Method for identifying bursaphelenchus xylophilus cellulase multi-copy gene
Through high-throughput sequencing and bioinformatics, the method identifies and characterizes multi-copy cellulase genes in pine wood nematodes, addressing the lack of theoretical basis for pathogenicity and aiding disease control.
Patent Information
- Application Number
- CN202411009050.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-26
- Publication Date
- 2024-12-24
AI Technical Summary
Existing methods fail to identify and utilize multi-copy genes related to pine wood nematode pathogenicity, lacking a reliable theoretical basis for copy number variation across different geographical origins.
A method involving high-throughput genome sequencing, bioinformatics analysis, and PCR amplification to identify and characterize multi-copy cellulase genes in pine wood nematodes from various geographical locations, using specific primers to verify copy number variations and tandem repeats.
Provides a reliable theoretical basis for understanding and regulating pine wood nematode pathogenicity, enabling effective disease prevention strategies.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic diversity analysis, in particular to a method for identifying multi-copy genes of pine wood nematode cellulase. Background Art
[0002] The pine wood nematode Bursaphelenchus xylophilusNickle originated in North America and is an invasive species. It is the pathogen of pine wilt disease. Pine wood nematodes have not caused major damage to native tree species in North America, but they pose a major threat to pine trees in Asia and Europe. According to statistics from my country's Forestry and Grassland Bureau, pine wood nematode disease outbreaks have occurred in more than 600 county-level administrative regions in 19 provinces (autonomous regions and municipalities directly under the central government), covering an area of 1.8092 million hm2 and killing 19.4703 million pine trees, causing indelible losses to my country's forestry industry.
[0003] In order to further understand the pine wood nematode, its whole genome sequencing results were published in 2011. Since the first publication of the genome assembly based on short-read sequencing, high-throughput sequencing has been frequently used to identify the pathogenicity of pine wood nematodes. The Glycoside Hydrolase45 Gene Family (GH45 gene family) endo-β-1,4-glucanase, which plays a role in degrading the cell wall during infection, is inherited from fungal genes. Copy number variation (CNV) refers to the change in the copy number fragment within the genomic sequence greater than 50bp between two individuals of a species due to insertion, deletion, duplication, complex multi-site variation, etc., causing the change of the main phenotype. Multi-copy genes are usually present in repetitive regions, such as segment duplications (SDs) and tandem repeats (TRs). TRs are unstable genomic elements that are repeated from beginning to end and are usually prone to mutation. Due to their unique polymorphism, TRs can be used to analyze new mutations and linkage inheritance and serve as genetic markers. Extensive studies have also revealed their potential roles in regulating pathogenic variation in microorganisms and human polygenic diseases.
[0004] At present, the multi-copy genes related to pine wood nematode pathogenicity have not been applied, and only other pathogenic genes have been studied. There is no reliable theoretical basis for the copy number variation of pine wood nematodes from different geographical origins. Summary of the invention
[0005] 1. Technical issues to be resolved
[0006] In view of the shortcomings of the prior art, the present invention provides a method for identifying multi-copy genes of pine wood nematode cellulase, which solves the problem that multi-copy genes related to pine wood nematode pathogenicity have not been applied, only other pathogenic genes have been studied, and there is no relevant reliable theoretical basis for the copy number variation of pine wood nematodes from different geographical origins.
[0007] (II) Technical solution
[0008] To achieve the above objectives, the present invention is implemented through the following technical scheme: a method for identifying multi-copy genes of pine wood nematode cellulase, first collecting pine wood nematode strains from different geographical locations, extracting their DNA and performing high-throughput sequencing; using bioinformatics software to analyze the whole genome information, obtain various tandem repeat (TR) sequence sites with CNV phenomena, and identify the types of multi-copy cellulase genes related to pathogenicity of pine wood nematode strains from different geographical origins.
[0009] Preferably, the method for identifying multiple copies of pine wood nematode cellulase genes comprises the following steps:
[0010] 1) Collect pine wood nematode-infected trees from different geographical sources, and separate the nematodes from the infected trees using the Baermann funnel method; collect the nematode fluid, expand the culture, and store the obtained nematode fluid in a 4°C refrigerator for later use;
[0011] 2) Extraction of pine wood nematode DNA;
[0012] 3) The qualified pine wood nematode DNA was subjected to high-throughput genome sequencing; the sequencing platform used was HiSeq4000, the sequencing method was non-strand-specific, 150bp double-end sequencing, the sequencing depth was >40x, and high-throughput sequencing of pine wood nematodes was performed;
[0013] 4) Use bioinformatics software to analyze the raw data returned by high-throughput sequencing to obtain CNV data, understand the distribution of CNVs in pine wood nematodes, and screen out pathogenicity-related CNV sequences;
[0014] 5) Design specific primers to identify CNV sequences and the TR sequences contained therein, and perform agarose gel electrophoresis verification;
[0015] 6) PCR amplification of pine wood nematode DNA from different geographical origins was performed using TR sequence-specific primers, the number of bands on agarose gel was observed, and classification was performed.
[0016] Preferably, in step 1, the nematode liquid is collected in a 15 mL centrifuge tube, 30 to 50 male and female pine wood nematodes in the nematode liquid are picked out under a microscope, inoculated into a PDA plate covered with Botrytis cinerea, and cultured at 25° C. until all the Botrytis cinerea is eaten; the cultured nematodes are collected in a 15 mL centrifuge tube using the Baermann funnel method, and centrifuged at 3500 rpm for 3 min; the supernatant is removed, 0.1% streptomycin sulfate is added in a 1:1 volume, shaken, allowed to stand for 5 to 10 min, centrifuged, and the supernatant is removed; the nematode liquid is repeatedly washed with sterile water for 3 times, and the obtained nematode liquid is stored in a 4° C. refrigerator for standby use.
[0017] Preferably, in step 2, the extraction method is: take 200 μL of the stored nematode solution into an Eppendorf tube, add 200 μL of nematode lysis solution, and mix; treat in a 65°C water bath for 1 hour, and shake once every 10 minutes; after cooling to room temperature, extract with an equal volume of phenol, centrifuge at 12000rpm for 20 minutes at 4°C, and take the supernatant; extract with an equal volume of phenol: chloroform: isoamyl alcohol, centrifuge; centrifuge with an equal volume of chloroform: isoamyl alcohol; add 1 / 10 volume of 3mmol / L sodium acetate and 2 times volume of anhydrous ethanol to the supernatant, and freeze at -80°C for 20 minutes; centrifuge at 12000rpm for 20 minutes, wash the precipitate twice with 70% ethanol, dry at room temperature, and air-dry the precipitate for 1 hour; add 100 μLTE to resuspend the precipitate, take 5 μL of it and dilute it 200 times with sterile water, determine the DNA concentration with a UV spectrophotometer, and set aside at -20°C.
[0018] Preferably, in steps 5-6, before sequencing, the concentration of the DNA extracted in 1.2 is first determined by Nanodrop2000, and then the DNA quality is detected by 1% agarose gel electrophoresis.
[0019] Preferably, in steps 5-6, among the 259 tested insect strains, CNVs are mainly divided into three categories, namely single copy, two copies and three copies, among which two copies account for the largest proportion, followed by single copy, and the least is three copies.
[0020] (III) Beneficial effects
[0021] The purpose of the present invention is to provide a method for identifying multi-copy genes of pine wood nematode cellulase, through Illumina high-throughput genome sequencing, using bioinformatics software to analyze the multi-copy sequence characteristics, to explore the copy number variation of different populations of pine wood nematodes from a molecular perspective, and to provide a reliable theoretical basis for the formulation of disease prevention and control strategies. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1GH45 gene identification, (a) visualization of GH45 genomic location, (b) PCR amplification results of four GH45 genes from AMA3 cDNA, (d) comparison of GH45 genes in AH1 and JP genomes; the missing copies GH45-2 and GH45-4 are marked with red rectangles;
[0023] Figure 2 .Identification of changes in tandem repeats (TRs) within the GH45 gene (a) Cloning and sequencing results of the TR region of the GH45_1 gene (b) The blue rectangle indicates the TR changes between GH45_1 and GH45_2 (c) The dotted double-headed arrow indicates the location where two copies of TRs (21 bp) were deleted;
[0024] Figure 3 .Sequence analysis of GH45 genes with different copy numbers, linear plot of ORF and TR positions between different GH45 copies - pink triangles indicate signal peptide cleavage sites;
[0025] Figure 4 .qPCR expression profile analysis of four GH45 copies (a) GH45_1 qPCR primer clone sequencing verification (b) GH45_2 qPCR primer clone sequencing verification;
[0026] Figure 5 .Copy number differences among different pine wood nematode strains (a) PCR reaction to confirm copy number changes (b) Illustration of different TRs among different pine wood nematode strains (c) Verification of different TR combinations among different pine wood nematode strains;
[0027] Figure 6 . Variation of cellulase gene copies in different populations of pine wood nematodes;
[0028] Figure 7 .Agarose gel electrophoresis was used to identify the copy numbers of TR sequences of GH45 gene of pine wood nematode in different populations. AH represents Anhui Province, CQ represents Chongqing City, FJ represents Fujian Province, SD represents Shandong Province, SC represents Sichuan Province, GD represents Guangdong Province, GZ represents Guizhou Province, GX represents Guangxi Zhuang Autonomous Region, HEN represents Henan Province, HN represents Hunan Province, HB represents Hubei Province, JS represents Jiangsu Province, SX represents Shaanxi Province, JX represents Jiangxi Province, LN represents Liaoning Province, ZJ represents Zhejiang Province, and YN represents Yunnan Province. DETAILED DESCRIPTION
[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] Example 1
[0031] 1) Collection and preservation of pine wood nematode strains
[0032] In this example, pine wood nematode diseased trees were collected from Guangdong Province and Sichuan Province (the specific geographical origins of the diseased trees are shown in Table 1), and the nematodes in the diseased trees were separated using the Baermann funnel method. The nematode liquid was collected in a 15 mL centrifuge tube, and 30 to 50 male and female pine wood nematodes in the nematode liquid were picked under a microscope, inoculated into a PDA plate covered with Botrytis cinerea, and cultured at 25°C until all the Botrytis cinerea were eaten (about 10 days).
[0033] The cultured nematodes were collected into a 15 mL centrifuge tube using the Baermann funnel method and centrifuged at 3500 rpm for 3 min; the supernatant was removed, 0.1% streptomycin sulfate was added in a 1:1 volume ratio, the mixture was shaken, allowed to stand for 5-10 min, centrifuged, and the supernatant was removed; the mixture was washed repeatedly with sterile water for 3 times, and the obtained nematode solution was stored in a 4°C refrigerator for later use.
[0034] 2) Extraction of pine wood nematode DNA
[0035] Take 200 μL of the stored nematode solution in an Eppendorf tube, add 200 μL of nematode lysis solution (200 mmol / LTris-HCl, 200 mmol / LNaCl, 100 mmol / LEDTA, 2% SDS, 2% β-mercaptoethanol, 200 μg / mL proteinase K), mix well; treat in a 65°C water bath for 1 hour, shake once every 10 minutes; after cooling to room temperature, extract with an equal volume (400 μL) of phenol, centrifuge at 12000 rpm for 20 minutes at 4°C, and take the supernatant; use an equal volume (400 μL) of phenol: chloroform: Extract with isoamyl alcohol (25:24:1) and centrifuge; use an equal volume (400 μL) of chloroform:isoamyl alcohol (24:1) and centrifuge; add 1 / 10 volume of 3 mmol / L sodium acetate (pH=4.6) and 2 volumes of anhydrous ethanol (-20°C) to the supernatant, and freeze at -80°C for 20 min; centrifuge at 12000 rpm for 20 min, wash the precipitate twice with 70% ethanol (pre-cooled at -20°C), dry at room temperature, and air-dry the precipitate for 1 h; add 100 μL of TE to resuspend the precipitate, take 5 μL of it and dilute it 200 times with sterile water, determine the DNA concentration with a UV spectrophotometer, and store at -20°C for use.
[0036] 3) High-throughput sequencing of pine wood nematodes
[0037] Before sequencing, the concentration of DNA extracted in 1.2 was first determined by Nanodrop2000, and then the DNA quality was tested by 1% agarose gel electrophoresis. The qualified pine wood nematode DNA was sent to Wuhan Future Group Biological Company for high-throughput genome sequencing. The sequencing platform used was HiSeq4000, and the sequencing method was non-strand-specific, 150bp double-end sequencing, with a sequencing depth of >40x.
[0038] 4) Data analysis
[0039] Identification of segmental duplications and tandem repeats was performed by BLAST based on result annotations. Multicopy gene comparisons were performed by DNAMAN (Lynnon, Quebec, Canada).
[0040] 5) Identification of multi-copy genes
[0041] At the genomic level, five primers were designed in the intergenic regions of the four GH45 genes and their presence was verified using different combinations. Appropriate primers were designed from the flanking sequences of the tandem repeats. The exact copy number can be assessed based on the length of the amplified product (upstream primer: CGTGGTGGAAGGTAAGAC, downstream primer: GTTGGAGTGGTGCTTGAT). The PCR program was as follows: 94°C for 2 minutes, 35 repeats of the next program (94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds), and finally 72°C for 10 minutes. The PCR product was verified using a 3% agarose electrophoresis gel.
[0042] The four GH45 genes were located in the same contig, adjacent to each other, with an interval of about 1.5 kb between genes. Based on the length assembly sequence (AH1), GH45-1 and GH45-2 were considered to be different copies of the same gene with a 42 bp insertion at the 3' end and a 19 bp variation at the 5' end. This difference may be caused by segmental duplication during evolution. GH45-3 and GH45-4 shared more variations at the 5' and 3' ends, and had no insertions and deletions. These two genes were judged to be two other copies of the GH45 gene. The existence of these four genes and the variation between the sequences were confirmed by PCR amplification and clone sequencing. GH45-1 has 6 copies of tandem repeat sequences, each with 21mers, while GH45-2 has only four copies. The open reading frames (ORFs) of the different copies are altered by nucleotide variations and tandem repeat sequences. The longest ORF of GH45-1 is 1056 bp, while the longest ORF of GH45-2 is only 1014 bp. TRs mutations found in GH45-1 and GH45-2 altered the ORF region, including two extra copies of the repeat polypeptide. In addition, the ORFs between GH45-3 (162 bp) and GH45-4 (288 bp) differed greatly. They shared the same start codon, but the stop codon was modified by the nuclear mutation. The location and type of the region were similar between GH45-1 and GH45-2, however, GH45-2 lacked a repeat region, which may be caused by the loss of two TRs.
[0043] Table 1. Overview of the different GH45 gene copies
[0044]
[0045] ＊ SP site indicates the signal peptide cleavage site.
[0046] Table 2. Summary of the copy numbers and geographical origins of different populations of pine nematodes
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[0048]
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[0058]
[0059]
[0060] It can be seen that the present application can divide 259 pine nematode strains from different geographical origins into three groups based on agarose gel electrophoresis technology, indicating that there is a copy number variation of the pathogenicity-related gene GH45 between pine nematode strains from different geographical origins, which is important for the study of the potential regulatory effect of pine nematode pathogenicity.
[0061] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions. The sentence "includes an element defined by ... does not exclude the existence of other identical elements in the process, method, article or device including the element".
[0062] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for identifying multiple copies of pine wood nematode cellulase genes, characterized in that: First, pine wood nematode strains from different geographical locations were collected, their DNA was extracted and high-throughput sequencing was performed; bioinformatics software was used to analyze the whole genome information, and the sites of various tandem repeat (TR) sequences with CNV phenomena were obtained, and the pathogenicity-related cellulase multi-copy gene types of pine wood nematode strains from different geographical origins were identified.
2. The method for identifying multiple copies of pine wood nematode cellulase genes according to claim 1, characterized in that: Here are the steps: 1) Collect pine wood nematode-infected trees from different geographical sources, and separate the nematodes from the infected trees using the Baermann funnel method; collect the nematode fluid, expand the culture, and store the obtained nematode fluid in a 4°C refrigerator for later use; 2) Extraction of pine wood nematode DNA; 3) The qualified pine wood nematode DNA was subjected to high-throughput genome sequencing; the sequencing platform used was HiSeq4000, the sequencing method was non-strand-specific, 150bp double-end sequencing, the sequencing depth was >40x, and high-throughput sequencing of pine wood nematodes was performed; 4) Use bioinformatics software to analyze the raw data returned by high-throughput sequencing to obtain CNV data, understand the distribution of CNVs in pine wood nematodes, and screen out pathogenicity-related CNV sequences; 5) Design specific primers to identify CNV sequences and the TR sequences contained therein, and perform agarose gel electrophoresis verification; 6) PCR amplification of pine wood nematode DNA from different geographical origins was performed using TR sequence-specific primers, the number of bands on agarose gel was observed, and classification was performed.
3. The method for identifying multiple copies of pine wood nematode cellulase genes according to claim 2, characterized in that: In step 1, the nematode liquid is collected in a 15 mL centrifuge tube, 30 to 50 male and female pine wood nematodes in the nematode liquid are picked out under a microscope, inoculated into a PDA plate covered with Botrytis cinerea, and cultured at 25° C. until all the Botrytis cinerea is eaten; the cultured nematodes are collected in a 15 mL centrifuge tube using a Baermann funnel method, and centrifuged at 3500 rpm for 3 min; the supernatant is removed, 0.1% streptomycin sulfate is added in a 1:1 volume, shaken, allowed to stand for 5 to 10 min, centrifuged, and the supernatant is removed; the nematode liquid is repeatedly washed with sterile water for 3 times, and the obtained nematode liquid is stored in a 4° C. refrigerator for later use.
4. The method for identifying multiple copies of pine wood nematode cellulase genes according to claim 2, characterized in that: In step 2, the extraction method is as follows: take 200 μL of the stored nematode solution into an Eppendorf tube, add 200 μL of nematode lysis solution, and mix; treat in a 65°C water bath for 1 hour, and shake once every 10 minutes; after cooling to room temperature, extract with an equal volume of phenol, centrifuge at 12000rpm for 20 minutes at 4°C, and take the supernatant; extract with an equal volume of phenol: chloroform: isoamyl alcohol, and centrifuge; centrifuge with an equal volume of chloroform: isoamyl alcohol; add 1 / 10 volume of 3mmol / L sodium acetate and 2 times volume of anhydrous ethanol to the supernatant, and freeze at -80°C for 20 minutes; centrifuge at 12000rpm for 20 minutes, wash the precipitate twice with 70% ethanol, dry at room temperature, and air-dry the precipitate for 1 hour; add 100 μL TE to resuspend the precipitate, take 5 μL of it and dilute it 200 times with sterile water, determine the DNA concentration with a UV spectrophotometer, and set aside at -20°C.
5. The method for identifying multiple copies of pine wood nematode cellulase genes according to claim 2, characterized in that: In step 5-6, before sequencing, the concentration of the DNA extracted in 1.2 was first determined using Nanodrop2000, and then the DNA quality was tested by 1% agarose gel electrophoresis.
6. The method for identifying multiple copies of pine wood nematode cellulase genes according to claim 2, characterized in that: In steps 5-6, among the 259 tested insect strains, CNVs were mainly divided into three categories, namely single copy, two copies and three copies, among which two copies accounted for the largest proportion, followed by single copy, and the least was three copies.