Application of SmCYP82D47 gene in improving resistance of eggplant to bacterial wilt

Abstract

The application discloses application of a SmCYP82D47 gene in improving resistance of eggplants to bacterial wilt, and belongs to the technical field of molecular biology. The nucleotide sequence is shown as SEQ ID NO. 2. On the basis of GWAS positioning of an eggplant bacterial wilt resistance linkage site, the application screens an eggplant bacterial wilt resistance candidate gene SmCYP82D47 through bioinformatics analysis such as gene sequence analysis and haplotype analysis; analyzes tissue expression specificity and hormone response expression characteristics of the candidate gene; and observes the disease resistance of a silencing plant and a wild type plant, and the disease resistance of a wild type plant and an overexpression plant by silencing the candidate gene in a resistant material S027 and overexpressing the candidate gene in a susceptible material S054, so as to not only verify the accuracy of the positioning result and determine the role of the candidate gene in regulating the bacterial wilt resistance of the eggplant, but also provide an important theoretical basis and technical means for eggplant disease resistance molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of the SmCYP82D47 gene in improving resistance to bacterial wilt in eggplant. Background Technology

[0002] Eggplant (Solanum melongena L.) belongs to the Solanaceae family and the Solanum genus. It can be cultivated as an annual or perennial plant, with its edible part being the berry. It is an important economic crop. Eggplant is widely distributed globally and has significant economic value. Due to the rich nutritional content of its fruit, it is now widely cultivated in temperate and subtropical regions. According to statistics from the Food and Agriculture Organization of the United Nations (FAO) in 2022, eggplant, with an annual total production of 59.3 million metric tons, ranks third in the world among Solanaceae crops, after potatoes and tomatoes. As an important vegetable crop, eggplant not only contains abundant protein, soluble sugars, dietary fiber, phenolic compounds, flavonoids, and vitamins A, C, and P, but also possesses health benefits such as significantly lowering cholesterol.

[0003] However, eggplant production is often threatened by various diseases, seriously affecting the safety of eggplant production. Bacterial wilt is the most serious solanaceous disease in the world and a devastating disease affecting the production of solanaceous crops in southern my country. Eggplant bacterial wilt is a relatively common global soil-borne bacterial disease that can directly lead to a significant reduction in eggplant yield and quality (up to about 80%). Currently, the identification of resistance genes and disease resistance mechanisms related to bacterial wilt in eggplant using forward genetics methods is still unclear. Because the pathogen can survive in the soil for a long time without a host plant, it is difficult to control through agronomic practices or chemical treatments. Therefore, studying resistance genes and breeding bacterial wilt-resistant varieties is the most economical and effective means of controlling bacterial wilt. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the SmCYP82D47 gene in improving resistance to bacterial wilt in eggplant, thereby addressing the problems existing in the prior art. The objective of this invention is to locate candidate genes for resistance to bacterial wilt in eggplant using forward genetics methods, clarify the mutation types of these candidate genes, and identify their mechanisms of action in regulating resistance to bacterial wilt in eggplant.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of the present invention is a SmCYP82D47 gene that regulates resistance to bacterial wilt in eggplant, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] The second technical solution of the present invention is that the protein encoded by the SmCYP82D47 gene has the amino acid sequence shown in SEQ ID NO.3.

[0008] The third technical solution of the present invention is a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the SmCYP82D47 gene.

[0009] The fourth technical solution of the present invention is the application of the SmCYP82D47 gene, the protein, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in improving resistance to bacterial wilt in eggplant.

[0010] The fifth technical solution of the present invention is the application of the SmCYP82D47 gene, the protein, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in the cultivation of transgenic eggplant varieties resistant to bacterial wilt.

[0011] The sixth technical solution of this invention is a method to improve the resistance of eggplant to bacterial wilt by overexpressing the SmCYP82D47 gene or upregulating the level of its encoded protein to improve the resistance of eggplant to bacterial wilt.

[0012] The seventh technical solution of this invention is a method for cultivating transgenic eggplant varieties resistant to bacterial wilt, wherein the SmCYP82D47 gene is introduced into the target plant to obtain the strain.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] Based on the GWAS mapping of eggplant bacterial wilt resistance linkage sites, this invention screened and obtained the candidate gene SmCYP82D47 for eggplant bacterial wilt resistance through bioinformatics analysis such as gene sequence analysis and haplotype analysis. The tissue expression specificity and hormone response expression characteristics of the candidate gene were analyzed. By silencing the candidate gene in the resistant material S027, the disease resistance of the silenced plants and wild-type plants was observed. This not only verifies the accuracy of the mapping results and clarifies the role of the candidate gene in regulating eggplant bacterial wilt resistance, but also provides an important theoretical basis and technical means for molecular breeding of eggplant disease resistance. Attached Figure Description

[0015] Figure 1 Comparative images of cross-sections of the lower stems of healthy plants and plants infected with Ralstonia solanacearum (a); phenotypic results of the incidence of bacterial wilt in 201 eggplant germplasms over many years at multiple locations (b).

[0016] Figure 2 To analyze the loci associated with resistance to bacterial wilt in eggplant using SNP-GWAS. GWAS mapping results were obtained from different years and batches, where a: 2022, batch 1; b: 2022, batch 2; c: 2022, batch 3; d: 2023, batch 1.

[0017] Figure 3 The relationship between the SmCYP82D47 TGA / TGG haplotype and disease incidence (a); tissue expression specificity of SmCYP82D47 in eggplant germplasm highly resistant to bacterial wilt S027 and highly susceptible to bacterial wilt S054 (b); expression pattern of SmCYP82D47 in response to Ralstonia solanacearum induction (c); expression pattern of SmCYP82D47 in response to SA (salicylic acid) induction (d); expression pattern of SmCYP82D47 in response to MeJA (methyl jasmonate) induction (e).

[0018] Figure 4 This study describes the SNP-CYP82D47-549 marker genotyping results of SNP-CYP82D47 in S054 (AA type), S027 (GG type), and 50 eggplant germplasm resources (natural populations). It can distinguish between the GG and AA haplotypes of SmCYP82D47 in S054 (AA type), S027 (GG type), and the 50 eggplant germplasm resources (natural populations). Green dots represent samples where only FAM signal was detected (GG), with the same genotype as S027; blue dots represent samples where only HEX signal was detected (AA), with the same genotype as S054; gray dots represent samples where neither FAM nor HEX fluorescence signals were detected.

[0019] Figure 5 The PTRV2-SmCYP82D47 eggplant VIGS vector was constructed. In the diagram, a shows the structure of the PTRV2-SmCYP82D47 vector; b shows the results of EcoV restriction enzyme digestion electrophoresis of the PTRV2-SmCYP82D47 plasmid, where M1 represents the Marker [DL5000]: 5000, 3000, 2000, 1000, 750, 500, 250, 100 bp; and 1 represents the EcoV restriction enzyme bands: 274, 657, 1923, 8278 bp.

[0020] Figure 6 The VIGS silencing gene SmCYP82D47 resulted in silenced plants exhibiting increased susceptibility to bacterial wilt. Here, a represents the expression level of SmCYP82D47 in wild-type plants and silenced plants; b represents the disease resistance phenotype of wild-type plants and SmCYP82D47 silenced plants after infection with Ralstonia solanacearum.

[0021] Figure 7The pBWA(V)HS-SmCYP82D47 eggplant overexpression vector was constructed. In the diagram, a shows the structure of the pBWA(V)HS-SmCYP82D47 vector; b shows the EcoRV restriction enzyme digestion electrophoresis results. For the pBWA(V)HS-SmCYP82D47 plasmid, M1 represents the Marker [DL5000]: 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100 bp; and 1 represents the EcoV restriction enzyme digestion bands: 952, 1134, 1879, 2691, 5324 bp.

[0022] Figure 8 Overexpression of the gene SmCYP82D47 resulted in transgenic lines exhibiting greater resistance to bacterial wilt. Here, a represents the expression level of SmCYP82D47 in wild-type plants and overexpression lines; b represents the disease resistance phenotype of wild-type plants and SmCYP82D47 overexpression lines after infection with Ralstonia solanacearum. Detailed Implementation

[0023] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0024] This invention provides a SmCYP82D47 gene that regulates resistance to bacterial wilt in eggplant, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0025] This invention also provides a protein encoded by the SmCYP82D47 gene, the amino acid sequence of which is shown in SEQ ID NO.3.

[0026] The present invention also provides a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria of the SmCYP82D47 gene.

[0027] This invention also provides the application of the SmCYP82D47 gene, the protein, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in improving resistance to bacterial wilt in eggplant.

[0028] This invention also provides the application of the SmCYP82D47 gene, the protein, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in the cultivation of transgenic eggplant varieties resistant to bacterial wilt.

[0029] This invention also provides a method for improving the resistance of eggplant to bacterial wilt by overexpressing the SmCYP82D47 gene or upregulating the level of its encoded protein.

[0030] This invention also provides a method for cultivating a transgenic eggplant line resistant to bacterial wilt, wherein the SmCYP82D47 gene is introduced into the target plant.

[0031] In some specific implementations, the SmCYP82D47 gene is introduced into the target plant via the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

[0032] The Vegetable Research Institute of the Guangdong Academy of Agricultural Sciences conducted whole-genome resequencing on 151 representative eggplant germplasm resources from both domestic and international sources to analyze their genetic diversity and structure. Following the root irrigation method described in the reference (Li Zhiliang, Li Zhenxing, He Zifu, Yu Hao. Identification of Bacterial Wilt Resistance in Eggplant Seedlings at the Field Stage. Guangdong Agricultural Sciences, 2006, 1:39-4.), artificial inoculation identification of the 151 eggplant germplasm resources with *Ralstonia solanacearum* was performed in four batches over two consecutive years (each batch using two completely randomized block designs). Based on GWAS genome-wide association analysis, the SmCYP82D47 gene locus (C / A SNP: Chr04.81374475) on chromosome 4 was identified as significantly associated with bacterial wilt resistance. This gene belongs to the cytochrome CYP450 family, and SmCYP82D47 was preliminarily identified as a candidate gene related to eggplant bacterial wilt resistance.

[0033] This study, through sequence variation analysis of SmCYP82D47, discovered a G / A SNP mutation in the first exon of the SmCYP82D47 gene in eggplant S054, a highly susceptible variety of bacterial wilt, which directly resulted in an advanced stop codon. Haplotype analysis revealed that the incidence of bacterial wilt in the TGA genotype was significantly higher than that in the normal TGG genotype. Tissue-specific expression analysis of this gene showed that its expression was tissue-specific in both highly resistant and highly susceptible germplasm. The role of the SmCYP82D47 gene in eggplant resistance to bacterial wilt was investigated by constructing a VIGS vector to silence the highly resistant material S027 and by overexpressing the SmCYP82D47 gene in the susceptible material S054. These studies not only validate the reliability of previous mapping results but also clarify the molecular mechanism by which the candidate gene SmCYP82D47 regulates eggplant resistance to bacterial wilt, providing important theoretical and guiding significance for future molecular breeding of eggplant for disease resistance.

[0034] The technical solution adopted in this invention is as follows: Analyzing significant loci within the GWAS mapping region to screen for gene loci related to disease resistance; identifying key variants and gene expression linked to disease resistance through haplotype analysis and tissue expression specificity analysis; clarifying the hormone-induced SmCYP82D47 gene expression characteristics through hormone treatment; constructing the PTRV2-SmCYP82D47VIGS silencing vector using the S027 SmCYP82D47 gene sequence information and injecting it into eggplant seedling leaves; constructing the pBWA(V)HS-SmCYP82D47 overexpression vector and performing genetic transformation using Agrobacterium-mediated transformation; and using a root irrigation method, infecting the roots of blank control, empty vector, and treated eggplants with bacterial wilt strains, observing the disease phenotype of empty vector and silent group seedlings, and detecting the expression of the candidate gene SmCYP82D47.

[0035] The molecular biology experimental techniques used in the following examples include DNA extraction, RNA extraction and reverse transcription, PCR amplification, enzyme digestion and ligation of PCR products and vectors, transformation of competent Escherichia coli DH5α and Agrobacterium GV3101, etc. Unless otherwise specified, they are generally performed according to conventional methods. For details, please refer to "Molecular Cloning: A Laboratory Manual" (3rd Edition) (translated by Sambrook J, Russell DW, Janssen K, Argentine J, Huang Peitang et al., 2002, Beijing: Science Press), or follow the conditions recommended by the manufacturer.

[0036] The eggplants used in this embodiment of the invention are eggplant germplasm resources preserved by the Vegetable Research Institute of Guangdong Academy of Agricultural Sciences.

[0037] Example 1

[0038] I. Location of candidate genes for resistance to bacterial wilt in eggplant

[0039] 1. Test materials

[0040] To locate the genomic regions associated with resistance to bacterial wilt in eggplant, the Vegetable Research Institute of the Guangdong Academy of Agricultural Sciences conducted resistance evaluations of 151 germplasm accessions (Table 1) in four batches (three batches in 2022 and one batch in 2023). 201 germplasm accessions were artificially inoculated with *Ralstonia solanacearum* using the root drenching method. One week after inoculation, disease incidence was recorded for each material (twice a week). Once the disease condition of each material stabilized, the incidence rate was statistically analyzed. Figure 1 (a, b)

[0041] Table 1 Eggplant Germplasm Resource Information

[0042]

[0043]

[0044]

[0045] 2. Results of candidate gene localization for resistance to bacterial wilt in eggplant

[0046] Using "S076" as the reference genome (https: / / db.cngb.org / cnsa / assembly / page / batch / sub062525 / view / , sample_accession: CNS1199600), SNP-GWAS analysis was performed. In multi-year, multi-batch GWAS results, strongly associated signals were primarily identified on Chr04. Figure 2 (a, b, c, d). Through analysis of significant sites of Chr04 (Peak point is C / A SNP: Chr04.81374475) and candidate genes in strongly linked LD regions, the results showed that the SNP with the highest P-value (Peak) is directly located in the CYP82D47 gene (evm.model.Chr04.2518), and this gene is also strongly linked within the LD region.

[0047] II. Sequence comparison and expression analysis of SmCYP82D47 gene in resistant and susceptible bacterial wilt materials

[0048] 1. Sequence comparison results of SmCYP82D47 gene in resistant and susceptible bacterial wilt materials

[0049] Based on the eggplant reference genome (S076), a comparison of the cds sequences of the SmCYP82D47 gene in eggplant S027 (SEQ ID NO.2), a highly resistant variety of bacterial wilt, and S054 (SEQ ID NO.1), a highly susceptible variety of bacterial wilt, revealed a G / A SNP variation in the first exon of the SmCYP82D47-1 gene. This variation directly resulted in the premature advance of a stop codon. Statistical analysis of the variation types (haplotype analysis) of 151 eggplant germplasms showed that the incidence of bacterial wilt in the TGA genotype was significantly higher than that in the normal TGG genotype. Figure 3 (a)

[0050] The cds sequence of the SmCYP82D47-1 mutant gene is 1431 bp, and its nucleotide sequence is shown in SEQ ID NO.1.

[0051] SEQ ID NO.1:ATGGATTTCTCACTGATTGCCCTCACTCTAGCTATCTCCTTCCT TGTTTTCTTCTTTCTTCACAAACTATTTTTCTCAGCTAAAATGCCAAGTAGAAATGTACCACAAGTCACCGGAGCATGGCCGATCATCGGCCATCTCCACCTCCTCAACGGACCTGAAATGCCTCACAAAATCTTCTCCGAAATGGCAGAAAAATACGGTCCAATTTTCCGATTAAAGCTCGGAGTAAATCAAGTAGTAATCGTGAGTGATCCCAAAATAGTCGAAGAATGTTTCACCACAAACGACAGGGCTTTTGCGAACCGGCCCAAAGCCATAGCTTCAGAGATCTTGGGCTACAATTACGCCATGTTTGGGTTTGGGCCTTACTGGCGAGAGATTAGAAAAATAGCCACAATTGAATTCCTTTCACCTCGGCGAATTGAGATGTCTAGCCACATTAGAGAATTTGAAGTAAAATCGGCTATCAAAGAGACGTATAATTACTGGTTGAAGAATAATAGTAGTAACTTAAATGGTGCTCTGAAGATGGAGATGAACGAA TGATTTGGAACTTTAGTTATCAACGCTATGTTGAAAATGCTATTTGGAATAAAATTTACAGATAAGGAAGATGAAGAAAGGAGTAAGGCTCACAAAGCAATCAAAAAATTCTTTGACTTATTGGGGGCCTCTGTTATAGCTGATTTTTTACCTTATTTAAGATGGTTGGATATTGAAGGCCATGAGAAGGCCATGAAAGAAACTGCTAAAGAAATTGACTTAATTGTTGAAGAATGGTTAGCAAAGCACAGAAGAAAGAGGGGAAATAAATGTGTGGGTGAAGAAGATTTCATGGATATCATGTTGTCCATTTGTGGAGATAAAAAAAATCTTCATGGTTTTGATGCTGATGATGTTATCAAATCCACATGTATGGCTCTTCAAACGGCAGGTTCTGATACCACAATAGTAACTCTAATATGGGCTCTATCTCTACTCCTAAACAACTACGATGCATTAAAAAAGGCCCAAGATGAACTAGACACTCATGTTGGCAAGAACAGATGGGTCCAAGAATCGGACATCAAGAATTTGGTTTATCTTCAAGCTATTATTAAAGAATCATTGCGTTTATATCCAGTCGTACCACTCTTAGTGCCCCACGAGTCGATCGAGGATTGTGTTGTTAGTGGCTACAATATACCGAAAGGGACTCGTCTATTAGTGAACGTATGGAAGCTGCATTCCGATCCTAAAATATGGCCTAATCCGCATAAGTTTAAGCCAGAGAGGTTCTTGACAACTCACAGAGATGTCGATGGGTTTAAAATTGAAAGACCTTCAGATGAACCAATTGATATGAGTGAGAGTCTTGGAGTGACACTTCGTAAAACCTTTCCACTCGAAGTTCACCTTACTCCACGCTTGGATTCTAATCTTTACTAA。

[0052] The cds sequence of the normal genotype of SmCYP82D47 is 1431 bp, and its nucleotide sequence is shown in SEQ ID NO.2.

[0053] SEQ ID NO.2:ATGGATTTCTCACTGATTGCCCTCACTCTAGCTATCTCCTTCCT TGTTTTCTTCTTTCTTCACAAACTATTTTTCTCAGCTAAAATGCCAAGTAGAAATGTACCACAAGTCACCGGAGCATGGCCGATCATCGGCCATCTCCACCTCCTCAACGGACCTGAAATGCCTCACAAAATCTTCTCCGAAATGGCAGAAAAATACGGTCCAATTTTCCGATTAAAGCTCGGAGTAAATCAAGTAGTAATCGTGAGTGATCCCAAAATAGTCGAAGAATGTTTCACCACAAACGACAGGGCTTTTGCGAACCGGCCCAAAGCCATAGCTTCAGAGATCTTGGGCTACAATTACGCCATGTTTGGGTTTGGGCCTTACTGGCGAGAGATTAGAAAAATAGCCACAATTGAATTCCTTTCACCTCGGCGAATTGAGATGTCTAGCCACATTAGAGAATTTGAAGTAAAATCGGCTATCAAAGAGACGTATAATTACTGGTTGAAGAATAATAGTAGTAACTTAAATGGTGCTCTGAAGATGGAGATGAACGAA TGGTTTGGAACTTTAGTTATCAACGCTATGTTGAAAATGCTATTTGGAATAAAATTTACAGATAAGGAAGATGAAGAAAGGAGTAAGGCTCACAAAGCAATCAAAAAATTCTTTGACTTATTGGGGGCCTCTGTTATAGCTGATTTTTTACCTTATTTAAGATGGTTGGATATTGAAGGCCATGAGAAGGCCATGAAAGAAACTGCTAAAGAAATTGACTTAATTGTTGAAGAATGGTTAGCAAAGCACAGAAGAAAGAGGGGAAATAAATGTGTGGGTGAAGAAGATTTCATGGATATCATGTTGTCCATTTGTGGAGATAAAAAAAATCTTCATGGTTTTGATGCTGATGATGTTATCAAATCCACATGTATGGCTCTTCAAACGGCAGGTTCTGATACCACAATAGTAACTCTAATATGGGCTCTATCTCTACTCCTAAACAACTACGATGCATTAAAAAAGGCCCAAGATGAACTAGACACTCATGTTGGCAAGAACAGATGGGTCCAAGAATCGGACATCAAGAATTTGGTTTATCTTCAAGCTATTATTAAAGAATCATTGCGTTTATATCCAGTCGTACCACTCTTAGTGCCCCACGAGTCGATCGAGGATTGTGTTGTTAGTGGCTACAATATACCGAAAGGGACTCGTCTATTAGTGAACGTATGGAAGCTGCATTCCGATCCTAAAATATGGCCTAATCCGCATAAGTTTAAGCCAGAGAGGTTCTTGACAACTCACAGAGATGTCGATGGGTTTAAAATTGAAAGACCTTCAGATGAACCAATTGATATGAGTGAGAGTCTTGGAGTGACACTTCGTAAAACCTTTCCACTCGAAGTTCACCTTACTCCACGCTTGGATTCTAATCTTTACTAA。

[0054] The amino acid sequence encoded by the SmCYP82D47 gene is shown in SEQ ID NO.3.

[0055] SEQ ID NO.3:MDFSLIALTLAISFLVFFFLHKLFFSAKMPSRNVPQVTGAWPIIG HLHLLNGPEMPHKIFSEMAEKYGPIFRLKLGVNQVVIVSDPKIVEECFTTNDRAFANRPKAIASEILGYNYAMFGFGPYWREIRKIATIEFLSPRRIEMSSHIREFEV KSAIKETYNYWLKNNSSNLNGALKMEMNEWFGTLVINAMLKMLFGIKFTDKEDEERSKAHKAIKKFFDLLGASVIADFLPYLRWLDIEGHEKAMKETAKEIDLIVEEW LAKHRRKRGNKCVGEEDFMDIMLSICGDKKNLHGFDADDVIKSTCMALQTAGSDTTIVTLIWALSLLLNNYDALKKAQDELDTHVGKNRWVQESDIKNLVYLQAIIKE SLRLYPVVPLLVPHESIEDCVVSGYNIPKGTRLLVNVWKLHSDPKIWPNPHKFKPERFLTTHRDVDGFKIERPSDEPIDMSESLGVTLRKTFPLEVHLTPRLDSNLY*;

[0056] The amino acid sequence encoded by the SmCYP82D47-1 gene is shown in SEQ ID NO.4.

[0057] SEQ ID NO.4: MDFSLIALTLAISFLVFFFLHKLFFSAKMPSRNVPQVTGAWPIIG HLHLLNGPEMPHKIFSEMAEKYGPIFRLKLGVNQVVIVSDPKIVEECFTTNDRAFANR PKAIASEILGYNYAMFGFGPYWREIRKIATIEFLSPRRIEMSSHIREFEVKSAIKETYNY WLKNNSSNLNGALKMEMNE*.

[0058] 2. Tissue-specific expression of the SmCYP82D47 gene

[0059] To understand the tissue specificity of SmCYP82D47 gene expression, qRT-PCR was used to analyze the expression of SmCYP82D47 gene in various tissues, including roots, stems, and leaves. Figure 3(b) The results showed that the expression of this gene was tissue-specific in both highly resistant and highly susceptible bacterial wilt germplasm. In the highly resistant bacterial wilt S027 material, SmCYP82D47 was highly expressed in leaves and upper stems, and less expressed in lower stems and roots; while in the highly susceptible bacterial wilt S054 material, SmCYP82D47 was highly expressed in leaves, and less expressed in stems and roots (extremely low expression in roots), indicating that SmCYP82D47 may play a role in bacterial wilt resistance in stems and leaves.

[0060] 3. Hormones and Ralstonia solanacearum induce SmCYP82D47 gene expression.

[0061] We treated eggplant varietals highly resistant to bacterial wilt (RS742 strain, from the Institute of Plant and Protection, Guangdong Academy of Agricultural Sciences), salicylic acid (SA), and methyl jasmonate (MeJA) at different time points with Ralstonia solanacearum (RS742 strain, from the Institute of Plant and Protection, Guangdong Academy of Agricultural Sciences). Seedlings of both strains at the 4-5 true leaf stage were inoculated with Ralstonia solanacearum using a root immersion in bacterial solution. Samples were taken at different time points (0, 1, 3, 6, 12, 18, and 24 hours) for RNA extraction, and the expression changes of the SmCYP82D47 gene were detected by qRT-PCR. The results showed that 1 hour after infection with Ralstonia solanacearum, the SmCYP82D47 gene could be induced to express in both resistant and susceptible materials, but the expression patterns differed. Figure 3 (c) The leaves of eggplant seedlings at the 4-5 true leaf stage (highly resistant to bacterial wilt S027 and highly susceptible to S054) were sprayed with exogenous hormones MeJA (1.0 mg / L) and SA (7.2 mg / L), respectively, until all leaves were covered with water droplets. Samples were taken at 0, 2, 6, 12, and 24 h after hormone treatment for RNA extraction, and the expression changes of the SmCYP82D47 gene were detected by qRT-PCR. The results showed that exogenous SA induced SmCYP82D47 gene expression in both resistant and susceptible materials 6 h after treatment. Figure 3 (d). In contrast, when MeJA was applied externally, the SmCYP82D47 gene was not induced in susceptible materials, but in resistant materials, the SmCYP82D47 gene was induced after 24 hours. Figure 3 (e).

[0062] 4. Development and application of SNP markers linked to the SmCYP82D47 gene

[0063] Based on the SmCYP82D47 sequence information, this invention has developed and validated molecular markers, as detailed below:

[0064] Primers were designed using Primer Premier 5 software based on the SmCYP82D47 sequence information. The primer pairs are as follows:

[0065] SNP-CYP82D47-549-Fg:

[0066] 5'-GAAGGTGACCAAGTTCATGCTTGAAGATGGAGATGAACGAATGG-3';

[0067] SNP-CYP82D47-549-Fa:

[0068] 5'-GAAGGTCGGAGTCAACGGATTCTGAAGATGGAGATGAACGAATGA-3';

[0069] SNP-CYP82D47-549-R: 5'-CCAAATAGCATTTTCAACATAGCG-3'.

[0070] After the design was completed, the specificity of the eggplant S054 and S027 genome sequences was determined by comparison.

[0071] The results are as follows Figure 4 As shown in Table 2, the GG and AA haplotypes of SmCYP82D47 from 52 eggplant germplasm resources (natural populations) that could be distinguished from S054 (AA type), S027 (GG type), and SmCYP82D47 were significantly higher than those from AA type plants. This is consistent with the phenotype of resistance to bacterial wilt. Therefore, this genotype was ultimately determined as a specific primer for amplifying A / GSNPs and can be used to detect eggplant materials resistant to bacterial wilt.

[0072] Table 2. Detection results of the SNP-CYP82D47-549 molecular marker in 52 eggplant germplasm resources.

[0073]

[0074]

[0075] Note: Samples with FAM signals represent the same genotype as S027, namely GG (resistant); samples with HEX signals represent the same genotype as S05, namely AA (susceptible).

[0076] Example 2

[0077] Effects of VIGS silencing of the SmCYP82D47 gene during eggplant seedling stage on bacterial wilt resistance in silenced plants and effects of transgenic lines overexpressing the SmCYP82D47 gene on bacterial wilt resistance.

[0078] 1. Construction of PTRV2-SmCYP82D47 eggplant VIGS vector

[0079] Using the CDS of infected eggplant S054 as a template, primers (F: gcctcgagacgcgtgagctcggtacc; R: taggatcggaatgcagcttcc; target fragment 274bp) were designed for PCR amplification to obtain a specific conserved fragment of the SmCYP82D47 gene. This specific SmCYP82D47 gene fragment was then ligated into the Ptrv2 vector. Enzyme digestion electrophoresis results showed that the PTRV2-SmCYP82D47 recombinant vector successfully constructed a 274bp target fragment after digestion. Figure 5 (a, b)

[0080] 2. Effects of VIGS silencing of the SmCYP82D47 gene during eggplant seedling stage on bacterial wilt resistance in silenced plants.

[0081] Using the VIGS technology system, the expression level of the candidate gene SmCYP82D47 in silenced plants was significantly lower than that in unsilent plants. Figure 6 (a) The results showed that the candidate gene SmCYP82D47 for resistance to bacterial wilt in eggplant was successfully silenced in S027 seedlings; after 5 days of infection with Ralstonia solanacearum, the silenced plants showed more wilting than the uninfected control plants. Figure 6 (b) The results showed that silencing the SmCYP82D47 gene in eggplant reduced its resistance to bacterial wilt. This result demonstrates that the SmCYP82D47 gene mutation reduces the plant's resistance to bacterial wilt, and that SmCYP82D47 may play an important role as one of the regulatory genes for eggplant resistance to bacterial wilt.

[0082] 3. Construction of eggplant overexpression vector

[0083] Using cDNA from eggplant material S027, which is highly resistant to bacterial wilt, as a template, the CDS of the SmCYP82D47 gene was amplified. Primers (6467(C+)F1: ggagagaacacgggggactttgcaac; R1: atggatttctcactgattgcc; 6467(C-)F2: ccactccctgaagcggccgctgtaca; R2: gtaaagattagaatccaagcgtg) were designed for PCR amplification, and the amplified DNA was ligated into the pBWA(V)HS vector containing the 35S promoter (an overexpression vector previously used by the project team). You, Q.,Li,H.,Wu,J.,Li,T.,Wang,YK,Sun,GW,Li,ZL,Sun,BJMapping and validation of epistatic D and Pgenes controlling anthocyanin biosynthesis in peel of eggplant(Solanummelongena L.).HorticultureResearch,2023,10:uhac268)( Figure 7 (a, b) Using the highly susceptible material S054 as the recipient, eggplant genetic transformation experiments were conducted to obtain lines transfected with the SmCYP82D47 gene, and bacterial wilt resistance was identified after seed harvest.

[0084] 4. Effects of SmCYP82D47 overexpression transgenic lines on bacterial wilt resistance

[0085] Using an eggplant overexpression technology system, the candidate gene SmCYP82D47 was overexpressed in S054. The expression level of SmCYP82D47 in the overexpressing transgenic lines was significantly higher than that in wild-type plants. Figure 8 (a) The results showed that the normal SmCYP82D47 gene was successfully overexpressed in S054; after 10 days of infection with Ralstonia solanacearum, the transgenic lines (9.7%) showed a lower incidence of disease compared to the wild-type plants (78%). Figure 8 (b) The results showed that overexpression of the SmCYP82D47 gene enhanced resistance to bacterial wilt. This result demonstrates that the normal SmCYP82D47 genotype increases plant resistance to bacterial wilt, and SmCYP82D47 may play an important role as one of the regulatory genes for bacterial wilt resistance in eggplant.

[0086] This invention discloses a candidate gene for resistance to bacterial wilt in eggplant, SmCYP82D47; it also discloses a mutant SmCYP82D47-1 gene in eggplant, which, compared to the cds sequence (SEQ ID NO.1 and SEQ ID NO.2) of the resistant eggplant SmCYP82D47 gene, contains one nonsense mutation, leading to premature termination of translation and a decrease in the resistance of eggplant to bacterial wilt. This invention also discloses the application of the candidate gene for resistance to bacterial wilt in eggplant resistance breeding.

[0087] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. SmCYP82D47 Gene, SmCYP82D47 Gene-encoded proteins or containing SmCYP82D47 The application of recombinant gene vectors, expression cassettes, transgenic cell lines, or recombinant bacteria in improving resistance to bacterial wilt in eggplant is characterized by, The SmCYP82D47 The nucleotide sequence of the gene is shown in SEQ ID NO.

2. SmCYP82D47 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

2. SmCYP82D47 Gene, SmCYP82D47 Gene-encoded proteins or containing SmCYP82D47 The application of recombinant gene vectors, expression cassettes, transgenic cell lines, or recombinant bacteria in the cultivation of transgenic eggplant varieties resistant to bacterial wilt, characterized in that... The SmCYP82D47 The nucleotide sequence of the gene is shown in SEQ ID NO.

2. SmCYP82D47 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

3. A method for improving resistance to bacterial wilt in eggplant, characterized in that, overexpression SmCYP82D47 The gene may upregulate the level of the protein it encodes, thereby increasing the resistance of eggplant to bacterial wilt. SmCYP82D47 The nucleotide sequence of the gene is shown in SEQ ID NO.

2. SmCYP82D47 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

4. A method for cultivating transgenic eggplant varieties resistant to bacterial wilt, characterized in that, Will SmCYP82D47 Genes are introduced into the target plant, and the desired result is obtained. SmCYP82D47 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

5. The method according to claim 4, characterized in that, The SmCYP82D47 Genes through containing SmCYP82D47 Gene recombinant vectors, expression cassettes, transgenic cell lines, or recombinant bacteria are introduced into target plants.

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