Pyriformospora indica and uses thereof

Abstract

The application discloses a pear anthracnose resistance gene MYC2- like and application, and belongs to the field of plant genetic engineering. MYC2‑like The nucleotide sequence of the gene is shown as SEQ ID NO:1, the protein sequence encoded by the gene is shown as SEQ ID NO:2, the gene is significantly up-regulated in response to MeJA and anthracnose fungus induction; the gene is located in the plant cell nucleus and has a conserved bHLH domain; a MYC2‑like MYC2‑like The constructed overexpression vector is used for transforming Solanum lycopersicum callus, so that the resistance of the callus to Colletotrichum gloeosporioides is increased, and the application provides an important gene resource for cultivating pear anthracnose resistance varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a pear anthracnose resistance gene. MYC2-like And its applications. Background Technology

[0002] In recent years, with the spread and occurrence of anthracnose in pear-producing areas of my country, it has become one of the main diseases causing early leaf drop in pears, a limiting factor for the development of the pear industry, and seriously affects its green and sustainable development. Chemical control is currently the main control measure, but long-term and large-scale application of chemical agents not only affects the safety of fruit consumption but also causes environmental pollution. Therefore, further exploration of plant resistance genes is expected to provide candidate genes for breeding pear anthracnose resistance. Studies have found that bHLH transcription factors play an important regulatory role in pear anthracnose resistance, but research on whether they participate in regulating pear anthracnose resistance has not yet been reported.

[0003] bHLH (basic Helix-Loop-Helix) transcription factors contain two conserved domains: an N-terminal basic DNA-binding domain responsible for recognizing E-box elements, and a C-terminal HLH domain mediating protein dimerization. These transcription factors are widely involved in plant secondary metabolic synthesis, hormone signal transduction, and responses to biotic and abiotic stresses. Currently, the function of bHLH transcription factors in pear anthracnose resistance has not been systematically studied. Therefore, elucidating the mechanism of action of these factors in pear's response to anthracnose can provide a theoretical basis and gene resources for molecular breeding of pear disease resistance, and has significant scientific value. Among them, members of the pear bHLH family... MYC2-like Its function and application in anthrax resistance have not been reported to date. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to propose an application of bHLH-type transcription factors in pear anthracnose resistance.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] The first aspect of the present invention proposes a pear MYC2-like The gene, whose coding region nucleotide sequence is shown in SEQ ID NO: 1.

[0007] The above pears MYC2-like The amino acid sequence encoded by the gene is shown in SEQ ID NO: 2.

[0008] The second aspect of the present invention proposes the above-mentioned pear MYC2-likeThe application of genes in regulating pear anthracnose resistance involves increasing pear resistance to anthracnose by promoting the expression of genes related to jasmonic acid synthesis and pear lignin synthesis.

[0009] The third aspect of the present invention proposes a pear MYC2-like Application of genes in the breeding of anthracnose-resistant pear varieties.

[0010] Fourth aspect of the invention: a pear MYC2-like The gene overexpression vector was obtained by digesting the fragment shown in SEQ ID NO: 1 into the pK7WG2D vector.

[0011] The fifth aspect of this invention proposes a method for cultivating anthracnose-resistant pear varieties, wherein the pear... MYC2-like Genes were introduced into the target plant to obtain transgenic pears with enhanced resistance to anthracnose.

[0012] Preferably, the pear variety includes, but is not limited to, any one of the following: eggplant pear, Dangshan crisp pear, Korla fragrant pear, Cuiguan pear, and Qiuyang pear.

[0013] Preferably, it includes the following steps: (1) Cloned pear MYC2-like Gene; (2) Constructing a pear MYC2-like Gene overexpression vectors; (3) Pear MYC2-like The gene overexpression vector was transformed into the target plant, and the transgenic pear with improved anthracnose resistance was obtained after identification.

[0014] Preferably, the method of transformation into the target plant includes, but is not limited to, any one of Agrobacterium-mediated transformation, gene gun transformation, etc.

[0015] The beneficial effects of this invention are as follows: 1. This invention proposes a pear bHLH-type transcription factor. MYC2-like The application of this gene in pear anthracnose resistance, along with its encoded amino acid sequence, is reported for the first time. RT-qPCR analysis showed that the gene expression was significantly upregulated in response to MeJA and anthracnose induction; the gene is located in the plant cell nucleus and has a conserved bHLH domain; overexpression of MYC2-like enhanced callus anthracnose resistance in pear and eggplant.

[0016] 2. The MYC2-like Gene overexpression significantly enhanced POD and SOD activity, reduced H2O2 content, and significantly promoted JA synthesis; in addition, overexpression... MYC2-like Genes can significantly promote disease resistance genes, disease course-related proteins, and resistance genes. PR1 Chitin synthase CHI and lignin ( PERThe expression of genes such as lignin-synthesizing genes promotes lignin synthesis. This invention provides valuable gene resources for the breeding of anthracnose-resistant pear varieties and is of great significance for elucidating the molecular mechanism of anthracnose resistance in pear.

[0017] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0018] Figure 1 In Embodiment 1 of the present invention PpMYC2-like Gene amplification and self-activation detection results, where A is... MYC2- like Gene amplification results, B is MYC2-like Gene self-activation detection results; Figure 2 In Embodiment 2 of the present invention PpMYC2-like Gene subcellular localization and amino acid sequence analysis, where A is PpMYC2-like Subcellular localization results, B is PpMYC2-like Conservative structural domain analysis, C is PpMYC2-like Evolutionary tree analysis; Figure 3 In Embodiment 3 of the present invention PpMYC2-like Gene expression pattern analysis, where A represents gene expression patterns at 6, 12, and 24 hours after inoculation with fruit anthracnose. PpMYC2-like Gene expression analysis, B represents the result of treatment with exogenous MeJA and SA. PpMYC2-like Gene expression analysis; the error bars in the figure represent the standard errors of three independent replicates. represent p The difference at the <0.0001 level was extremely significant.

[0019] Figure 4 In Embodiment 4 of the present invention PpMYC2-like Evaluation of transgenic anthrax resistance, where A represents overexpression. PpMYC2-like Phenotypic characteristics of callus inoculated with anthrax bacteria at 3 and 5 days; B indicates overexpression. PpMYC2-like Callus identification; C represents overexpression PpMYC2-like Lesion diameters on callus inoculated with *Bacillus anthracis* at 3 and 5 days post-inoculation. EV: control line; 1-5 represent five different overexpression lines. Data were analyzed using one-way ANOVA. Error bars in the figure represent the standard errors of three independent replicates. represent p The difference was statistically significant at levels <0.05. represent p <0.01 indicates a highly significant difference. represent p The difference at the <0.001 level was extremely significant. represent p The difference at the <0.0001 level was extremely significant.

[0020] Figure 5 In Embodiment 5 of the present invention PpMYC2-like- Analysis of hormone and enzyme activity in strains 1, 2, and 3, where AB represents overexpression. PpMYC2-like JA and SA levels at 0 h, 6 h, and 24 h after callus inoculation with *Anthracis cirrhosa* spores; CE was an overexpression. PpMYC2-like POD and SOD activities and H2O2 content in callus inoculated with *Anthracis fruitii* spores at 0 h, 6 h, and 24 h. Data were analyzed using one-way ANOVA. Error bars in the figure represent three independent variances. p The difference at the <0.001 level was extremely significant. represent p The difference at the <0.0001 level was extremely significant.

[0021] Figure 6 In Embodiment 6 of the present invention PpMYC2-like- Expression analysis of resistance-related genes in lines 1, 2, and 3, Figure AF: qRT-PCR analysis of nine differentially expressed genes. Data were analyzed using one-way ANOVA; error bars in the figure represent the standard errors of three independent replicates. represent p The difference at the <0.01 level was highly significant. represent p The difference at the <0.001 level was extremely significant. represent p The difference at the <0.0001 level was extremely significant. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0023] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0024] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0025] Example 1 PpMYC2-like Genes (pear) MYC2-like Gene amplification and self-activation detection This invention utilizes bioinformatics methods combined with PCR-specific amplification to clone genes. Specifically, it is based on predictions from the 'Crown' genome database (https: / / bigd.big.ac.cn / gwh). MYC2-like Primers were designed based on the coding region sequence (EVM0040969). The gene amplification primer sequences are shown below: MYC2-like Forward primer ( MYC2-like -F): 5'-ATGGAGGAAATTATATCCTCTTG-3', (SEQ ID NO: 3) MYC2-like Reverse primer ( MYC2-like -R): 5'-CTAGTTGTACCATCTGTTCAA-3' (SEQ ID NO: 4).

[0026] This invention uses cDNA from 'Cuiguan' tissue culture seedlings as a template for PCR amplification. The PCR amplification system is as follows: 2X Phanta Max Buffer 25 μL, cDNA 1 μL, dNTP Mix (10 mM) 1 μL, forward / reverse primers 2 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and ddH2O added to a final volume of 50 μL. Figure 1As shown, the amplified MYC2-like CDS fragment (SEQ ID NO: 1) is 1446 bp in length and encodes 481 amino acids (SEQ ID NO: 2). Verification using a yeast self-activation detection system revealed... PpMYC2-like The full-length gene has transcriptional activation activity.

[0027] Example 2 PpMYC2-like Subcellular localization and amino acid sequence analysis of genes according to PpMYC2-like Based on the cDNA sequence, subcellular localization vector primers were designed: 1300-MYC2-like-F: 5'-GAGAACACGGGGGACTCTAGAATGGAGGAAATTATATC-3'; (SEQ ID NO: 5) 1300-MYC2-like-R: 5'-GCCCTTGCTCACCATGGATCCGTTGTACCATCTGTTCAA-3' (SEQ ID NO: 6) In order to determine PpMYC2-like The specific localization within the plant was investigated using the pCAMBIA1300-GFP vector, and the results are as follows: Figure 2 As shown in Figure A, leaves co-injected with Agrobacterium pCAMBIA1300 and Agrobacterium nucleatum exhibit green cells under green fluorescence and red nuclei under red fluorescence; the nuclei overlap in the fused image. pCAMBIA1300- PpMYC2-like Tobacco cells co-injected with Agrobacterium and Agrobacterium nucleatum showed only green nuclei under green fluorescence and red nuclei under red fluorescence; the nuclei overlapped in the fused image. This indicates... PpMYC2-like It is a nuclear localization protein.

[0028] cloned PpMYC2-like The gene amino acid sequence was submitted to the NCBI database for sequence alignment. Conserved Domain Search software was used to download amino acid sequences of genes with high homology to other species. MEGA and TBtools software were then used for amino acid sequence evolution analysis and conserved domain alignment analysis (e.g., ...). Figure 2 (B and C), the results are shown in the figure: PpMYC2-like Egg white and apple MdMYC4-like、 European pear PbMYC4-like The conserved structural domains are most similar, and based on the results of amino acid evolution analysis, it was further named... PpMYC2-like .

[0029] Example 3 PpMYC2-like Gene expression patterns and promoter sequence analysis To investigatePpMYC2-like Gene expression patterns were further investigated in this study after 'Green Crown' was inoculated with anthracnose. PpMYC2- like Gene expression was analyzed. The results are as follows: Figure 3 As shown in A: PpMYC2-like The expression was significantly upregulated at 6 h and 12 h after anthrax inoculation, indicating that... PpMYC2-like The response to pathogen induction was observed. Simultaneously, 'Cuiguan' pear tissue culture seedlings were treated with exogenous MeJA at a concentration of 0.1 mM, and samples were taken at 6, 12, and 24 hours for analysis. PpMYC2-like Gene expression. The results are shown in the figure: PpMYC2-like MeJA-treated groups showed significantly higher expression levels at 6, 12, and 24 h compared to the control group, while the SA-treated group... PpMYC2-like The expression level was not significantly different from the control (e.g. Figure 3 (As shown in B).

[0030] Extracted from the 'Cuiguan' pear genome file PpMYC2-like The promoter sequence was determined, and primers were designed using Primer 5.0 to clone 'Cuiguan' leaf DNA using a template. Plant Care software was used for further analysis. PpMYC2-like The promoter element was analyzed. The results are as follows: Figure 3 As shown in C: PpMYC2-like The promoter contains multiple MYB, MYC, ABA, and G-box binding sites, indicating that... PpMYC2-like The differences in expression may be related to upstream regulation.

[0031] Example 4 PpMYC2-like Evaluation of transgenic anthrax resistance The 1443 bp coding sequence (excluding the stop codon) of the amplified PpMYC2-like gene was ligated into the overexpression vector pK7WG2D plasmid using homologous recombination. Positive clones were selected for sequencing, and the correctly sequenced plasmids were transformed into Agrobacterium strain GV3101. The obtained single clones were identified as positive by PCR. Agrobacterium-mediated transformation was then used to transform 'Pear and Eggplant' callus.

[0032] For the obtained PpMYC2-like Analysis of overexpression of 'eggplant-pear' callus revealed five overexpression lines. PpMYC2-like Gene expression was more than 40 times that of wild-type callus (e.g. Figure 4 (As shown in Figure A). Further evaluation of the overexpression of callus anthrax resistance was conducted, and the results are as follows: Figure 4 Figures B and C show the overexpression lines. PpMYC2-like The diameter of lesions in strains -1, 2, 3, and 5 inoculated with anthrax was significantly smaller than that of wild-type callus at 3 and 5 days after inoculation. PpMYC2-like -4 strain showed significantly smaller lesion diameters than wild-type callus at 5 days after inoculation.

[0033] Example 5: Analysis of Hormone and Enzyme Activity Indicators in PpMYC2 Transgenic Lines In order to determine PpMYC2-like Changes in physiological and biochemical indicators of overexpression lines were observed in this study. PpMYC2-like- Three transgenic lines, 1, 2, and 3, were tested. The results showed that the jasmonic acid (JA) content in the overexpression lines was higher than that in the wild type at 0 h, but the difference was not statistically significant; however, the JA content in the overexpression lines was significantly higher than that in wild-type callus (e.g., *Anthracis cirrhosa*) at 24 h after inoculation. Figure 5 (As shown in Figure A); SA content was higher in callus 24 h after inoculation than in wild-type inoculated callus, but the difference was not statistically significant (e.g., Figure 5 (As shown in Figure B). Explanation PpMYC2-like It can regulate SA content to a certain extent.

[0034] The results of enzyme activity and other index measurements showed that the activities of peroxidase (POD) and superoxide dismutase (SOD) were higher than those of wild-type callus before and after inoculation in all three transgenic lines. Furthermore, the POD activities of the three transgenic lines showed a significant difference 24 hours after inoculation (e.g., ...). Figure 5 As shown in Figure C), SOD activity reached a significant level 6 h after inoculation (e.g., Figure 5 (As shown in D). The hydrogen peroxide content was lower in the three transgenic lines than in the wild-type callus (e.g., ...). Figure 5 As shown in Figure E), the content decreased 6 hours after inoculation. These results indicate that: PpMYC2-like Genes play an important regulatory role in the JA signaling pathway and reactive oxygen species metabolism pathway.

[0035] Example 6 PpMYC2-like - Expression analysis of resistance-related genes in lines 1, 2, and 3 The expression of lignin and resistance-related genes was screened and analyzed. The primer sequences for quantitative fluorescence analysis are as follows: The internal reference primer used was pear actin, and the primer sequence is as follows: Actin-qF:5'-TGGTGTCATGGTTGGTATGG-3' (SEQ ID NO: 7) Actin-qR:5'-CAGGAGCAACGAAGTTCA-3' (SEQ ID NO: 8) qCHI5-1F: 5'-CTGCCCGTTGTGTCCGTTGAG-3' (SEQ ID NO: 9) qCHI5-1R: 5'-CTTAGGTGGTCAGTCAGCTTCGC-3' (SEQ ID NO: 10) qPrx52-R: 5'-TGTCTGGTCCTGGCAAATGAAGC-3' (SEQ ID NO: 11) qPrx52-F: 5'-AGGTTCCGAGCTGTAGGTCTCTC-3' (SEQ ID NO: 12) qPR1-F: 5'-GAGCAGCAGTAGGCGTTGGTCCC-3' (SEQ ID NO: 13) qPR1-R: 5'-TAGTCGGCTTTCTCCGCCACCCA-3' (SEQ ID NO: 14) qOMT1-F: 5'-AACCAAGACCCCTGACGGACC-3' (SEQ ID NO: 15) qOMT1-R: 5'-ACCTTACGCCAGCCTTTTACG-3' (SEQ ID NO: 16) qCCR1-F: 5'-ATGGCGGTGAGGCGGCTGAT-3' (SEQ ID NO: 17) qCCR1-R: 5'-CTTGATGTCTCCTGGTTGCCT-3' (SEQ ID NO: 18) qPER4-F: 5'-CCAAACGCTCTTATCACTATC-3' (SEQ ID NO: 19) qPER4-R: 5'-GGCAGACCTTCTCCACTTCAG-3' (SEQ ID NO: 20) Leaves from 'Cuiguan' tissue culture seedlings that had been subcultured for approximately 30 days were inoculated separately with sterile water and anthracnose spore solution, with a spore solution concentration of 1×10⁻⁶. 8 CFU / mL, one well was punctured on each side of the vein of 'Cuiguan' pear leaves using an inoculation needle. 5 μL of sterile water or spore solution was added to each well. Samples were collected at 6 h, 12 h, and 24 h post-inoculation. RNA was extracted using the Omega Plant RNA Extraction Kit (OMEGA, USA). First-strand cDNA synthesis was performed using the Maxima H Minus First Strand cDNA kit (Thermo Fisher Scientific, Shanghai, China). The qPCR reaction system consisted of: 5 μL SYBR Green PCR Master Mix, 1 μL cDNA, 0.8 μL each of forward and reverse primers, and 2.4 μL sterile water. The qPCR program was: 95℃, 30 s; 95℃, 10 s; 60℃, 30 s, 40 cycles. Results are as follows: Figure 6 As shown.

[0036] MYC2-like Genes derived from crown Pyrus pyrifolia MYC2-like Gene coding region sequence (SEQ ID NO: 1) The amino acid sequence encoded by the MYC2-like gene of the crown jasmine (SEQ ID NO: 2) MEEIISSCSSQNFGQENSPTQQRLQFLLQNRPERWIYSIFWEASKHSNDLVSLSWAGGHFQSTRDLTSQKTYNKISHHQPKLGFDVLERKRAISREVEALFHEDMDVDGGDVTDAEWFYF YTVSLTQSFTAGHGNSNILGRTFCSGGFVWLAGDHELQFYECERVKEARMHGIQTLVCIATSCGVLELASLDVIKENWGLVQLSKSLFESDNTTTTTDKVSKQLSSRLENHVPLLESGMF SGADKKRVGNTRWYKEAAPVNVGGSSSDSGPSDSVGNLTSKNTENIRLKKRGRSSNRETGRPESPINHVEAERQRREKLNCRFYALRSVVPNVSRMDKASLLSDAVVYINELKARVGQLE SKIQAQLQKPGVDIGGQTSSIVDHHQTRPRLSSSYNNRAVGDMEMDVKIVGSEAMLRVQCPDCNYPNARLMNALKDLELQVYHASISSVKELMIQDVVVRVPHGFTSEEAMRTAILNRWYN The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of pear MYC2-like Genes, characterized by, Its coding region nucleotide sequence is shown in SEQ ID NO:

1.

2. The pear according to claim 1 MYC2-like Genes, characterized by, The encoded amino acid sequence is shown in SEQ ID NO:

2.

3. The pear according to claim 1 MYC2-like The application of genes in regulating resistance to pear anthracnose is characterized by, By promoting the synthesis of jasmonic acid and the expression of genes related to pear lignin synthesis, the resistance of pear to anthracnose can be increased.

4. The pear according to claim 1 MYC2-like Application of genes in the breeding of anthracnose-resistant pear varieties.

5. A type of pear MYC2-like Gene overexpression vector, characterized in that, It was obtained by digesting the fragment shown in SEQ ID NO:1 into the pK7WG2D vector.

6. A method for cultivating anthracnose-resistant pear varieties, characterized in that, The pear as described in claim 1 MYC2-like Genes were introduced into the target plant to obtain transgenic pears with enhanced resistance to anthracnose.

7. The cultivation method according to claim 6, characterized in that, The pear variety is any one of the following: eggplant pear, Dangshan crisp pear, Korla fragrant pear, Cuiguan pear, and Qiuyang pear.

8. The cultivation method according to claim 7, characterized in that, The pear variety mentioned is the eggplant pear.

9. The cultivation method according to claim 7, characterized in that, Specifically, the following steps are included: (1) Cloning the pear as described in claim 1 MYC2-like Gene; (2) Constructing a pear MYC2-like Gene overexpression vectors; (3) Pear MYC2-like The gene overexpression vector was transformed into the target plant, and the transgenic pear with improved anthracnose resistance was obtained after identification.

10. The cultivation method according to claim 9, characterized in that, The method of transformation into the target plant can be either Agrobacterium-mediated transformation or gene gun transformation.

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