEPS1 exist Pst DC3000 Induced expression at different time points after treatment; Figure 4 C display gene cluster BGC2 component genes SlCoAOMT2 exist Pst DC3000 Induced expression at different time points after treatment; Figure 4 D shows the BGC2 component genes in the gene cluster SlCoOOMT3 exist Pst DC3000 Induced expression at different time points after treatment; Figure 4 E displays the BGC2 component genes in the gene cluster SlCoAOMT4 exist Pst DC3000 Induced expression at different time points after treatment.

[0019] Figure 5 Construction and analysis of tomato overexpression and knockout lines of acyltransferase SlEPS1: Figure 5 A in the figure shows the expression level identification of the T2 generation SlEPS1 overexpression strain; Figure 5 B shows the analysis of SA content of metabolites in the T2 generation SlEPS1 overexpression strain; Figure 5 C shows the metabolite Fer-Spd and diFer-Spd analysis of the T2 generation SlEPS1 overexpression lines, the control variety is tomato Micro Tom, marked as WT; Figure 5 D in the figure shows the schematic diagram of the knockout structure of SlEPS1 in the T2 generation; Figure 5 E in the figure shows the analysis of the SA content of metabolites in the T2 generation SlEPS1 knockout line; Figure 5F in the figure shows the metabolite Fer-Spd and diFer-Spd analysis of the T2 generation SlEPS1 knockout line. The control variety is tomato Micro Tom, marked as WT, and the standard deviation is based on three biological replicates. Each sample is a mixed sample of 3 materials. The significance analysis is represented by *P<0.05, **P<0.01 and ***P<0.001 (t-test).

[0020] Figure 6 For T2 generation SlEPS1 Gene overexpression and knockout inoculation Pst DC3000 Post-phenotype, colony count and in vitro antibacterial analysis of phenolamine metabolites: Figure 6 Figures A and C in the figure are SlEPS1 Gene overexpression and knockout lines Pst DC3000 The phenotype after Figure 6 Figures B and D in the figure are SlEPS1 Statistics of colony numbers after inoculation of gene overexpression lines and knockout lines, standard deviation based on three biological replicates; Figure 6 Figures E and F show Pst DC3000 The situation and statistics of Fer-Spd culture at different concentrations. Pst DC3000 Bacteria were cultured in R2A medium supplemented with 0, 1, 2, or 4 mM Fer-Spd; Figure 6 Figures G and H in the figure show the different concentrations of Fer-Spd. Pst DC3000 Four Fer-Spd concentrations were tested for growth inhibition in King B agar medium: 0, 1, 2, and 4 mM Fer-Spd. The control variety was tomato Micro Tom, marked as WT. The significance analysis was indicated as *P<0.05, **P<0.01, and ***P<0.001 (t-test), respectively. DETAILED DESCRIPTION

[0021] The following examples further define the present invention and describe the genes (including SlEPS1, SlPBS3, SlCoAOMT2, SlCoOOMT3 and SlCoAOMT4) contained in the metabolic gene cluster for separating phenolamine and salicylic acid biosynthetic pathway, in vitro enzyme activity assay, tobacco transient expression, genetic transformation and application in disease resistance. Based on the following description and these examples, those skilled in the art can determine the basic characteristics of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for a variety of uses and conditions.

[0022] Example 1: Discovery and definition of the phenolamine and salicylic acid bifunctional gene cluster To elucidate the genetic basis of natural variation in tomato phenolamines, the present inventors detected the levels of these compounds in leaves of 401 core tomato varieties, including 258 BIG, 115 CER, and 28 PIM varieties. During domestication and improvement, the abundance of most phenolamines gradually decreased. Subsequently, a genome-wide association study (GWAS) was performed using 2,318,614 single nucleotide polymorphisms (SNPs) with a minor allele frequency above 0.05, identifying a SNP on chromosome 2 that was significantly associated with diFer-Spd variation (SF0254126808, P = 0.09 × 10 -8 )( Figure 1 A). Four genes related to phenolamine content near this SNP were identified, including three methyltransferases (Solyc02g093230, SlCoAOMT2 ;Solyc02g093250, SlCoAOMT3 ; and Solyc02g93270, SlCoAOMT4 ); and an acyltransferase gene ( SlEPS1 , Solyc02g093180)( Figure 1 A), highly homologous to the acyltransferase EPS1 involved in salicylic acid biosynthesis in Arabidopsis thaliana, and classified into the 1B branch ( Figure 1 B). In addition, SlEPS1 Genes related to the salicylic acid biosynthesis pathway were observed 324 kb upstream ( SlPBS3 , Solyc02g092820)( Figure 1 A). This feature meets the definition of plant gene clusters, so the inventors named this metabolic gene cluster "phenolamine and salicylic acid biosynthesis gene cluster BGC2" based on its final products being phenolamine and salicylic acid.

[0023] Example 2: Genes of each component of the phenolamine and salicylic acid biosynthetic gene cluster ( SlEPS1 , SlPBS3 , SlCoAOMT2 , SlCoOOMT3 and SlCoAOMT4 ) cloning and protein expression vector construction The present invention technicians used TRIZOL reagent (purchased from Invitrogen) to extract total RNA from different tissues of the sequenced tomato variety Micro Tom (the extraction method was based on the TRIZOL reagent instructions), and used the reverse transcription kit Supermix (purchased from Beijing Quanshijin Company) to reverse transcribe the RNA into cDNA, with the reaction conditions of 42°C for 30min and 80°C for 5sec. qRT-PCR was further used to detect SlEPS1 The expression levels in different tissues were found to be higher in flowers and leaves. SlEPS1, SlPBS3 , SlCoAOMT2 , SlCoOOMT3 and SlCoAOMT4 The coding sequence of the gene, the present technicians used the flower cDNA as a template, and according to the location and structure of these genes in the tomato genome (tomato genome database SGN), predicted the full-length open reading frame sequence of these candidate genes, and designed PCR specific primers with homologous recombination adapters (Table 1, synthesized by Qingke Biological Company) using ultra-fidelity KOD enzyme (Dalian Baoriyi Bioengineering Co., Ltd.) to amplify the full-length CDS fragments of the above 5 genes. PCR reaction conditions: 94℃ pre-denaturation for 5min; 98℃ 10sec, 60℃ 30sec, 68℃ 2min, 32 cycles; 68℃ extension for 5min. The amplified PCR product was connected to the pDonr207 entry vector through the BP reaction of GATEWAY cloning technology, and transformed into Escherichia coli DH5α competent cells. Colony PCR verification was performed to obtain positive clones, and sequencing was performed to obtain correct clones without mutations containing candidate genes (Haikou Qingke Biotechnology Co., Ltd.), and entry vectors of pDonr207-SlEPS1, SlPBS3, SlCoAOMT2, SlCoOOMT3 and SlCoAOMT4 genes were obtained respectively, and then connected into the protein expression vector pGEX-6P-1 vector (Invitrogen, USA) through the LR reaction of GATEWAY cloning technology.

[0024] Table 1 Primers used in constructing protein expression vectors in the present invention

[0025] Example 3: Components of the phenolamine and salicylic acid biosynthetic gene clusters ( SlEPS1 , SlCoAOMT2 , SlCoOOMT3 and SlCoAOMT4 ) The protein expression vector construction method is as follows: first, the positive clone pGEX-6p-1-SlEPS1, SlCoAOMT2, SlCoOOMT3 and SlCoAOMT4 plasmids obtained in Example 2 were respectively transformed into Escherichia coli competent cells BL21 (Shanghai Weidi Biological), and the Escherichia coli was induced to express the proteins encoded by the candidate genes.

[0026] Specific steps: (1) E. coli culture: The plasmid containing the target gene fragment is transformed into E. coli BL21 competent cells, and a single colony is picked and inoculated into 5 ml LB medium containing 50 μg / ml ampicillin antibiotics, and cultured at 37°C overnight. The next day, the bacterial solution is inoculated into 250 ml LB medium containing 50 μg / ml ampicillin antibiotics at a ratio of 1:50, and cultured at 37°C and 220 rpm for 3 hours until the OD600 of the bacterial solution is about 0.5. Then, 1 mol / L IPTG (isopropylthiogalactoside, Shanghai Sangon Co., Ltd.) is added to the bacterial solution to induce protein expression. After adding IPTG, the bacterial solution is placed in a shaking incubator at 16°C and 160 rpm for overnight culture. (2) E. coli collection: The cells are collected in a 500 ml centrifuge tube and centrifuged at 4°C and 5000 rpm for 8 minutes. The supernatant is discarded, and 50 ml Lysis buffer is added to suspend the cells and placed on ice. Use a high-pressure cell disruptor (Suzhou Antos Nanotechnology Co., Ltd.) to disrupt the suspended bacteria. (3) SDS-PAGE: The disrupted bacteria are always kept on an ice-water mixture. Take 20ul of total protein and add 5ul of 5×SDS Loading buffer. Take another 1ml of total protein and centrifuge at 12000rpm and 4℃ for 10min to separate the supernatant and precipitate. Take 20ul of supernatant and add 5ul of 5×SDS Loading buffer. Heat in a dry bath at 100℃ for 10min. After the heated sample is cooled to room temperature, take 15ul and spot it for SDS-PAGE detection to see if there is target protein in the total protein and supernatant. The remaining protein is stored at -80℃ for later use. (4) Protein purification: Use Lysis buffer to fully wash the purification column containing glutathione agarose 4B agarose (GE Healthcare, USA), add the supernatant containing the target protein, collect the effluent, and pass it through the column again. Wash the column fully with Lysis buffer (5 times column volume). After sufficient washing, add 15mmol of reduced glutathione eluent (Shanghai Bioengineering Co., Ltd.) to the purification column, add 1ml each time, and collect 1ml of the flow-through solution and repeat 5-6 times. Confirm whether the collected flow-through solution contains the target protein by SDS-PAGE. The inventors successively obtained well-purified SlEPS1, SlCoAOMT2, SlCoOOMT3 and SlCoAOMT4 protein fusion GST tag protein recombinant proteins, and stored the above proteins in a -80℃ refrigerator for use ( Figure 2 A).

[0027] Example 4: Phenolamine and salicylic acid biosynthetic gene cluster components ( SlEPS1 , SlPBS3 , SlCoAOMT2 , SlCoOOMT3 and SlCoAOMT4 In vitro enzyme activity assay 1. In vitro enzyme activity assay of SlEPS1 protein In order to verify the function of the candidate gene SlEPS1, the inventors used the above-mentioned purified SlEPS1 protein for in vitro enzyme activity assay. The in vitro enzyme activity reaction system of SlEPS1 protein is 10ul, 1 mol / L Tris-HCl buffer (pH 7.4) 1ul, 50 mmol / L MgCl2 1ul, 1 mmol / L Fer-CoA 1ul, 5 mmol / L Spd 1ul and 500ng purified protein, ddH2O is made up to 10ul, incubated at 37°C for 30 min, and 50ul pre-cooled methanol is added to terminate the reaction. The reaction mixture was centrifuged at 4°C and 12000rpm for 10min, and 50ul of the supernatant was taken for LC-MS detection and analysis. The LC-MS detection results showed that Fer-Spd, diFer-Spd and triFer-Spd were detected in the reaction products of SlEPS1 protein, indicating that the enzyme encoded by the SlEPS1 gene is responsible for the most critical catalytic reaction ( Figure 2 B).

[0028] 2. In vitro enzyme activity assay of SlCoAOMT2, SlCoAOMT3 and SlCoAOMT4 proteins To validate candidate genes SlCoAOMT2 , SlCoAOMT3 and SlCoAOMT4 The inventors used the purified SlCoAOMT2, SlCoAOMT3 and SlCoAOMT4 recombinant proteins to perform in vitro enzyme activity assays. The in vitro enzyme activity reaction system of methyltransferase is 10ul, containing 5 mmol / L methyl donor SAM 1ul, 1 mmol / L Caf-CoA 1ul, 500ng purified protein, 1 mol / L Tris-HCl buffer (pH 7.4) 1ul and 50 mmol / L MgCl2 1ul, ddH2O is made up to 10ul, incubated at 30°C for 30min, and 50ul pre-cooled methanol is added to terminate the reaction. The reaction mixture is centrifuged at 4°C and 12000rpm for 10min, and 50ul of the supernatant is taken for LC-MS detection and analysis. The results of LC-MS detection showed that Fer-CoA was detected in the reaction products of SlCoAOMT2, SlCoAOMT3 and SlCoAOMT4 proteins, indicating that these three enzymes have the function of catalyzing the synthesis of Fer-CoA from Caf-CoA ( Figure 2 C).

[0029] Example 5: Phenolamine and salicylic acid biosynthetic gene cluster components ( SlEPS1 and SlPBS3 ) in tobacco transient expression In order to further verify the functions of the gene cluster components SlEPS1 and SlPBS3. The present technicians connected the pDonr207-SlEPS1 and SlPBS3 obtained in Example 2 to pJF754-pEAQ-SlEPS1 and SlPBS3 through the LR reaction of GATEWAY cloning technology. After the plasmid sequencing was correct, it was used for transient transformation of tobacco. pEAQ-HT containing the candidate tomato gene or GFP (negative control) was transformed into Agrobacterium tumefaciens (GV3101) and cultured at 30°C for 3 days on LB plates containing 50 mg / mL kanamycin and 30 mg / mL gentamicin. Positive clones were screened and plated on LB plates containing 50 μg·ml -1 Cultures were grown in Luria-Bertani medium (Luria-Bertani, 10 ml) with kanamycin to an OD600 of 2.0, washed with wash buffer (10 mM MES, pH 5.6), and resuspended in MMA buffer (10 mM MES (pH 5.6), 10 mM magnesium chloride, 100 mM acetosyringone) to an OD600 of 1.0. The cultures were incubated at room temperature for 2 h, and 1 ml of the culture was infiltrated into the underside of 6-week-old Nicotiana benthamiana leaves using a 1 ml needleless syringe. Leaves from different plants (n=3) were harvested 3 days after infiltration, frozen in liquid nitrogen, and stored at -80°C. Metabolite analysis revealed that the expression of AtEPS1, AtPBS3, SlEPS1, or SlPBS3 alone did not lead to the synthesis of SA ( Figure 3 A). When AtICS1-AtPBS3, AtPBS3-AtEPS1, SlICS1-SlPBS3, or SlPBS3-SlEPS1 were co-expressed, a slight increase in SA levels was observed ( Figure 3 However, co-expression of SlICS1-SlPBS3-SlEPS1 resulted in significant accumulation of SA, which was consistent with the results of AtICS1-AtPBS3-AtEPS1 ( Figure 3 A). These experiments in tobacco highlight the key role of SlEPS1 in SA biosynthesis. We further detected the phenolamine content of AtEPS1 and SlEPS1, respectively, and found that SlEPS1 can couple Fer-CoA and Spd, leading to the accumulation of Fer-Spd, diFer-Spd, and triFer-Spd ( Figure 3 B). In contrast, AtEPS1 showed no such activity, supporting the theory that SlEPS1 plays a dual role in phenolamine and salicylic acid biosynthesis ( Figure 3 C).

[0030] Example 6: Analysis of expression patterns of phenolamine and salicylic acid biosynthetic gene cluster components The present invention technicians used the cDNA from different tissues to perform qRT-PCR to detect the expression level of each component and found that the genes of each component were highly co-expressed mainly in leaves. Pst DC3000 To determine whether it would induce the expression of spermidine derivatives and salicylic acid gene cluster components, the present invention technicians conducted a test on Micro tom tomato seedlings that were about 4 weeks old. Pst DC3000 Treatment, tomato leaf tissues were sampled at different time points after 48 h of treatment to extract total RNA.

[0031] The total RNA extracted above was reverse transcribed. The specific steps of reverse transcription are as follows: 3-5 g of total RNA was treated with DNaseI (Invitrogen, USA) for 15 min to remove genomic DNA contamination, and reverse transcription was performed using oligo(dT)18 oligo primer and M-MLV reverse transcriptase (Promega, USA). Real-time quantitative PCR analysis kit Green PCRMaster Mix (Takara Biotechnology Dalian Co., Ltd.) was used according to the instructions of the kit to perform real-time quantitative PCR reaction on the ABI 7500 RealTime PCR system instrument (Applied Biosystems, USA). Gene expression was quantified using the relative quantitative method. The expression level of the tomato housekeeping gene Ubiquitin3 gene was used to measure and normalize the sample RNA content.

[0032] qRT-PCR analysis showed that all four components of the spermidine derivative and salicylic acid gene cluster were affected. Pst DC3000 Different degrees of induced expression, with the highest level at 8h ( Figure 4 BE), indicating that these four component genes may be involved in the defense process of tomato against pathogens.

[0033] The specific PCR primers for the five component genes in the qRT-PCR analysis are shown in Table 2 .

[0034] Table 2 Primers used for fluorescence quantification in the present invention

[0035] Example 7: Phenolamine and salicylic acid biosynthetic gene cluster components ( SlEPS1 ) Construction and genetic transformation of overexpression transgenic materials and knockout line materials, as well as expression and metabolite analysis 1. Construction of transgenic plants overexpressing SlEPS1 To further verify SlEPS1The present invention technicians connected the pDonr207-SlEPS1 obtained in Example 2 to pBI- SlEPS1 After the plasmid was sequenced correctly, it was used for genetic transformation of tomatoes. First, it was introduced into the tomato variety Micro Tom through the tomato genetic transformation system mediated by Agrobacterium LBA4404 (the strain was from Shanghai Weidi Biological Company). After pre-cultivation, infection, co-cultivation, screening of callus with kanamycin resistance, differentiation, rooting, transplantation, and identification, transgenic plants were obtained.

[0036] 2. Transgenic plants SlEPS1 Gene expression analysis and metabolite analysis Real-time quantitative PCR analysis was used to detect the expression of the gene in the plant. The overexpressed plants were propagated to obtain T2 transgenic plants. The results showed that compared with the control wild-type (non-transgenic tomato) Micro Tom, the expression of the gene in the positive transformed plants was significantly higher than that in the wild-type (non-transgenic tomato) Micro Tom. SlEPS1 The gene expression was significantly increased ( Figure 5 A).

[0037] In order to further verify the function of candidate genes in regulating metabolite synthesis in tomatoes, the inventors selected strains with higher overexpression multiples from the T2 generation of SlEPS1 transgenic materials and performed LC-MS detection and analysis on tomato leaves. The test results showed that compared with wild-type materials, SlEPS1 In the overexpression plants, the contents of SA, Fer-Spd and diFer-Spd were significantly increased, indicating that SlEPS1 The gene is mainly responsible for the synthesis of SA, Fer-Spd and diFer-Spd in tomatoes ( Figure 5 B and C).

[0038] 3. Construction of SlEPS1 knockout transgenic plants In order to further verify the function of the SlEPS1 gene. The present technicians used CRISPR-Ca9 technology to construct a transgenic knockout vector of SlEPS1, and the plasmid was used for tomato genetic transformation after correct sequencing. First, it was introduced into the tomato variety Micro Tom through the tomato genetic transformation system mediated by Agrobacterium LBA4404 (the strain is from Shanghai Weidi Biological Company), and after pre-cultivation, infection, co-cultivation, screening of callus with kanamycin resistance, differentiation, rooting, transplanting, and identification, transgenic plants were obtained.

[0039] 4. Transgenic plants SlEPS1 Gene sequencing analysis and metabolite analysis DNA was extracted from the transgenic plants and PCR analysis was used to detect the editing of genes in the plants. The positive knockout plants were propagated to obtain T2 transgenic plants ( Figure 5 D).

[0040] In order to further verify the function of candidate genes in regulating metabolite synthesis in tomatoes, the inventors selected SlEPS1 The knockout strains in the T2 generation of transgenic materials were tested and analyzed by LC-MS in tomato leaves. The test results showed that compared with wild-type materials, SlEPS1 In the knockout lines of the gene, the contents of SA, Fer-Spd, and diFer-Spd were significantly reduced, indicating that SlEPS1 The gene is mainly responsible for the synthesis of SA, Fer-Spd and diFer-Spd in tomatoes ( Figure 5 E and F).

[0041] Example 8: Disease resistance analysis of acyltransferase SlEPS1 gene overexpression transgenic strains and knockout strains and in vitro antibacterial analysis of metabolites To validate candidate genes SlEPS1 Whether it can improve the disease resistance of tomatoes, the researchers of the present invention studied the effect of SlEPS1 Genetically modified material Pst DC3000 deal with.

[0042] Pst DC3000 Inoculation experiment: Pst DC3000 The strain was inoculated on King's B solid medium containing 25 mg / L rifampicin and cultured at 28°C for 2 days. A single colony was selected and inoculated into King's B liquid medium containing 25 mg / L rifampicin and cultured at 28°C and 200 rpm for 8 to 12 hours. After that, centrifuge at 4000g for 5 minutes at 4°C, discard the supernatant, wash twice with 10 mmol / L MgCl2 solution and resuspend, and adjust the bacterial concentration to OD600 = 0.05. The bacterial suspension containing 0.03% silicone oil was evenly sprayed on tomato leaves as the treatment group, while the control group was sprayed with 10 mmol / L MgCl2 solution containing 0.03% silicone oil. After inoculation Pst DC3000 Tissue samples were collected at 0, 2, 4, 8, 12, 24, and 48 hours after the experiment, and total RNA was extracted for qRT-PCR analysis. The experiment was repeated at least three times, and similar results were obtained each time.

[0043] Colony count: On the 3rd and 4th day after inoculation with Pst DC3000, fully expanded functional leaves were randomly collected using a cork punch, sterilized in 70% v / v ethanol for 10 seconds, washed twice with sterile water, and thoroughly ground in 500 μL of 10 mmol / L MgCl2 solution. Three biological replicates were set for each treatment. The ground solution was then stirred for 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Serial dilutions were made and 10 μL of the original liquid and the dilutions were spotted on Kings' B solid medium containing 25 mg / L rifampicin. The plates were incubated at 28°C for 2 days. Each dilution concentration was replicated three times.

[0044] The specific operation of the in vitro liquid antibacterial test is as follows: select a PstDC3000 Colonies were cultured overnight at 28°C and 180 rpm in R2A medium, and then centrifuged at 600g for 10 minutes at 4°C to collect the bacteria. The collected bacteria were resuspended in R2A medium to OD600 = 0.01, and then sterile 1M Fer-Spd solution (dissolved in ethanol and filtered through a 0.22μm filter) was added to the culture medium to achieve different concentrations. As a control, an equal volume of ethanol solvent (filtered through a 0.22μm filter) was added. 0, 1, 2, and 4mM Fer-Spd were added to the R2A medium to observe the effect of Fer-Spd on Pst DC3000 Effects on cell growth.

[0045] The specific operation of the in vitro solid antibacterial test is as follows: the previously sterilized agar medium is heated until it is completely dissolved. After the medium is cooled to about 50°C, 1% of OD600 = 0.01 is added. PstDC3000 Resuspended liquid, mixed thoroughly, poured evenly into the culture dish, 15mL per dish and allowed to solidify, Oxford cup placed vertically on the surface of the culture medium, gently pressed to ensure close contact with the culture medium without leaving gaps. Add different concentrations of Fer-Spd solution to the Oxford cup, 200μL per cup, ensuring no overflow, and incubated at 37℃ for 16-18 hours.

[0046] The results showed that vaccination Pst DC3000 On the 3rd and 4th days after PCR, compared with the wild-type control, SlEPS1 The overexpression lines of the gene showed a strong disease resistance phenotype ( Figure 6 A). Colony counts showed that the number of lesions on the leaves of tomato overexpression plants after inoculation was much smaller than that on the leaves of wild-type tomato plants, indicating stronger disease resistance ( Figure 6 B) Compared with the wild-type plant control, SlEPS1The knockout strains of the gene showed a strong disease phenotype ( Figure 6 C). Colony counts showed that the number of lesions on the leaves of the knockout tomato plants after inoculation was much higher than that on the leaves of the wild-type tomato plants, indicating that the disease resistance was weaker ( Figure 6 D) The results of in vitro antibacterial experiments of Fer-Spd show that a specific concentration of Fer-Spd can effectively inhibit PstDC3000 When the concentration of Fer-Spd reached 2 mM, PstDC3000 The growth of Figure 6 EH).

[0047] Combined with the above results, it is shown that overexpression of key components of the phenolamine and salicylic acid biosynthesis gene clusters SlEPS1 The gene can significantly enhance the accumulation of spermidine derivatives and salicylic acid in tomato plants, thereby enhancing Pst DC3000 Therefore, this approach can be used to breed disease-resistant tomatoes.

[0048] The present invention is described in detail above. Definitions of Terms Related to the Present Invention Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

[0049] The term "protein" is used interchangeably herein to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0050] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0051] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0052] For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application, and the changes made with conventional techniques known in the art.

Claims

1. A bifunctional gene cluster for phenolamine and salicylic acid biosynthesis, characterized in that: The gene cluster includes five genes, which are SlEPS1 , SlPBS3 , SlCoAOMT2 , SlCoOOMT3 and SlCoAOMT4 ,in, SlEPS1 The nucleotide sequence of the gene is shown in SEQ ID No.

1. SlPBS3 The nucleotide sequence of the gene is shown in SEQ ID No.

3. SlCoAOMT2 The nucleotide sequence of the gene is shown in SEQ ID No.

5. SlCoOOMT3 The nucleotide sequence of the gene is shown in SEQ ID No. 7, SlCoAOMT4 The nucleotide sequence of the gene is shown in SEQ ID No.

9.

2. The gene cluster according to claim 1, characterized in that Said SlEPS1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

2. SlPBS3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

4. SlCoAOMT2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

6. SlCoOOMT3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

8. SlCoAOMT4 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

10.

3. The gene cluster according to claim 1 or 2 is useful in improving tomato resistance PstDC3000 The application on pathogenic bacteria is characterized by: The method of application is to overexpress gene cluster components SlEPS1 Gene, enhancer Pst DC3000 Resistance to pathogens.

4. The use according to claim 3, characterized in that: The overexpressed gene cluster components SlEPS1 Gene, enhancer Pst DC3000 The ability to resist pathogens includes the following steps: (1) Extract total RNA from tomato flowers, reverse transcribe to obtain cDNA, use cDNA as template, and use primers SlEPS1 -F and SlEPS1 -R for PCR amplification; (2) The PCR product obtained in step (1) was connected to the pDonr207 entry vector through the BP reaction of the GATEWAY cloning technology, and the positive clones were screened and sequenced to obtain SlEPS1 The full-length cDNA of the gene was then linked into the overexpression vector pBI121 through the LR reaction of GATEWAY cloning technology to construct pBI- SlEPS1 Carrier; (3) The pBI- SlEPS1 The vector was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

5. The use according to claim 4, characterized in that: The primer SlEPS1 -F and primers SlEPS1 The primer sequences for -R are as follows: SlEPS1 -F:5'-AAAAAGCAGGCTTAATGACAAAACTTCGAGTAGTATCAG-3'; SlEPS1 -R:5'-AGAAAGCTGGGTATTAAGCGATTTCAATTTTTACTGAC-3'。 6. Components of the gene cluster according to claim 1 or 2 SlEPS1 The use of a gene for enhancing the accumulation of spermidine derivatives and salicylic acid in tomato plants is characterized in that: The application method is to overexpress in tomato SlEPS1 Gene.

Images