A bifunctional gene cluster for the biosynthesis of phenol amide and salicylic acid and its application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a bifunctional gene cluster for the biosynthesis of phenylethylamine and salicylic acid and its application. The gene cluster includes five genes, and the five genes are SlEPS1 , SlPBS3 , SlCoAOMT2 , SlCoOOMT3 and SlCoAOMT4 , and 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. Overexpression of the gene cluster component SlEPS1 gene can significantly enhance the accumulation of spermidine derivatives and salicylic acid in tomato plants, thereby enhancing the resistance to Pst DC3000 , which lays a foundation for the genetic improvement of tomato disease resistance by using this gene cluster, and also provides technical reference for the excavation of related metabolic gene clusters in other crops and in 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 phenolamines and salicylic acid and applications thereof. Background Art

[0002] Plants are sessile and immobile. To adapt to their ever-changing environments, such as abiotic stresses like drought, salinity, high temperatures, ultraviolet radiation, and chilling damage, biotic stresses like pests and diseases, and animal predation, as well as human activities, plants produce a variety of metabolites. These metabolites are often not essential for normal cellular activity, but are nonetheless crucial for plant adaptation. This process is called plant secondary metabolism, and these metabolites are collectively referred to as plant secondary metabolites.

[0003] tomato( SolanumlycopersicumL. ) is a widely consumed vegetable crop worldwide and a key model organism for scientific research due to its agricultural significance. During tomato domestication and improvement, an overemphasis on high yield has led to a significant increase in the susceptibility of modern cultivated tomatoes to pathogens, including Pseudomonas syringae, which can result in reduced yield and quality. The wild tomato (Solanum pimpinellifolium, PIM), cerasiforme (CER), and S. lycopersicum (BIG), all descended from tomato varieties and exhibiting significant genetic diversity, 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). These polyamines are biosynthesized by hydroxycinnamoyltransferases (HCTs), key enzymes in the initial stage of the phenolamine biosynthesis pathway. Furthermore, 159 phenolamines have been reported to play beneficial roles in plant defense against biotic stresses. However, research on the effects of phenolamines on tomato biotic resistance, particularly tolerance to bacterial diseases, remains limited.

[0005] Salicylic acid (SA) is a key plant hormone in plant immunity and is essential for resistance to pathogens 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 determined to be independent of the phenylalanine pathway. Furthermore, SA not only activates local defense responses but also initiates systemic defenses. 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. While these findings reveal the interaction between SA and PAs in plant defense mechanisms, the synergistic regulatory mechanism with SA during biosynthesis requires further exploration.

[0006] Therefore, it is of great significance to analyze the bifunctional gene cluster for phenolamine and salicylic acid biosynthesis and further explore its disease resistance mechanism. It will 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 present invention aims to enrich the current pool of plant disease resistance genes and substances. The authors isolated and cloned a bifunctional gene cluster, BGC2, involved in the biosynthesis of spermidine derivatives and salicylic acid in tomatoes. Using in vitro enzyme activity assays, transient expression in tobacco, and overexpression in transgenic tomatoes, the authors verified that this gene cluster participates in the biosynthesis of phenolamines and salicylic acid, and that its products confer disease resistance in tomatoes. This invention lays the foundation for utilizing this gene cluster for genetic improvement of tomato disease resistance.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] 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.

[0010] 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.

[0011] The second aspect of the present invention provides the above gene cluster in improving tomato resistance PstDC3000 Application on pathogenic bacteria, wherein the application approach is to overexpress gene cluster components SlEPS1 Gene, enhancer Pst DC3000 Ability to resist pathogens.

[0012] Furthermore, the overexpressed gene cluster components SlEPS1 Gene, enhancer Pst DC3000 The ability to resist pathogens includes the following steps:

[0013] (1) Extract the total RNA of tomato flowers, reverse transcribe to obtain cDNA, use cDNA as template, and use primers SlEPS1 -F and SlEPS1 -R for PCR amplification;

[0014] (2) The PCR product obtained by amplification 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 the GATEWAY cloning technology to construct the pBI- SlEPS1 carrier;

[0015] (3) The pBI- SlEPS1 The vector was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

[0016] Furthermore, the primer SlEPS1 -F and primers SlEPS1 The primer sequences for -R are as follows:

[0017] SlEPS1 -F: 5'-AAAAAGCAGGCTTAATGACAAAACTTCGAGTAGTATCAG-3';

[0018] SlEPS1 -R: 5'-AGAAAGCTGGGTATTAAGCGATTTCAATTTTTACTGAC-3'.

[0019] 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 achieved by overexpression in tomatoes SlEPS1 Gene.

[0020] Beneficial effects of the present invention:

[0021] This invention cloned a metabolic gene cluster that controls the biosynthesis of phenolamines and salicylic acid in tomatoes for the first time. In vitro enzyme activity assays, tobacco transient expression, and tomato overexpression transgenic methods verified that this gene cluster is responsible for the biosynthesis of phenolamines and salicylic acid. Its final products, spermidine derivatives and salicylic acid, can enhance the plant's disease resistance. This lays the foundation for using this gene cluster for genetic improvement of tomato resistance, and also provides technical reference for the discovery of related metabolic gene clusters in other crops and for resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Identification of the phenolamine and salicylic acid biosynthesis 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. 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.

[0023] Figure 2 Preliminary verification of the components of the phenolamine and salicylic acid biosynthesis gene cluster BGC2: Figure 2 A 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 Panel B shows the chromatogram showing the enzyme activity results of the reaction of recombinant proteins SlEPS1, SlEPS1-mut and AtEPS1 with donor Fer-CoA and substrate Spd; Figure 2 Figure C shows that the chromatograms sequentially display the enzyme activity results of the recombinant proteins SlCoAOMT2, SlCoAOMT3 and SlCoAOMT4 reacting with the substrate Caf-CoA and the donor SAM.

[0024] Figure 3 Dual-function validation of the gene cluster component SlEPS1: Figure 3 Panel A shows the results of SA synthesis by transiently expressing SlEPS1, SlPBS3, SlICS1, AtEPS1, AtPBS3, and AtICS1 in tobacco; Figure 3 Panel B shows the results of transient expression of SlEPS1 and AtEPS1 in tobacco to synthesize Fer-Spd and diFer-Spd; Figure 3 Figure C shows the metabolic pathway for the synthesis of phenolamines and SA.

[0025] 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 display gene cluster BGC2 component genes SlCoOOMT3 exist Pst DC3000 Induced expression at different time points after treatment; Figure 4 E displays gene cluster BGC2 component genes SlCoAOMT4 exist Pst DC3000 Induction expression at different time points after treatment.

[0026] Figure 5 Construction and analysis of tomato overexpression and knockout lines of the acyltransferase SlEPS1: Figure 5 A in the figure shows the expression level identification of T2 generation SlEPS1 overexpression lines; Figure 5 B shows the analysis of SA content of metabolites in the T2 generation SlEPS1 overexpression strain; Figure 5 Figure C shows the metabolite Fer-Spd and diFer-Spd analysis of the T2 generation SlEPS1 overexpression line, with the tomato variety Micro Tom as the control, marked as WT; Figure 5 D in the figure shows the schematic diagram of the T2 generation SlEPS1 knockout structure; Figure 5 Panel E shows the analysis of SA content of metabolites in the T2 generation SlEPS1 knockout line; Figure 5Figure F shows the metabolite Fer-Spd and diFer-Spd analysis of the T2 generation SlEPS1 knockout line. The control variety was tomato Micro Tom, marked as WT. The standard deviation was based on three biological replicates. Each sample was a mixture of three materials. The significance analysis was represented by *P<0.05, **P<0.01, and ***P<0.001 (t-test).

[0027] Figure 6 T2 generation SlEPS1 Gene overexpression and knockout line inoculation Pst DC3000 Post-incubation phenotype, colony counts, and in vitro antibacterial analysis of phenolamine metabolites: Figure 6 Figures A and C in the figure are SlEPS1 Inoculation of gene overexpression and knockout lines Pst DC3000 The phenotype after Figure 6 Figures B and D in the figure are SlEPS1 The colony counts of gene overexpression and knockout lines after inoculation were statistically analyzed, and the standard deviation was 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 show the effects of different concentrations of Fer-Spd. Pst DC3000 Growth inhibition effect in King B agar medium was tested at four Fer-Spd concentrations: 0, 1, 2, and 4 mM Fer-Spd. The control variety was tomato Micro Tom, marked as WT. Significance analysis is indicated as *P<0.05, **P<0.01, and ***P<0.001 (t-test), respectively. DETAILED DESCRIPTION

[0028] The following examples further define the present invention and describe the genes contained in the metabolic gene cluster for the biosynthetic pathways of phenolamines and salicylic acid (including SlEPS1, SlPBS3, SlCoAOMT2, SlCoOOMT3, and SlCoAOMT4), as well as their use in in vitro enzyme activity assays, transient expression in tobacco, genetic transformation, and disease resistance. Based on the following description and these examples, those skilled in the art will be able to ascertain the essential features of the present invention and, without departing from the spirit and scope of the invention, may make various changes and modifications to adapt the invention to a variety of uses and conditions.

[0029] Example 1: Discovery and definition of a bifunctional gene cluster for phenolamines and salicylic acid

[0030] To elucidate the genetic basis of natural variation in tomato phenolamines, the present inventors measured the levels of these compounds in leaves of 401 core tomato varieties, including 258 BIG, 115 CER, and 28 PIM varieties. The abundance of most phenolamines gradually decreased during domestication and improvement. Subsequently, a genome-wide association study (GWAS) using 2,318,614 single nucleotide polymorphisms (SNPs) with a minor allele frequency above 0.05 identified 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 clade 1B ( 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 a plant gene cluster, so the inventors named this metabolic gene cluster "phenolamine and salicylic acid biosynthesis gene cluster BGC2" based on its final products being phenolamines and salicylic acid.

[0031] Example 2: Component genes of the phenolamine and salicylic acid biosynthesis gene clusters ( SlEPS1 , SlPBS3 , SlCoAOMT2 、 SlCoOOMT3 and SlCoAOMT4 ) cloning and protein expression vector construction

[0032] The present inventors 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). The RNA was reverse transcribed into cDNA using the reverse transcription kit Supermix (purchased from Beijing Quanshijin Company) under the following reaction conditions: 42°C for 30 minutes and 80°C for 5 seconds. qRT-PCR was then used to detect the RNA. SlEPS1 The expression levels in different tissues were found to be higher in flowers and leaves. SlEPS1 , SlPBS3 , SlCoAOMT2 、 SlCoOOMT3 and SlCoAOMT4 To identify the coding sequences of these genes, researchers used flower cDNA as a template and, based on the location and structure of these genes in the tomato genome (SGN), predicted the full-length open reading frame sequences of these candidate genes. They then designed specific PCR primers with homologous recombination linkers (Table 1, synthesized by Qingke Biotechnology Co., Ltd.). Ultra-fidelity KOD enzyme (Dalian Baoriyi Bioengineering Co., Ltd.) was used to amplify the full-length CDS fragments of the five genes. PCR reaction conditions included: pre-denaturation at 94°C for 5 minutes; 32 cycles of 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 2 minutes; and extension at 68°C for 5 minutes. The amplified PCR product was ligated into the pDonr207 entry vector via a BP reaction using GATEWAY cloning technology and transformed into competent E. coli DH5α cells. Positive clones were obtained by colony PCR verification, and correct clones without mutations containing candidate genes were obtained by sequencing (Haikou Qingke Biological Company). 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 Company, USA) through LR reaction of GATEWAY cloning technology.

[0033] Table 1 Primers used in constructing protein expression vectors in the present invention

[0034]

[0035] Example 3: Components of the phenolamine and salicylic acid biosynthetic gene clusters ( SlEPS1 , SlCoAOMT2 、 SlCoOOMT3 and SlCoAOMT4 ) prokaryotic protein expression

[0036] 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 Biotechnology), and the Escherichia coli was induced to express the proteins encoded by the candidate genes.

[0037] Specific steps: (1) E. coli culture: The plasmid containing the target gene fragment was transformed into E. coli BL21 competent cells, and a single colony was picked and inoculated into 5 ml of LB medium containing 50 μg / ml ampicillin antibiotics, and cultured at 37°C overnight. The next day, the bacterial solution was inoculated into 250 ml of 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 was about 0.5. Then, 1 mol / L IPTG (isopropylthiogalactoside, Shanghai Sangon Co., Ltd.) was added to the bacterial solution to induce protein expression. After adding IPTG, the bacterial solution was placed in a shaking incubator at 16°C and 160 rpm for overnight culture. (2) E. coli collection: The cells were collected in a 500 ml centrifuge tube and centrifuged at 4°C and 5000 rpm for 8 minutes. The supernatant was discarded, and 50 ml of Lysis buffer was added to suspend the cells and placed on ice. Use a high-pressure cell disruptor (Suzhou Antos Nanotechnology Co., Ltd.) to disrupt the suspended cells. (3) SDS-PAGE: The disrupted cells were 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. Separate the supernatant and precipitate. Take 20ul of the supernatant and add 5ul of 5×SDS Loading buffer. Heat in a dry bath at 100℃ for 10min. After the heated sample cools to room temperature, take 15ul of the sample and spot it. Perform SDS-PAGE to check whether the target protein is present in the total protein and supernatant. The remaining protein was stored at -80℃ for future use. (4) Protein purification: Wash the purification column containing glutathione agarose 4B (GE Healthcare, USA) thoroughly with Lysis buffer, add the supernatant containing the target protein, collect the effluent, and pass it through the column again. Wash the column thoroughly with Lysis buffer (5 times column volume). After sufficient washing, 15 mmol of reduced glutathione eluent (Shanghai Sangon Co., Ltd.) was added to the purification column, 1 ml at a time, and 1 ml of flow-through was collected and repeated 5-6 times. The collected flow-through was confirmed by SDS-PAGE to determine whether the target protein was present. The inventors successively obtained well-purified recombinant proteins of SlEPS1, SlCoAOMT2, SlCoOOMT3, and SlCoAOMT4 proteins fused with GST-tagged proteins, and the above proteins were aliquoted and stored in a -80°C refrigerator until use ( Figure 2 A).

[0038] Example 4: Phenolamine and salicylic acid biosynthetic gene cluster components ( SlEPS1 , SlPBS3 , SlCoAOMT2 、 SlCoOOMT3 and SlCoAOMT4 ) in vitro enzyme activity assay

[0039] 1. In vitro enzyme activity assay of SlEPS1 protein

[0040] In order to verify the function of the candidate gene SlEPS1, the inventors used the above-mentioned purified SlEPS1 protein to perform in vitro enzyme activity assays. 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 added 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).

[0041] 2. In vitro enzyme activity assay of SlCoAOMT2, SlCoAOMT3, and SlCoAOMT4 proteins

[0042] To validate candidate genes SlCoAOMT2 、 SlCoAOMT3 and SlCoAOMT4 To investigate the function of SlCoAOMT2, SlCoAOMT3, and SlCoAOMT4 recombinant proteins purified above, the inventors performed in vitro enzyme activity assays. The in vitro methyltransferase activity reaction system consisted of a 10-μl reaction mixture containing 1 μl of 5 mmol / L methyl donor SAM, 1 μl of 1 mmol / L Caf-CoA, 500 ng of purified protein, 1 μl of 1 mol / L Tris-HCl buffer (pH 7.4), and 1 μl of 50 mmol / L MgCl2. The mixture was then filled to 10 μl with ddH2O. After incubation at 30°C for 30 minutes, the reaction was terminated by adding 50 μl of pre-chilled methanol. The reaction mixture was centrifuged at 4°C, 12,000 rpm, for 10 minutes, and 50 μl of the supernatant was collected for LC-MS analysis. LC-MS detection results 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).

[0043] Example 5: Phenolamine and salicylic acid biosynthetic gene cluster components ( SlEPS1 and SlPBS3 ) transient expression in tobacco

[0044] 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 cultured 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 expressing AtEPS1, AtPBS3, SlEPS1, or SlPBS3 alone did not result in SA synthesis ( 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 a 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 examined the phenolamine content of AtEPS1 and SlEPS1 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 did not exhibit this activity, supporting the theory that SlEPS1 plays a dual role in phenolamine and salicylic acid biosynthesis ( Figure 3C).

[0045] Example 6: Analysis of expression patterns of phenolamine and salicylic acid biosynthetic gene cluster components

[0046] 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 can induce the expression of spermidine derivatives and salicylic acid gene cluster components, the present invention technicians conducted a 4-week-old Micro tomato seedling experiment. Pst DC3000 Treatment, after 48 hours of treatment, tomato leaf tissues were sampled at different time points to extract total RNA.

[0047] The total RNA extracted above was reverse transcribed. The specific steps for reverse transcription were as follows: 3-5 g of total RNA was treated with DNase I (Invitrogen, USA) for 15 minutes to remove genomic DNA contamination. Reverse transcription was then performed using oligo(dT)18 oligoprimer and M-MLV reverse transcriptase (Promega, USA). Real-time quantitative PCR analysis kit Green PCR Master Mix (Takara Biotechnology Dalian Co., Ltd.) was used according to the kit's instructions, and real-time quantitative PCR reactions were performed on an ABI 7500 RealTime PCR system (Applied Biosystems, USA). Gene expression was quantified using relative quantification methods. Sample RNA content was measured and normalized using the expression level of the tomato housekeeping gene Ubiquitin3.

[0048] qRT-PCR analysis showed that the four components of the spermidine derivative and salicylic acid gene cluster were affected. Pst DC3000 Different degrees of induced expression were observed, with the highest expression at 8 h ( Figure 4 BE), indicating that these four component genes may be involved in the defense process of tomato against pathogens.

[0049] The specific PCR primers for the five component genes in the qRT-PCR analysis are shown in Table 2 .

[0050] Table 2 Primers used for fluorescence quantification in the present invention

[0051]

[0052] 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

[0053] 1. Construction of SlEPS1-overexpressing transgenic plants

[0054] To further verify SlEPS1 The present invention technicians used the LR reaction of the GATEWAY cloning technology to connect the pDonr207-SlEPS1 obtained in Example 2 to the 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 via the Agrobacterium tumefaciens LBA4404 (strain from Shanghai Weidi Biotechnology Co., Ltd.) through a tomato genetic transformation system. After pre-cultivation, infection, co-cultivation, selection of calli with kanamycin resistance, differentiation, rooting, and transplanting, transgenic plants were obtained after identification.

[0055] 2. Transgenic plants SlEPS1 Gene expression analysis and metabolite analysis

[0056] 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 wild-type (non-transgenic tomato) Micro Tom control, the expression of the gene in the positive transformed plants was significantly higher than that in the wild-type (non-transgenic) Micro Tom control. SlEPS1 The gene expression was significantly increased ( Figure 5 A).

[0057] 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 gene 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).

[0058] 3. Construction of SlEPS1 knockout transgenic plants

[0059] To further verify the function of the SlEPS1 gene, the present inventors used CRISPR-Ca9 technology to construct a transgenic knockout vector for SlEPS1. After plasmid sequencing was confirmed, the vector was used for genetic transformation of tomatoes. First, the vector was introduced into the tomato variety Micro Tom using the Agrobacterium tumefaciens LBA4404 (strain obtained from Shanghai Weidi Biotechnology Co., Ltd.) through a tomato genetic transformation system. After pre-cultivation, infection, co-cultivation, screening for kanamycin-resistant calli, differentiation, rooting, and transplanting, transgenic plants were obtained after identification.

[0060] 4. Transgenic plants SlEPS1 Gene sequencing analysis and metabolite analysis

[0061] DNA was extracted from the transgenic plants and PCR analysis was used to detect the editing of the gene in the plant. The positive knockout plants were propagated to obtain T2 transgenic plants ( Figure 5 D).

[0062] In order to further verify the function of candidate genes in regulating metabolite synthesis in tomatoes, the inventors selected SlEPS1 The knockout lines in the T2 generation of transgenic materials were detected 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).

[0063] Example 8: Disease resistance analysis of transgenic strains and knockout strains overexpressing the acyltransferase SlEPS1 gene and in vitro antibacterial analysis of metabolites

[0064] 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.

[0065] 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. Afterwards, centrifuged at 4000g for 5 minutes at 4°C, the supernatant was discarded, washed twice with 10 mmol / L MgCl2 solution and resuspended, and the bacterial concentration was adjusted 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. This experiment was repeated at least three times, and similar results were obtained each time.

[0066] Colony count: On the 3rd and 4th day after inoculation with Pst DC3000, fully expanded functional leaves were randomly collected using a hole 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 washed for 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Serial dilutions were made and 10 μL of the original solution 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 in triplicate.

[0067] The specific operation of the in vitro liquid antibacterial test is as follows: select a PstDC3000 Colonies were cultured in R2A medium at 28°C and 180 rpm overnight, 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, and the effect of Fer-Spd on the Pst DC3000 Effects on cell growth.

[0068] 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, and after the medium is cooled to about 50°C, 1% of OD600 = 0.01 is added. PstDC3000 Resuspend the solution, mix thoroughly, and evenly pour 15 mL into each culture dish. Allow to solidify. Place the Oxford cup vertically on the culture medium surface, pressing gently to ensure close contact with the culture medium without leaving any gaps. Add 200 μL of Fer-Spd solution of varying concentrations to the Oxford cup, ensuring no overflow. Incubate at 37°C for 16-18 hours.

[0069] The results showed that vaccination Pst DC3000 On the 3rd and 4th day after filtration, compared with the wild-type plant control, SlEPS1 The gene overexpression strains showed a strong disease resistance phenotype ( Figure 6 A). Colony counts showed that the number of lesions on the leaves of the tomato overexpression plants after inoculation was much smaller than that on the leaves of the wild-type tomato plants, indicating that the disease resistance was stronger ( Figure 6 B) Compared with wild-type plants, SlEPS1 The 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 the in vitro antibacterial experiment of Fer-Spd showed that a specific concentration of Fer-Spd can effectively inhibit PstDC3000 When the concentration of Fer-Spd reaches 2mM, PstDC3000 The growth of Figure 6 EH).

[0070] Combined with the above results, it was shown that overexpression of key components of 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.

[0071] The present invention has been 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.

[0072] 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 is a non-naturally encoded amino acid. 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.

[0073] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0074] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0075] 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, can be equivalent parameters, concentration and conditions, implement the present invention in a wide range. Although the present invention provides special embodiments, it should be understood that the present invention can be further improved. In a word, by the principle of the present invention, the application is intended to include any variation, purposes or improvements to the present invention, including departing from the disclosed range in the application, and the changes performed with conventional techniques known in the art.

Claims

1. SlEPS1 Genes in improving tomato resistance PstDC3000 The application on pathogenic bacteria is characterized by: The application approach is overexpression SlEPS1 Gene, enhancer Pst DC3000 The ability to resist pathogens, SlEPS1 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

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

2.

3. The use according to claim 1, characterized in that The overexpression SlEPS1 Gene, enhancer Psst DC3000 The ability to resist pathogens includes the following steps: (1) Extract the total RNA of tomato flowers, reverse transcribe to obtain cDNA, use cDNA as template, and use primers SlEPS1 -F and SlEPS1 -R for PCR amplification; The primers SlEPS1 -F and primers SlEPS1 The primer sequences for -R are as follows: SlEPS1 -F:5'-AAAAAGCAGGCTTAATGACAAAACTTCGAGTAGTATCAG-3'; SlEPS1 -R:5'-AGAAAGCTGGGTATTAAGCGATTTCAATTTTTACTGAC-3'; (2) The PCR product obtained by amplification 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 the GATEWAY cloning technology to construct the pBI- SlEPS1 carrier; (3) The pBI- SlEPS1 The vector was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

4. The method according to claim 1 SlEPS1 The invention relates to a method for enhancing the accumulation of spermidine derivatives Fer-Spd, spermidine derivatives diFer-Spd and salicylic acid in tomato plants, wherein: The application approach is to overexpress in tomato SlEPS1 Gene.

Images