Method for identifying variable shear of pine wood nematodes

Abstract

The invention discloses a method for identifying variable shearing of bursaphelenchus xylophilus, and relates to the technical field of genetic diversity analysis. The method comprises the following steps: analyzing whole genome information by using bioinformatics software which autonomously participates in writing, designing specific primers about 200bp before and after a pectate lyase fragment for predicting existence of the variable shear phenomenon, amplifying a product through PCR (Polymerase Chain Reaction), and purifying. And introducing the purified fragment and a target fragment product connected with the carrier into escherichia coli competent (DH5alpha), carrying out mass replication and cloning, and then introducing into an LB culture medium with ampicillin antibiotic for overnight growth. A single colony with a fragment sequence is selected for sequencing and checking whether a sequence is consistent with a transcriptome template sequence or not. A sequencing result is imported into bioinformatics analysis software DNAMAN-Multiplex Sequence Aligient to carry out multi-sequence alignment, and it is found through type analysis that different insect strains contain different types of variable cleavage. The invention takes the design of specific primers as a breakthrough point.

Description

Technical Field

[0001] The invention relates to the technical field of genetic diversity analysis, in particular to a method for identifying variable shearing of pine wood nematodes. Background Art

[0002] Pine wilt disease (PWD), also known as pine wilt, is a systemic infectious disease caused by the pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle). It mainly harms Pinus plants, but can also harm a few non-Pinus conifers, such as Larix olgensis. North America is considered to be the origin of pine wood nematodes. However, the pathogenicity of pine wood nematodes to native pine trees is not serious, and it only causes death to some introduced pine trees.

[0003] China is currently the country with the most serious pine wilt disease epidemic. In 1982, pine wilt disease was first discovered in the Sun Yat-sen Mausoleum in Nanjing, Jiangsu Province, causing the death of 265 black pines. In the same year, pine wilt disease was also confirmed on Masson pine (Pinus massoniana) in Hong Kong, China. In 1983, pine wilt disease occurred on sulfur pine in Taipei County, Taiwan Province, China. Over the past 40 years, the disease has continued to develop in China. In 2021, there were outbreaks in 742 county-level administrative regions in 19 provinces (autonomous regions, municipalities) across the country, covering an area of ​​1.7165 million hm2.

[0004] Pectin is a negatively charged acidic heteropolysaccharide that is mainly found in the cell walls of higher plants. When it cross-links with cellulose, lignin, etc. in plant tissues, it can give the cell wall rigidity and become a natural barrier to the invasion of pathogenic microorganisms. Therefore, for plant pathogens, the large number of cell wall degrading enzymes (including pectinases, cellulases, and proteases) present in nature enable them to break through the plant cell wall barrier and enter the host cell. In the process of interaction between microorganisms and plants, pectate lyases secreted by pathogenic microorganisms play an important role in degrading plant cell walls. They are usually secreted in the early stages of infection and are a potential virulence factor.

[0005] Alternative splicing, also known as alternative splicing (AS), refers to the process of producing different mRNA isomers in an mRNA precursor through different splicing methods. It is a common biological event in eukaryotes and an important mechanism for organisms to produce a variety of proteins. The existence of alternative splicing greatly enriches the diversity of genes. About 95% of gene sequences in the human genome have alternative splicing. Alternative splicing is often closely related to cancer or some genetic diseases. Therefore, alternative splicing is also an important direction for our research on the pathogenicity of pine wood nematodes.

[0006] However, it is worth noting that from the relatively low-level prokaryotes to the relatively complex high-level multicellular eukaryotes, the proportion of intron length in the entire organism's genes gradually increases, suggesting that introns may play an important role in regulating and inducing complex genomics, or a regulatory means to enrich the gene expression of organisms. The proportion of genes containing introns to the total number of genes in different organisms varies greatly. For example, in some relatively low-level organisms: Drosophila melanogaster, Arabidopsis thaliana and Caenorhabditis elegans, genes containing introns exceed 80% of the total number of genes. Previous studies on intron retention events by relevant scholars mainly focused on various model species and humans, but with the extension of time and in-depth technology for intron research, the important role of intron has become increasingly significant. The regulation of gene expression by introns was first reported in animal studies in 1983. Banerji et al. found that the enhancer of mouse immunoglobulin is located in intron, and the activity of this enhancer is cell-specific. Summary of the invention

[0007] 1. Technical issues to be resolved

[0008] In view of the deficiencies in the prior art, the present invention provides a method for identifying alternative splicing of the pectate lyase gene of pine wood nematode. The exon and intron regions of the gene are determined by comparing the whole genome sequence and the transcriptome analysis file, and the sequence of the existing insect strain is obtained by designing specific primers, amplifying gene fragments and sequencing. The sequence comparison and analysis is performed by Bioedit and DNAMAN software to determine whether there is an alternative splicing phenomenon, thereby providing a reliable innovative thinking method for the pathogenic mechanism of pine wood nematode, the pathogen of pine wilt disease.

[0009] (II) Technical solution

[0010] 1. To achieve the above objectives, the present invention is implemented by the following technical solutions: A method for identifying variable shearing of pine wood nematodes, specifically comprising the following steps:

[0011] S1. Use bioinformatics software to analyze the whole genome information and compare it with the transcriptome information to identify the exon and intron regions of the pectate lyase gene;

[0012] S2, extracting RNA of pine wood nematode and reversely transcribing it into cDNA;

[0013] S3. By analyzing the genomic data, specific primers were designed at the upstream and downstream of the pectate lyase gene, and the fragment of the pectate lyase gene was amplified by PCR;

[0014] S4, run the obtained PCR product on agarose gel to verify whether the band size is the same as predicted;

[0015] S5. After verification, the bands with the same size as the predicted result are cut and recovered, and the purified PCR products are connected to the PMD19-T vector;

[0016] S6. The product of the ligated vector was transferred into competent E. coli (DH5α), incubated at 37°C, 200 rpm for 1 hour, centrifuged at 8000 rpm for 3 minutes, and the obtained colony precipitate was poured into a bacterial culture medium with ampicillin added, and incubated at 37°C overnight;

[0017] S7, performing PCR amplification on the single colony on the bacterial culture medium, performing 30 cycles of amplification at 98°C for 5 min, 98°C for 30 s, 58°C for 30 s and 72°C for 5 min, and then performing an extension at 72°C for 10 min;

[0018] S8. Use 1% agarose electrophoresis gel to run the gel for verification. For 1% agarose electrophoresis gel, use 0.25g of agarose powder, add 25ml of 1×TAE solution, heat in a microwave oven for 30s, pour into the plate with the comb inserted, and after the temperature cools down, run the gel to verify whether the amplified PCR product meets the expectations.

[0019] S9. Send the colonies whose band sizes match the expected results to the company for sequencing, use Bioedit to cut the vector sequence, and use DNAMAN to align the sequences to observe whether there is variable splicing in the intron region.

[0020] Preferably, the gel running verification of the obtained PCR product in step S4 is performed by using agarose electrophoresis gel for gel running verification.

[0021] Preferably, the gel excision and recovery in step S5 is processed using a Vazyme gel excision and recovery kit.

[0022] Preferably, the method for analyzing the alternative splicing types of pine wood nematodes specifically comprises the following steps:

[0023] A1. Perform multiple sequence alignment between template sequence and sequencing sequence using DNAMAN software;

[0024] A2. Observe whether there is a phenomenon of full or partial retention of introns in the intron region of the sequencing sequence;

[0025] A3. The retained intron sequence is formed into a separate TXT file, which is compared with the intron sequence of the whole genome to observe the type of intron retention.

[0026] Preferably, the method for identifying the alternative splicing expression pattern of the pine wood nematode pectate lyase gene specifically comprises the following steps:

[0027] B1. Based on the reverse transcription gene file, specific upstream and downstream primers are designed in the normal exon region of the pectate lyase gene, and specific primers are designed in the upstream and downstream of the exon region retained by alternative splicing. Specific primers are also designed in the region between the normal exon and the exon retained by alternative splicing. These three pairs of primers can respectively amplify the region of the normal exon, the region retained by alternative splicing to become an exon to perform functions, and the region where the two work together;

[0028] B2. PCR amplify this part and verify whether the band size is consistent with the prediction by running agarose electrophoresis gel to determine whether the primer is specific;

[0029] B3. A semi-quantitative test was performed using Takara's QPCR reagent to determine the expression pattern of the pectate lyase gene.

[0030] Preferably, the method for extracting RNA of pine wood nematode in step S2 specifically comprises the following steps:

[0031] C1. Separate the nematodes cultured on the Botrytis cinerea medium using the Baermann funnel method, collect the nematode solution in a 10 ml centrifuge tube, and wash with 0.05% streptomycin sulfate to remove foreign bacteria;

[0032] C2. Place the cleaned pine wood nematodes in a 1.5 ml EP tube, add 100 ul of Trizol protective solution to the sample, and freeze it in liquid nitrogen;

[0033] C3. Grind the sample into powder using an electric grinding rod. During this period, it should be continuously put into liquid nitrogen for quick freezing to keep the temperature low so as to prevent high temperature from affecting the quality of the sample.

[0034] C4. Add 900ul Trizol to the powdered sample and let it stand for 10min.

[0035] C5. Centrifuge at 4°C and 12,000 rpm for 5 min. Transfer the supernatant to a new EP tube, add 200 ul of chloroform, shake well, and let stand for 5 min.

[0036] C6, centrifuge at 4°C and 12000 rpm for 15 min, take the supernatant into a new 1.5 ml EP tube, add an equal volume of isopropanol, pour and mix, and let stand for 10 min;

[0037] C7, centrifuge 4 at 4 ° C, 12000 rpm for 15 min to obtain a white precipitate, and discard the supernatant;

[0038] C8. Add 1 ml of 75% anhydrous ethanol, gently rotate the EP tube to clean and remove the influence of the isopropanol reagent, and centrifuge at 4°C and 14,000 rpm for 5 minutes;

[0039] C9, repeat the above steps C1-C8 and dry naturally for 5 minutes;

[0040] C10, add appropriate amount of DEPC water and dissolve.

[0041] Preferably, the method of reverse transcribing into cDNA in step S2 specifically comprises the following steps:

[0042] D1. The extracted RNA was quality tested by NanoDrop to ensure that its 230 / 260 and 260 / 280 ratios were within 1.8-2.0, and the integrity of the bands was verified by running agarose electrophoresis gel.

[0043] D2, determine the concentration of RNA, and divide it by 500 to obtain the mass of the initial RNA used for reverse transcription;

[0044] D3, using Takara kit, 4×gDNA wipe Mix-4ul, initial RNA and RNase free ddHH 2 O totaled 16ul, pipetted and mixed, and then placed in 42℃ PCR for 2min to eliminate whole genome contamination;

[0045] D4. Add 4ul of 5×HiScript QRT PCR to the reagents in step D3, perform PCR amplification at 50°C for 15min, and then perform PCR amplification at 85°C for 5s.

[0046] (III) Beneficial effects

[0047] The present invention provides a method for identifying variable shearing of pine wood nematodes. Compared with the prior art, it has the following beneficial effects: the method for identifying variable shearing of pine wood nematodes predicts variable shearing by using a prediction database (https: / / bioinformatics.mdanderson.org / TCGASpliceSeq / ); secondly, using the assembled pine wood nematode genome as a template, designing primers about 200bp before and after the site where variable shearing exists, PCR amplification to obtain products with variable shearing sequences, connecting the PMD19-T vector, introducing the DH5α competent state to obtain a large number of cloned strains, and sending them to the company for sequencing, different types of variable shearing sequences can be obtained, and the bioinformatics software written by independent participation can be used to analyze the whole genome information, and the primers are designed by expanding about 200bp before and after the pectate lyase fragment where the variable shearing phenomenon is predicted, and the product is amplified by PCR. The product is cut and recovered to obtain a purified fragment gene. In order to successfully transform and improve the transformation efficiency, the target fragment product connected to the vector is introduced into the competent state of Escherichia coli (DH5α), a large number of replication clones are performed, and then introduced into the LB medium with ampicillin for overnight growth. The single colony with the fragment sequence is sequenced to check whether the sequence is consistent with the template sequence. The sequencing results contain different types of variable splicing sequences. Through BLAST comparison, type analysis is performed to provide a basis for inferring the pathogenicity of different variable splicing insect strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic diagram of the design of primers specific for amplifying the IR region of the present invention;

[0049] Figure 2 A primer design diagram for the intron portion retained by alternative splicing of the present invention;

[0050] Figure 3 A schematic diagram of the identification of sequences retained for the introns of the present invention;

[0051] Figure 4 It is the species variable splicing type obtained by comparing and analyzing the sequencing results in the present invention. DETAILED DESCRIPTION

[0052] 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.

[0053] See also Figure 1-4The embodiment of the present invention provides three technical solutions: a method for identifying variable shearing of pine wood nematodes, specifically including the following embodiments:

[0054] Embodiment 1: A method for identifying variable shearing of pine wood nematodes, comprising the following steps:

[0055] S1. Use bioinformatics software to analyze the whole genome information and compare it with the transcriptome information to identify the exon and intron regions of the pectate lyase gene;

[0056] S2, extracting RNA of pine wood nematode and reversely transcribing it into cDNA;

[0057] S3. By analyzing the genomic data, specific primers were designed at the upstream and downstream of the pectate lyase gene, and the fragment of the pectate lyase gene was amplified by PCR;

[0058] S4, running the obtained PCR product on gel to verify whether the band size is the same as the predicted one, and running the obtained PCR product on gel is to use agarose electrophoresis gel for running verification;

[0059] S5. After verification, the bands with the same size as the predicted result are cut and recovered by gel excision. The purified PCR products are connected to the PMD19-T vector. The gel excision recovery is processed by Vazyme gel excision recovery kit;

[0060] S6. The product of the ligated vector was transferred into competent E. coli (DH5α), incubated at 37°C, 200 rpm for 1 hour, centrifuged at 8000 rpm for 3 minutes, and the obtained colony precipitate was poured into a bacterial culture medium with ampicillin added, and incubated at 37°C overnight;

[0061] S7, performing PCR amplification on the single colony on the bacterial culture medium, performing 30 cycles of amplification at 98°C for 5 min, 98°C for 30 s, 58°C for 30 s and 72°C for 5 min, and then performing an extension at 72°C for 10 min;

[0062] S8. Use 1% agarose electrophoresis gel to run the gel for verification. For 1% agarose electrophoresis gel, use 0.25g of agarose powder, add 25ml of 1×TAE solution, heat in a microwave oven for 30s, pour into the plate with the comb inserted, and after the temperature cools down, run the gel to verify whether the amplified PCR product meets the expectations.

[0063] S9. Send the colonies whose band sizes match the expected results to the company for sequencing, use Bioedit to cut the vector sequence, and use DNAMAN to align the sequences to observe whether there is variable splicing in the intron region.

[0064] In the embodiment of the present invention, the method for extracting RNA of pine wood nematode in step S2 specifically comprises the following steps:

[0065] C1. Separate the nematodes cultured on the Botrytis cinerea medium using the Baermann funnel method, collect the nematode solution in a 10 ml centrifuge tube, and wash with 0.05% streptomycin sulfate to remove foreign bacteria;

[0066] C2. Place the cleaned pine wood nematodes in a 1.5 ml EP tube, add 100 ul of Trizol protective solution to the sample, and freeze it in liquid nitrogen;

[0067] C3. Grind the sample into powder using an electric grinding rod. Keep it in liquid nitrogen for quick freezing to keep the temperature low so that high temperature will not affect the quality of the sample.

[0068] C4. Add 900ul Trizol to the powdered sample and let it stand for 10min.

[0069] C5. Centrifuge at 4°C and 12,000 rpm for 5 min. Transfer the supernatant to a new EP tube, add 200 ul of chloroform, shake well, and let stand for 5 min.

[0070] C6, centrifuge at 4°C and 12000 rpm for 15 min, take the supernatant into a new 1.5 ml EP tube, add an equal volume of isopropanol, pour and mix, and let stand for 10 min;

[0071] C7, centrifuge 4 at 4 ° C, 12000 rpm for 15 min to obtain a white precipitate, and discard the supernatant;

[0072] C8. Add 1 ml of 75% anhydrous ethanol, gently rotate the EP tube to clean and remove the influence of the isopropanol reagent, and centrifuge at 4°C and 14,000 rpm for 5 minutes;

[0073] C9, repeat the above steps C1-C8 and dry naturally for 5 minutes;

[0074] C10, add appropriate amount of DEPC water and dissolve.

[0075] In the embodiment of the present invention, the method for reverse transcription into cDNA in step S2 specifically comprises the following steps:

[0076] D1. The extracted RNA was quality tested by Nano Drop to ensure that its 230 / 260 and 260 / 280 ratios were within 1.8-2.0, and the integrity of the bands was verified by running agarose electrophoresis gel.

[0077] D2. After determining the concentration, 500 μg RNA of each sample was used for reverse transcription reaction to obtain cDNA;

[0078] D3, using Takara kit, 4×gDNA wipe Mix-4ul, initial RNA and RNase free ddHH 2 O totaled 16ul, pipetted and mixed, and then placed in 42℃ PCR for 2min to eliminate whole genome contamination;

[0079] D4. Add 4ul of 5×HiScript QRT PCR to the reagent in step D3, place it at 50°C for PCR amplification for 15 minutes, and then place it at 85°C for PCR amplification for 5 seconds.

[0080] Embodiment 2: The difference between the embodiment of the present invention and the embodiment 1 is that the method for analyzing the alternative splicing type of pine wood nematode specifically comprises the following steps:

[0081] A1. Perform multiple sequence alignment between template sequence and sequencing sequence using DNAMAN software;

[0082] A2. Observe whether there is a phenomenon of full or partial retention of introns in the intron region of the sequencing sequence;

[0083] A3. The retained intron sequence is formed into a separate TXT file, which is compared with the intron sequence of the whole genome to observe the type of intron retention.

[0084] Example 3: The difference between the embodiment of the present invention and the embodiment 2 is that the method for identifying the alternative splicing expression pattern of the pine wood nematode pectate lyase gene specifically comprises the following steps:

[0085] B1. Based on the reverse transcription gene file, specific upstream and downstream primers are designed in the normal exon region of the pectate lyase gene, and specific primers are designed in the upstream and downstream of the exon region retained by alternative splicing. Specific primers are also designed in the region between the normal exon and the exon retained by alternative splicing. These three pairs of primers can respectively amplify the region of the normal exon, the region retained by alternative splicing to become an exon to perform functions, and the region where the two work together;

[0086] B2. PCR amplify this part and verify whether the band size is consistent with the prediction by running agarose electrophoresis gel to determine whether the primer is specific;

[0087] B3. A semi-quantitative test was performed using Takara's QPCR reagent to determine the expression pattern of the pectate lyase gene.

[0088] In this example, the numbers of alternative splicing types in different insect strains obtained by comparing and analyzing the sequencing results are shown in Table 1.

[0089] Table 1 Data on the types of alternative splicing in different insect strains

[0090]

[0091]

[0092] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0093] Due to the presence of IR, the PEL gene will produce multiple transcripts during the transcription process. Under normal transcription (N-IR), only Exon-1 and Exon-2 exist, but due to the presence of intron retention, the PEL gene consists of three parts: Exon-1, Intron, and Exon-2. The existence of variable splicing is randomly generated and not controlled by humans. When sequencing using Pel-F and Pel-R, it is easy to amplify transcript sequences that do not contain introns, which seriously reduces the identification efficiency. In order to facilitate sequencing experiments in multiple insect strains, this study used the specific primer design principles, took the intron-containing transcript sequence as a template, and designed primers in the upstream and downstream regions of IR. Specific primers that only amplify the IR region were redesigned, namely IR-F, IR-R (such as Figure 2 As shown), the primer sequences are: IR-F: AACTTTGATGTAGGTTG; IR-R: CTTGTCCCTGTTTGTAA.

[0094] The primer design of the IR part site uses the transcript with intron retention as the template. The downstream primer should contain the common sequence of Exon-1 and Intron, so that the primer can only bind to the sequence containing Intron during amplification to ensure the accuracy of intron gene expression. Because there are introns of different lengths in variable splicing, when designing primers for amplifying the intron retention part, it should be noted that the upstream and downstream primers should be set in the stable regions upstream and downstream of the retention region, respectively, to ensure the stability and effectiveness of amplification. The primer sequences are as follows, Intron-F: GCAGCCGATGGTATC, Intron-R: CAACCTACATCAAAGTT.

[0095] 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.

[0096] 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 variable shearing of pine wood nematodes, characterized by: The specific steps include: S1. Use bioinformatics software to analyze the whole genome information and compare it with the transcriptome information to identify the exon and intron regions of the pectate lyase gene; S2, extracting RNA from pine wood nematodes and reverse-transcribing it into cDNA; S3. By analyzing genomic data, specific primers were designed upstream and downstream of the pectate lyase gene, and amplified the pectate lyase gene fragment by PCR; S4. Run the obtained PCR product on a gel to verify whether the band size is the same as the predicted one; S5. After verification, the bands with the same size as the predicted result were cut from the gel and recovered, and the purified PCR products were ligated to the PMD19-T vector; S6. Transform the product ligated with the vector into competent E. coli, incubate at 37°C, 200 rpm, shake for 1 hour, centrifuge at 8000 rpm for 3 minutes, pour the obtained colony precipitate onto bacterial culture medium supplemented with ampicillin, and incubate at 37°C overnight. S7. PCR amplification was performed on the single colony on the bacterial culture medium, and 30 cycles of amplification were performed in sequence: 98°C for 5 min, 98°C for 30 s, 58°C for 30 s, and 72°C for 5 min, followed by extension at 72°C for 10 min. S8. Run the gel on a 1% agarose gel for verification. For a 1% agarose gel, add 0.25 g of agarose powder to 25 ml of 1× TAE solution, microwave for 30 seconds, and pour into the plate with the comb inserted. After the temperature cools, run the gel to verify whether the amplified PCR product meets the expectations. S9. Send the colonies whose band sizes match the expected results to the company for sequencing, use Bio-edit to cut the vector sequence, and use DNAMAN to perform sequence alignment to observe whether there is variable splicing in the intron region.

2. The method for identifying variable shearing of pine wood nematodes according to claim 1, characterized in that: In step S4, the PCR product obtained is run on agarose electrophoresis gel for verification.

3. The method for identifying variable shearing of pine wood nematodes according to claim 1, characterized in that: In step S5, the gel excision and recovery is performed using a Vazyme gel excision and recovery kit.

4. The method for identifying variable shearing of pine wood nematodes according to claim 1, characterized in that: The method for analyzing the alternative splicing types of pine wood nematodes specifically comprises the following steps: A1. Perform multiple sequence alignment between the template sequence and the sequencing sequence using DNAMAN software; A2. Observe whether there is any complete or partial retention of introns in the intron region of the sequenced sequence; A3. Create a separate TXT file for the retained intron sequence and compare it with the intron sequence of the whole genome to observe the type of intron retention.

5. The method for identifying variable shearing of pine wood nematodes according to claim 1, characterized in that: The method for identifying the alternative splicing expression pattern of the pine wood nematode pectate lyase gene specifically comprises the following steps: B1. Based on the reverse transcription gene file, design specific upstream and downstream primers for the normal exon regions of the pectate lyase gene, design specific primers for the upstream and downstream regions of the exon region retained by alternative splicing, and design specific primers for the region between the normal exon and the exon retained by alternative splicing. These three primer pairs can respectively amplify the region of the normal exon, the region retained as an exon by alternative splicing to perform functions, and the region where both exons play a role; B2. PCR amplify this part and run the gel on agarose electrophoresis to verify whether the band size is consistent with the prediction to determine whether the primer is specific; B3. Semi-quantitative test was performed using Takara's QPCR reagent to determine the expression pattern of the pectate lyase gene.

6. The method for identifying variable shearing of pine wood nematodes according to claim 1, characterized in that: The method for extracting RNA of pine wood nematode in step S2 specifically comprises the following steps: C1. Isolate nematodes cultured on Botrytis cinerea medium using the Baermann funnel method, collect the nematode solution in a 10 ml centrifuge tube, and wash with 0.05% streptomycin sulfate to remove contaminants. C2. Place the cleaned pine wood nematodes in a 1.5 ml EP tube, add 100 μl of Trizol protective solution to the sample, and freeze it in liquid nitrogen; C3. Grind the sample into powder using an electric grinding rod. During this process, continuously freeze the sample in liquid nitrogen to maintain a low temperature to prevent high temperatures from affecting the quality of the sample. C4. Add 900 μl of Trizol to the powdered sample and let it stand for 10 minutes. C5. Centrifuge at 4°C and 12,000 rpm for 5 minutes. Transfer the supernatant to a new EP tube, add 200 μl of chloroform, shake well, and let stand for 5 minutes. C6. Centrifuge at 4°C and 12,000 rpm for 15 min. Transfer the supernatant to a new 1.5 ml EP tube, add an equal volume of isopropanol, pour over to mix, and let stand for 10 min. C7, centrifuge 4 at 4 ° C, 12000 rpm for 15 min to obtain a white precipitate, and discard the supernatant; C8. Add 1 ml of 75% anhydrous ethanol, gently rotate the EP tube to clean and remove the effects of the isopropanol reagent, and centrifuge at 4°C and 14,000 rpm for 5 minutes; C9. Repeat steps C1-C8 above and dry naturally for 5 minutes; C10, add appropriate amount of DEPC water and dissolve.

7. The method for identifying variable shearing of pine wood nematodes according to claim 1, characterized in that: The method for reverse transcription into cDNA in step S2 specifically comprises the following steps: D1. Measure the quality of the extracted RNA using a NanoDrop to ensure that its 230 / 260 and 260 / 280 ratios are within 1.8-2.0, and verify the integrity of the bands by running them on an agarose gel. D2. Determine the concentration of RNA and divide it by 500 to obtain the mass of the initial RNA used for reverse transcription; D3: Use the Takara kit, 4 μl of 4× gDNA wipe mix, 16 μl of initial RNA and RNase-free ddHH2O, pipette and mix thoroughly, then place in a 42°C PCR incubator for 2 minutes to eliminate genome-wide contamination. D4. Add 4 μl of 5×HiScript QRT PCR to the reagents in step D3, perform PCR amplification at 50°C for 15 minutes, and then perform PCR amplification at 85°C for 5 seconds.

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