CN116656702B

CN116656702B - cDNA sequence for coding black pine leucine-rich repetitive sequence type receptor protein kinase and amino acid sequence and application thereof

Info

Publication number: CN116656702B
Application number: CN202310630413.7A
Authority: CN
Inventors: 张廷婷; 张宇; 李荣贵; 冯忠良; 张文靖; 董国颂
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2023-03-30
Filing date: 2023-05-30
Publication date: 2024-03-12
Anticipated expiration: 2043-05-30
Also published as: CN116656702A

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a cDNA sequence for coding black pine PthLRK1 gene, and an amino acid sequence and application thereof. The cDNA sequence is separated from black pine through a rapid amplification cDNA terminal path and named as PthLRK1 gene of black pine, the full length of the cDNA sequence is 3081bp, the cDNA sequence contains 2679bp open reading frame ORF, the cDNA sequence encodes 892 amino acids, the theoretical molecular weight is 99.63kDa, and the theoretical isoelectric point (pI) is 5.46; when the black pine is infected by pine wood nematodes, the expression level of the PthLRK1 gene is obviously changed, and the PthLRK1 gene has disease resistance; the cloning and functional analysis of the gene lay a foundation for researching the response mechanism of the black pine to the pine wood nematode stress and the regulation and control of the molecular level thereof, and have important significance for genetic engineering breeding of the black pine, prevention and control of pine wood nematode disease and detection and judgment of whether the black pine plant is infected with the pine wood nematode disease.

Description

cDNA sequence for coding black pine leucine-rich repetitive sequence type receptor protein kinase and amino acid sequence and application thereof

Technical field:

the invention belongs to the technical field of genetic engineering, relates to cloning and functional verification of genes related to pine wood nematode disease resistance of black pine seedlings, and particularly relates to a cDNA sequence for coding Leucine-rich repetitive sequence type receptor protein kinase (Leucine-rich-repeat receptor like kinases, LRR-RLKs, LRK) of black pine and an amino acid sequence and application thereof.

The background technology is as follows:

pine wood nematode disease (Bursaphelenchus xylophilus) is a worldwide serious quarantine forest disease. The pine wood nematode disease has fast disease development speed after infection of pine tree in Asia, great harm, difficult prevention and control and serious economic loss. There are 51 pine species, 16 non-pine species, which can be infected by pine nematode disease in nature.

Leucine-rich repetitive sequence type receptor-like protein kinase (Leucine-rich-repeat receptor like kinases, LRR-RLKs, LRK) is the largest type of transmembrane receptor kinase detected in plants, comprises an extracellular domain, a single transmembrane domain and an intracellular kinase domain, and can activate the phosphorylation/dephosphorylation activity of the intracellular kinase domain by combining the extracellular domain with an external signal molecule related to pathogenic bacteria and hormone signals, complete the transmembrane transport of signals, and further regulate the growth and development of plants, participate in hormone signal pathways and respond processes to external stresses. The biosynthetic pathway of leucine repeat type receptor-like protein kinases has been fully elucidated in Arabidopsis, but in Pinus koraiensis, no cloning of the LRK gene has been studied. Therefore, the gene of key enzyme involved in the process of resisting pine wood nematode disease of black pine is researched, cloning and functional research are carried out on the gene, so that the method is favorable for understanding the response mechanism of resisting pine wood nematode disease of black pine and the regulation and control of the molecular level of the gene, and has important significance for genetic engineering breeding of black pine, prevention and control of pine wood nematode disease and detection and judgment of whether black pine plants are infected with pine wood nematode disease.

The invention comprises the following steps:

the invention aims to overcome the defects of the prior art and provide a gene (PthLRK 1) sequence related to pine wood nematode disease resistance of black pine seedlings and application thereof, in particular to a cDNA sequence for coding leucine-rich repeat type receptor protein kinase (LRK) of black pine and an amino acid sequence and application thereof.

In order to achieve the above object, the present invention provides a cDNA sequence encoding a leucine-rich repeat type receptor-like protein kinase (LRK) of Pinus thunbergii and an amino acid sequence thereof, which are operated according to the following methods:

1. cloning of cDNA partial sequence of Pinus koraiensis PthLRK1 Gene

Extracting total RNA of Pinus koraiensis by referring to Trizol extraction kit (B511321, shanghai) and taking 0.5-2 μg total RNA as template, and synthesizing cDNA first strand by using first strand cDNA synthesis kit (B532435, shanghai) and freezing at-20deg.C for use.

According to the known LRK sequence data and the known black pine transcriptome sequencing data of the coastal pine of the near-edge species of the black pine, three pairs of primers are designed to carry out PCR amplification respectively, wherein the three pairs of primers are as follows:

Y1'-F：5′-GAACCGAACTGCAGAAAACAATAT-3′；

Y1'-R：5′-AACTTGGTTTTGATAGCCTCCAG-3′。

Y2'F：5′-AGATCCTTTTCCGTGAGAATAAATG-3′；

Y2'R：5′-TTAGTTTGGAATGATGTGCATTAGG-3′。

Y3F：5′-CCGTCAAATTGCTCTCACAGTTC-3′；

Y3R：5′-CCCAAGATTTGACGAGATGGTTAC-3′。

PCR amplification is carried out by taking a first strand of cDNA as a template, and the reaction system is as follows: primer F (10. Mu.M) 0.5. Mu.L, primer R (10. Mu.M) 0.5. Mu.L, dNTP (10 mM) 0.2. Mu.L, cDNA first strand template 1. Mu.L, taq enzyme (5U/. Mu.L)L)0.2μL，ddH ₂ O10.1. Mu.L. PCR procedure: pre-denaturation at 95℃for 3min,94℃for 30s,58℃for 30s,72℃for 90s, and cycle for 33 times, and extension at 72℃for 10min; after the obtained PCR products are separated by 1% agarose gel electrophoresis (as shown in figure 1A, B, C), target DNA is respectively recovered by using a SanPrep column type DNA gel recovery kit (B518131, manufactured and bioengineered (Shanghai) Co., ltd.), and respectively connected to T-Vector to construct T-PthLRK, and the vectors connected with the PCR products are respectively transformed into E.coli DH5 alpha, and positive clones are selected and sequenced; and respectively carrying out PCR amplification by using the three pairs of primers to obtain fragments with the lengths of 1017bp, 1222bp and 1099bp, and splicing to obtain the large fragment of the black pine PthLRK1 gene cDNA sequence.

2. Cloning of cDNA sequence at 3' end of Pinus koraiensis PthLRK1 gene

3'-RACE method is adopted for cloning the cDNA sequence at the 3' -end, according to the specification of a 3'-RACE kit (B605101, manufactured and bioengineered (Shanghai) Co., ltd.) and a proper amount of total RNA is used as a template, 3'adaptor GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTT is used as a primer, and under the action of Reverse Transcriptase Mix (RNase H-) reverse transcriptase, the cDNA is incubated for 30min at 50 ℃ and heated for 1min at 85 ℃ to synthesize a first cDNA strand, and the first cDNA strand is preserved at-20 ℃; primers (primers were synthesized by Biotechnology (Shanghai) Co., ltd.) were designed based on the large fragment cDNA sequence of the Pinus koraiensis PthLRK1 gene, and two rounds of PCR were performed. The primer sequences were as follows:

NF9：5′-TCTCTTTATCTGTGGATTCGACTGC-3′；

NF10：5′-GAGGAATGGAGTGGAGTGACAAC-3′。

5.3′outer：5′-GCTGTCAACGATACGCTACGTAAC-3′；

5.3′inner：5′-GCTACGTAACGGCATGACAGTG-3′。

the first strand of cDNA obtained by using the 3' adapter as a reverse transcription primer is used as a template, and two rounds of PCR amplification are carried out by using two primers, wherein the reaction system is as follows:

PCR procedure: the first round is a cool down PCR: pre-denatured 60s at 94 ℃, 30s at 94 ℃,70 ^*1 C (1 represents a 1 ℃ drop per cycle) for 30s,72 ℃ for 60s, 10 cycles; a second wheel: 94 ℃ for 30s,60 ℃ for 30s,72 ℃ for 60s, 25 times of circulation and 72 ℃ for 480s; after the obtained PCR product was separated by 1% agarose gel electrophoresis (as in FIG. 1D), the target DNA was recovered by using SanPrep column type DNA gel recovery kit (B518131, shanghai) and ligated to T-Vector to construct T-PthLRK, the Vector ligated with the PCR product was transformed into E.coli DH 5. Alpha., and the positive clone was selected and sequenced, the sequence size was 375bp.

3. Cloning of cDNA sequence of 5' end of Pinus koraiensis PthLRK1 gene

Cloning of the 5 '-end cDNA sequence was performed by using a 5' -RACE method according to the instructions of a 5'-RACE kit (B605102, shanghai manufacturing company) and using a proper amount of total RNA as a template, using a 5' -RACE reverse transcription specific primer (NRT 1), incubating at 50℃for 30min under the action of Reverse Transcriptase Mix (RNase H-) reverse transcriptase, heating at 85℃for 1min to synthesize a first cDNA strand, subjecting the first cDNA strand to RNase H digestion, cDNA purification recovery, tdT and C tail treatment, and preserving at-20 ℃.

Primers required for cloning the 5' -end cDNA sequence were designed based on the large fragment cDNA sequence of the Pinus koraiensis PthLRK1 gene (primers were synthesized by Biotechnology (Shanghai) Co., ltd.) and two rounds of PCR were performed. The primer sequences were as follows:

5′adaptor：GCTGTCAACGATACGCTACGTAACGGCATGACAGTGGGIIGGGIIGGGIIG。

NRT1：5′-GTCTCTTGACAATTCACAGGCTG-3′。

NR1：5′-CAGCAATCTGCCAGAGCTAACC-3′。

NR2：5′-TTTATCTGAGACCGCTACGTTTCC-3′。

5.3′outer：5′-GCTGTCAACGATACGCTACGTAAC-3′。

the first strand of the cDNA treated by TdT and C tail is used as a template, and two pairs of primers are used for carrying out two-round PCR amplification, and the reaction system is as follows:

PCR procedure: the first round is a cool down PCR: pre-denatured 60s at 94 ℃, 30s at 94 ℃,70 ^*1 30s at C (1 ℃ C. For each cycle), 60s at 72 ℃ C., 10 cycles; a second wheel: 94 ℃ for 30s,60 ℃ for 30s,72 ℃ for 60s, 25 times of circulation and 72 ℃ for 480s; after the obtained PCR product was separated by 1% agarose gel electrophoresis (FIG. 1E), the target DNA was recovered by using SanPrep column type DNA gel recovery kit (B518131, shanghai) and ligated to T-Vector to construct T-PthLRK, the Vector ligated with the PCR product was transformed into E.coli DH 5. Alpha., and the positive clone was selected and sequenced, the sequence size was 404bp.

4. Sequencing of full-length cDNA sequence of Pinus thunbergii PthLRK1 Gene

The 5 sequences (1017 bp, 1222bp, 1099bp, 375bp and 404 bp) obtained above were spliced to infer the full-length cDNA sequence of the Pinus koraiensis PthLRK1 gene, which is 3081bp in total. The ORF Finder (https:// www.ncbi.nlm.nih.gov/orffinder /) is used to search the open reading frame of the spliced full-length cDNA sequence, the ORF region of the black pine PthLRK1 gene is 2679bp, is positioned at 78-2756bp, and codes 892 amino acids. Three-dimensional structure prediction is carried out on the black pine PthLRK1 gene coding protein through a Phyre2 program, and the result shows that the protein has clear alpha-helix, beta-sheet and random coil.

Sequence 1: full-length cDNA sequence of Pinus koraiensis PthLRK1 gene

TAATTTCTGGTCTTGTGGAAGGATTTTTGAGTATCGACTGCGGAGGGAAAACGAACCGAACTGCAGAAAACAATATAATGTGGGTCACTGATGATAATTATATAGACGTGGGTCACAGAGGAGAGATCGGAAACGCTAGTGCTTACGGTTCTTACTTGCACACCTTGCGAGTTTTCCCAAAGCCTCTCAACAAGTCTTGCTATCAGTTGCCTGTGGTCCCTGATGTGCCTTATCTCTTAAGGTTATGGTTTGAAGTTGGAAATTACAGTGGATTCAAACAACTTCCAAGCTTTGCTTTCTCTATTGAGACAGAGGGTATGTTAGCTATGGGAAACGTGACGATCTCAGACTGTGACACACCATGTTATGATGAAATAATATTTGTTAGTTCTGGCAGAGTCCTGTACATTTGCTTGATCAGAACCTCGGAGTCTGATGATCCCTTCGTCTCTGCTATCGAGTTGAGAACGCTTCAACAAGGCATGTATGGACTGGCTAAGCCAGGAACAATGTTGATGCTAGTATGGAGATATGATGCTGGTGGAAATATTACCATTAGGTATCCTCAAGACATGTTCGACCGGATCTGGGATTACGAATTGAGAGTAACTTTTGGCAATCATTATCCTGTACAGCCTGTGAATTGTCGAGAGACCATTTCAACCAGCAATACCACAGAACTTCCTCCAAGTGCTGTGATGCAAACAGCGGCGGTTATTATTGACCCGGCACGTGCGTTCGATGTCGGATCGACTAATGGTCATAGATTATTATTGCTGTTATATGTTGCAGAGATCGAGCAGCTAAATATGTCTGAACATAGATCCTTTTATGTGACAATAAATGATGAGAAACGGTCTGAGGCCATTACTTCTCTGAGCGATTATTCTACCCGGGAGCTAAAATTTATATCCAATCCAACAGATGATTTCGATTTTGCCTTGGTCAACACTAGGGATGCAACCAGTGATCCCATAATGAACGCCTTTGAGGCTTACGAAATAATTGACACCCAACC

GGCAACATATGCGCAAGATAACAAAGCTCTTGAGGCTATCAAAAGCAGGTTTGGCGTAA

AGGATTGGATTTCTGATCCTTGTTTTTTGATCCAGTGGAATGGAATTGTGTGTGAAAGCA

GCACTTACCCCATAAGAATTTCGGAAATTGATTTGTCAGGAAAGAATCTTACAGGATTGG

TGCCCGACGATATTAGACAGTTGACGGCATTAGTTAAAGTGTCGCTTTACAATAATCATTT

GATAGGGCAATTGCCAAATTTGTCCAGTTTAACCATGTTGGAGAGATTGTATCTACAAAA

CAACAACCTGAGCGGTACTCTTCCGTCCTGGCTATGTGAACTGAAAAACCTAAAAGAAT

TGAATATAGAGAATAATAATTTCAGCGGTGTAATACCTGTCCAACTTCTCAATGGATCATT

GAAATTTAGTTACTGTGGAAACCCCTACTTACCTATGCATAAGGGGAAATGCACCCTGCA

TACTACAAATAAGAATAAATTGAAGATTATACTCGGAATAACACTAGGCGGAATATTAATC

ATCGCTTTAGCGCTGATAGTAGCTATTATTATGTATCGCAATAAATTCGGGCGAAAAGAAC

AGGGTATTGACAGCAGAAGAAGTGGACTGGGAATGGAACCTAGATCTTACTTGTACCAA

GACTACTCAATGGTTACCGTGCCAAATTCAACAAAATCCCGTTCCTTCACTCTAGATGAG

ATGATAGCGGCCACACAAGGCTTTAGCCAGGAGATCGGACGAGGAGGTTTTGGAGCGG

TGTTTTTAGGTAAATTGCCCGAAGGAAAATACATAGCCGTCAAATTGCTCTCACAGTTCT

CCCAACAAGGGGTTCAAGAATTCTTGAATGAGGTTGATCTTCTTTCCAGAATCCATCATA

AGAACTTGGTATCTTTACTGGGATATTGTAACGAATCAAGAGAAATTATGCTTATCTACGA

CTATATGGCAGAAGGGTCTTTAAGGGATCATCTTTCTGGTCCTAACGCACATCATTCCAA

ACTGACTTGGAGAGCCAGACTCAAAATAACTTTAGATGCAGCTCAAGGACTGGAATACC

TGCACGTTGGTTGCACCCCCAAAATAATTCACAGGGATATCAAGACCGCCAACATCTTGT

TAGACAGCGATTTGAATGGAAAACTAGGCGATTTCGGTCTTTCCAAGATGACAACTGAT

GGGGAGGCTACTCATGTTACCACTGCCGTAAAAGGAACTGCAGGATACCTGGATCCAGA

GTATTTCAACACTCAAATGTTGACGGAGAAGAGTGATGTGTACAGTTTTGGAGTGGTTT

TGCTGGAGATCATATGTGGTAGACAACCCATAGATCTAAAACTACCTGCAGAAGAACTG

AACATCGTTAGATGGGTGAAGCCGTATGTAGTGGAGAATGATAATCCTAGCAGAATATCA

GAAATCATTGACAAGAGGTTAGGTGGAGATTACGACATGACATCTATCACTAGTGTTGCC

AAAGTAGCGATGAGATGTGTTCACGCTGAACCGTGGTCTAGGCCGAACGTGAGCGAGAT

AGTGGCCGAGCTGAAAGAGGCTATCAAACATGAGGATCATCGTGCTTCTGTTTCAATTTC

AGAGGAAGCTGGTATTCAAAGTAGCGATTTGTCGTCGGGGCCAGTCTCTTTATCTGTGGA

TTCGACTGCACACGGAGGAATGGAGTGGAGTGACAACTCCAATATTTCTCAATTGGGAA

GGTAGGTTGGCACGTTGCTGATATGGATTGAAGGGAAGTTGGCCAAGAAGTCAAACCTA

CAGGTACTAGTGATGGCATGATTTACAATATGGGTTGATGTCGTGCATTTGACATAATTGT

GTCGCACACATCTGAAAAAATTGGTGAAACTTAAACTGTAACCATCTCGTCAAATCTTG

GGGTGGTACGTGGATGTATATACACGATCATACATTATGCTGAAACTAATCTTTATTAAATA

GGTATTGTGTATTTTAAATCTTCATATCAGTTTTAGAATTTTCCAGGTTAAAATGTTTACAA

ATTAATAATTTCAAAAAAAAAAAAAAA(SEQ ID NO:1)

Sequence 2: amino acid sequence encoded by black pine PthLRK1 gene ORF

MWVTDDNYIDVGHRGEIGNASAYGSYLHTLRVFPKPLNKSCYQLPVVPDVPYLLRLWFEVGNYSGFKQLPSFAFSIETEGMLAMGNVTISDCDTPCYDEIIFVSSGRVLYICLIRTSESDDPFVSAIELRTLQQGMYGLAKPGTMLMLVWRYDAGGNITIRYPQDMFDRIWDYELRVTFGNHYPVQPVNCRETISTSNTTELPPSAVMQTAAVIIDPARAFDVGSTNGHRLLLLLYVAEIEQLNMSEHRSFYVTINDEKRSEAITSLSDYSTRELKFISNPTDDFDFALVNTRDATSDPIMNAFEAYEIIDTQPATYAQDNKALEAIKSRFGVKDWISDPCFLIQWNGIVCESSTYPIRISEIDLSGKNLTGLVPDDIRQLTALVKVSLYNNHLIGQLPNLSSLTMLERLYLQNNNLSGTLPSWLCELKNLKELNIENNNFSGVIPVQLLNGSLKFSYCGNPYLPMHKGKCTLHTTNKNKLKIILGITLGGILIIALALIVAIIMYRNKFGRKEQGIDSRRSGLGMEPRSYLYQDYSMVTVPNSTKSRSFTLDEMIAATQGFSQEIGRGGFGAVFLGKLPEGKYIAVKLLSQFSQQGVQEFLNEVDLLSRIHHKNLVSLLGYCNESREIMLIYDYMAEGSLRDHLSGPNAHHSKLTWRARLKITLDAAQGLEYLHVGCTPKIIHRDIKTANILLDSDLNGKLGDFGLSKMTTDGEATHVTTAVKGTAGYLDPEYFNTQMLTEKSDVYSFGVVLLEIICGRQPIDLKLPAEELNIVRWVKPYVVENDNPSRISEIIDKRLGGDYDMTSITSVAKVAMRCVHAEPWSRPNVSEIVAELKEAIKHEDHRASVSISEEAGIQSSDLSSGPVSLSVDSTAHGGMEWSDNSNISQLGR(SEQ ID NO:2)

The full-length cDNA sequence of the Pinus koraiensis PthLRK1 gene is a complete cDNA sequence, is separated from Pinus koraiensis by a rapid amplification cDNA end approach (RACE) and is named as Pinus koraiensis PthLRK1. The full-length cDNA sequence is 3081bp, contains 2679bp Open Reading Frame (ORF), encodes 892 amino acids, and has a theoretical molecular weight of 99.63kDa and a theoretical isoelectric point (pI) of 5.46.

Using SignalP-5.0 programhttps://services.healthtech.dtu.dk) The predicted signal peptide effect indicates that the protein does not have a signal peptide region.

TMHMM-2.0 procedurehttps://services.healthtech.dtu.dk) The prediction of the transmembrane domain shows that the PthLRK1 gene coding protein has a single transmembrane region at 484-507 amino acids (figure 2A), and extends from outside to inside (figure 2B), belonging to a transmembrane protein.

The functional domain of the protein was predicted on the InterPro website (FIG. 3), and the result shows that a Malectin domain exists at the 2-309 amino acid positions, and is a membrane anchoring protein on the endoplasmic reticulum; the amino acid position 317-472 contains an LRR (Leucine-rich repeat domain) superfamily domain with disease resistance characteristics; at amino acid positions 562-386 there is a Serine-threonine, tyrosine-Protein kinase (Serine-threonine/tyrosine-Protein kinase) catalytic domain, which overlaps partially with this domain, with the superfamily of Protein kinases (Protein kinase-like domain) located at amino acid positions 543-836 and 560-840. These domains and the cognate superfamily constitute the main functional structure of the PthLRK1 protein, and function together as PthLRK1 genes.

Using BLAST programs, the nucleotide sequence of the Pinus thunbergii PthLRK1 gene was analyzed for homology to the nucleotide sequence of Pinus thunbergii (P.tabuliformis, KI 711073.1), picea glauca (BT 118800.1), pinus taeda (JQ 019187.1), picea megacephala (Picea sitchensis, BT 123520.1), ji Hudie orchid (Phalaenopsis equestris, XM_ 020737730.1), juglans regia (Carya illinoinensis, XM_ 043109997.1), lactuca sativa (Lactuca sativa, XM_ 023908240.3), and oil palm (Elaeis guineensis, XM_ 029268492.1), and as a result, the nucleotide sequence of Pinus thunbergii PthLRK1 gene was found to be the highest in homology to that of Pinus thunbergii (P.tabuliformis, KI 711073.1). The analysis result of the phylogenetic tree shows (figure 4) that the gene and the Chinese pine are on the same branch, the genetic relationship is relatively close, and the homology analysis result of the phylogenetic tree result and the nucleotide sequence keeps relatively high consistency.

The predicted protein (https:// www.predictprotein.org /) predicts the secondary structure of the protein, indicating 60.56% random coil, 25.08% alpha-helix and 14.36% beta-sheet.

Three-dimensional structure prediction is carried out on PthLRK1 gene coding protein through Phyre2 program, and the result shows that the protein has clear alpha-helix, beta-sheet and random coil (figure 5).

Leucine-rich repetitive sequence type receptor-like protein kinase (Leucine-rich-repeat receptor like kinases, LRR-RLKs, LRK) is the largest type of transmembrane receptor kinase detected in plants, comprises an extracellular domain, a single transmembrane domain and an intracellular kinase domain, and can activate the phosphorylation/dephosphorylation activity of the intracellular kinase domain by combining the extracellular domain with an external signal molecule related to pathogenic bacteria and hormone signals, complete the transmembrane transport of signals, and further regulate the growth and development of plants, participate in hormone signal pathways and respond processes to external stresses.

Compared with the prior art, the invention discovers that PthLRK1 gene participates in the disease resistance reaction of the black pine by researching the gene with the expression level obviously changed after the black pine is infected by the pine wood nematode, which indicates that the gene has disease resistance and successfully clones the gene; the method is helpful for understanding the response mechanism of the black pine to the pine wood nematode and the regulation and control of the molecular level thereof, and can be used for genetic engineering breeding of the black pine, prevention and control of pine wood nematode and detection and judgment of whether the black pine plant is infected with the pine wood nematode.

The drawings in the specification:

FIG. 1 is a cloning electrophoresis diagram of a PthLRK1 gene cDNA fragment of Pinus koraiensis, wherein A-C is a cloning electrophoresis diagram of a partial sequence of a PthLRK1 gene cDNA fragment of Pinus koraiensis, the primer of A is Y1'-F/R, the primer of B is Y2' F/R, and the primer of C is Y3F/R; d is a 3' -RACE amplification product electrophoretogram; e is an electrophoresis chart of a 5' -RACE amplification product; m:3000bp DNA Marker.

FIG. 2 is a predicted transmembrane domain of the PthLRK1 gene-encoded protein of Pinus koraiensis, wherein A is the single transmembrane region; b is an extension from extracellular to intracellular.

FIG. 3 is a diagram showing the prediction of the functional domain of the PthLRK1 gene-encoded protein of Pinus massoniana.

FIG. 4 shows a phylogenetic tree of the nucleotide sequences of the PthLRK1 gene of Pinus massoniana.

FIG. 5 is a three-dimensional structure diagram of the PthLRK1 gene encoded protein of Pinus massoniana.

FIG. 6 is a schematic representation of the expression of the PthLRK1 gene of black pine at various times after infection with pine wood nematodes.

The specific embodiment is as follows:

the invention is further described below with reference to specific embodiments and figures.

Example 1,

Cloning of the Pinus koraiensis PthLRK1 gene from Pinus koraiensis. FIGS. 1A-C are partial sequence cloning electrophoresis diagrams of the PthLRK1 gene cDNA of Pinus koraiensis; FIG. 1D is an electrophoretogram of the 3' -RACE amplification product (M: 3000bp DNA Marker); FIG. 1E is an electrophoretogram of the 5' -RACE amplification product (M: 3000bp DNA Marker).

The operation steps are as follows:

1. cloning of partial cDNA sequence of Pinus koraiensis PthLRK1 gene

Y1'-F：5′-GAACCGAACTGCAGAAAACAATAT-3′；

Y1'-R：5′-AACTTGGTTTTGATAGCCTCCAG-3′。

Y2'F：5′-AGATCCTTTTCCGTGAGAATAAATG-3′；

Y2'R：5′-TTAGTTTGGAATGATGTGCATTAGG-3′。

Y3F：5′-CCGTCAAATTGCTCTCACAGTTC-3′；

Y3R：5′-CCCAAGATTTGACGAGATGGTTAC-3′。

PCR amplification is carried out by taking a first strand of cDNA as a template, and the reaction system is as follows: primer F (10. Mu.M) 0.5. Mu.L, primer R (10. Mu.M) 0.5. Mu.L, dNTP (10 mM) 0.2. Mu.L, cDNA first strand template 1. Mu.L, taq enzyme (5U/. Mu.L) 0.2. Mu.L, ddH ₂ O10.1. Mu.L. PCR procedure: pre-denaturation at 95℃for 3min,94℃for 30s,58℃for 30s,72℃for 90s, and cycle for 33 times, and extension at 72℃for 10min; the obtained PCR products are separated by 1% agarose gel electrophoresis (as shown in figure 1A, B, C), target DNA is respectively recovered by using a SanPrep column type DNA gel recovery kit (B518131, shanghai) and respectively connected to a T-Vector to construct T-PthLRK, the vectors connected with the PCR products are respectively transformed into E.coli DH5 alpha, positive clones are selected and sequenced, PCR amplification is carried out by using the three pairs of primers to obtain fragments 1-3 with the lengths of 1017bp, 1222bp and 1099bp respectively, and the large fragment of the black pine PthLRK1 gene cDNA sequence is obtained after splicing.

Cloned partial cDNA sequence:

segment 1:

GAACCGAACTGCAGAAAACAATATAATGTGGGTCACTGATGATAATTATATAGACGTGGGTCACAGAGGAGAGATCGGAAACGCTAGTGCTTACGGTTCTTACTTGCACACCTTGCGAGTTTTCCCAAAGCCTCTCAACAAGTCTTGCTATCAGTTGCCTGTGGTCCCTGATGTGCCTTATCTCTTAAGGTTATGGTTTGAAGTTGGAAATTACAGTGGATTCAAACAACTTCCAAGCTTTGCTTTCTCTATTGAGACAGAGGGTATGTTAGCTATGGGAAACGTGACGATCTCAGACTGTGACACACCATGTTATGATGAAATAATATTTGTTAGTTCTGGCAGAGTCCTGTACATTTGCTTGATCAGAACCTCGGAGTCTGATGATCCCTTCGTCTCTGCTATCGAGTTGAGAACGCTTCAACAAGGCATGTATGGACTGGCTAAGCCAGGAACAATGTTGATGCTAGTATGGAGATATGATGCTGGTGGAAATATTACCATTAGGTATCCTCAAGACATGTTCGACCGGATCTGGGATTACGAATTGAGAGTAACTTTTGGCAATCATTATCCTGTACAGCCTGTGAATTGTCGAGAGACCATTTCAACCAGCAATACCACAGAACTTCCTCCAAGTGCTGTGATGCAAACAGCGGCGGTTATTATTGACCCGGCACGTGCGTTCGATGTCGGATCGACTAATGGTCATAGATTATTATTGCTGTTATATGTTGCAGAGATCGAGCAGCTAAATATGTCTGAACATAGATCCTTTTATGTGACAATAAATGATGAGAAACGGTCTGAGGCCATTACTTCTCTGAGCGATTATTCTACCCGGGAGCTAAAATTTATATCCAATCCAACAGATGATTTCGATTTTGCCTTGGTCAACACTAGGGATGCAACCAGTGATCCCATAATGAACGCCTTTGAGGCTTACGAAATAATTGACACCCAACCGGCAACATATGCGCAAGATAACAAAGCTCTTGAGGCTATCAAAAGCAGGTT(SEQ ID NO:3)

fragment 2:

AGATCCTTTTATGTGACAATAAATGATGAGAAACGGTCTGAGGCCATTACTTCTCTGAGCGATTATTCTACCCGGGAGCTAAAATTTATATCCAATCCAACAGATGATTTCGATTTTGCCTTGGTCAACACTAGGGATGCAACCAGTGATCCCATAATGAACGCCTTTGAGGCTTACGAAATAATTGACACCCAACCGGCAACATATGCGCAAGATAACAAAGCTCTTGAGGCTATCAAAAGCAGGTTTGGCGTAAAGGATTGGATTTCTGATCCTTGTTTTTTGATCCAGTGGAATGGAATTGTGTGTGAAAGCAGCACTTACCCCATAAGAATTTCGGAAATTGATTTGTCAGGAAAGAATCTTACAGGATTGGTGCCCGACGATATTAGACAGTTGACGGCATTAGTTAAAGTGTCGCTTTACAATAATCATTTGATAGGGCAATTGCCAAATTTGTCCAGTTTAACCATGTTGGAGAGATTGTATCTACAAAACAACAACCTGAGCGGTACTCTTCCGTCCTGGCTATGTGAACTGAAAAACCTAAAAGAATTGAATATAGAGAATAATAATTTCAGCGGTGTAATACCTGTCCAACTTCTCAATGGATCATTGAAATTTAGTTACTGTGGAAACCCCTACTTACCTATGCATAAGGGGAAATGCACCCTGCATACTACAAATAAGAATAAATTGAAGATTATACTCGGAATAACACTAGGCGGAATATTAATCATCGCTTTAGCGCTGATAGTAGCTATTATTATGTATCGCAATAAATTCGGGCGAAAAGAACAGGGTATTGACAGCAGAAGAAGTGGACTGGGAATGGAACCTAGATCTTACTTGTACCAAGACTACTCAATGGTTACCGTGCCAAATTCAACAAAATCCCGTTCCTTCACTCTAGATGAGATGATAGCGGCCACACAAGGCTTTAGCCAGGAGATCGGACGAGGAGGTTTTGGAGCGGTGTTTTTAGGTAAATTGCCCGAAGGAAAATACATAGCCGTCAAATTGCTCTCACAGTTCTCCCAACAAGGGGTTCAAGAATTCTTGAATGAGGTTGATCTTCTTTCCAGAATCCATCATAAGAACTTGGTATCTTTACTGGGATATTGTAACGAATCAAGAGAAATTATGCTTATCTACGACTATATGGCAGAAGGGTCTTTAAGGGATCATCTTTCTGGTCCTAACGCACATCATTCCAAACTGA(SEQ ID NO:4)

fragment 3:

CCGTCAAATTGCTCTCACAGTTCTCCCAACAAGGGGTTCAAGAATTCTTGAATGAGGTTGATCTTCTTTCCAGAATCCATCATAAGAACTTGGTATCTTTACTGGGATATTGTAACGAATCAAGAGAAATTATGCTTATCTACGACTATATGGCAGAAGGGTCTTTAAGGGATCATCTTTCTGGTCCTAACGCACATCATTCCAAACTGACTTGGAGAGCCAGACTCAAAATAACTTTAGATGCAGCTCAAGGACTGGAATACCTGCACGTTGGTTGCACCCCCAAAATAATTCACAGGGATATCAAGACCGCCAACATCTTGTTAGACAGCGATTTGAATGGAAAACTAGGCGATTTCGGTCTTTCCAAGATGACAACTGATGGGGAGGCTACTCATGTTACCACTGCCGTAAAAGGAACTGCAGGATACCTGGATCCAGAGTATTTCAACACTCAAATGTTGACGGAGAAGAGTGATGTGTACAGTTTTGGAGTGGTTTTGCTGGAGATCATATGTGGTAGACAACCCATAGATCTAAAACTACCTGCAGAAGAACTGAACATCGTTAGATGGGTGAAGCCGTATGTAGTGGAGAATGATAATCCTAGCAGAATATCAGAAATCATTGACAAGAGGTTAGGTGGAGATTACGACATGACATCTATCACTAGTGTTGCCAAAGTAGCGATGAGATGTGTTCACGCTGAACCGTGGTCTAGGCCGAACGTGAGCGAGATAGTGGCCGAGCTGAAAGAGGCTATCAAACATGAGGATCATCGTGCTTCTGTTTCAATTTCAGAGGAAGCTGGTATTCAAAGTAGCGATTTGTCGTCGGGGCCAGTCTCTTTATCTGTGGATTCGACTGCACACGGAGGAATGGAGTGGAGTGACAACTCCAATATTTCTCAATTGGGAAGGTAGGTTGGCACGTTGCTGATATGGATTGAAGGGAAGTTGGCCAAGAAGTCAAACCTACAGGTACTAGTGATGGCATGATTTACAATATGGGTTGATGTCGTGCATTTGACATAATTGTGTCGCACACATCTGAAAAAATTGGTGAAACTTAAACTGTAACCATCTCGTCAAATCTTGGG(SEQ ID NO:5)

2. cloning of cDNA sequence at 3' end of Pinus koraiensis PthLRK1 gene

The 3 '-end cDNA sequence cloning is carried out by adopting a 3' -RACE method and referring to the specification of a 3'-RACE kit (B605101, manufactured and bioengineering (Shanghai) Co., ltd.) and using a proper amount of total RNA as a template and 3'adaptor GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTT as a primer, under the action of Reverse Transcriptase Mix (RNase H-) reverse transcriptase, the first strand of cDNA is synthesized by incubation at 50 ℃ for 30min and heating at 85 ℃ for 1min, and the first strand is preserved at-20 ℃.

Primers (primers were synthesized by Biotechnology (Shanghai) Co., ltd.) were designed based on the large fragment cDNA sequence of the Pinus koraiensis PthLRK1 gene, and two rounds of PCR were performed. The primer sequences were as follows:

NF9：5′-TCTCTTTATCTGTGGATTCGACTGC-3′；

NF10：5′-GAGGAATGGAGTGGAGTGACAAC-3′。

5.3′outer：5′-GCTGTCAACGATACGCTACGTAAC-3′；

5.3′inner：5′-GCTACGTAACGGCATGACAGTG-3′。

the first round is a cool down PCR: pre-denatured 60s at 94 ℃, 30s at 94 ℃,70 ^*1 C (1 represents a 1 ℃ drop per cycle) for 30s,72 ℃ for 60s, 10 cycles; a second wheel: 94 ℃ for 30s,60 ℃ for 30s,72 ℃ for 60s, 25 times of circulation and 72 ℃ for 480s; after the obtained PCR product was separated by 1% agarose gel electrophoresis (as in FIG. 1D), the target DNA was recovered by using SanPrep column type DNA gel recovery kit (B518131, shanghai) and ligated to T-Vector to construct T-PthLRK, the Vector ligated with the PCR product was transformed into E.coli DH 5. Alpha., and the positive clone was selected and sequenced, the sequence size was 375bp.

3' race derived sequence:

GAGGAATGGAGTGGAGTGACAACTCCAATATTTCTCAATTGGGAAGGTAGGTTGGCACGTTGCTGATATGGATTGAAGGGAAGTTGGCCAAGAAGTCAAACCTACAGGTACTAGTGATGGCATGATTTACAATATGGGTTGATGTCGTGCATTTGACATAATTGTGTCGCACACATCTGAAAAAATTGGTGAAACTTAAACTGTAACCATCTCGTCAAATCTTGGGGTGGTACGTGGATGTATATACACGATCATACATTATGCTGAAACTAATCTTTATTAAATAGGTATTGTGTATTTTAAATCTTCATATCAGTTTTAGAATTTTCCAGGTTAAAATGTTTACAAATTAATAATTTCAAAAAAAAAAAAAAA(SEQ ID NO:6)

3. cloning of cDNA sequence of 5' end of Pinus koraiensis PthLRK1 gene

5′adaptor：GCTGTCAACGATACGCTACGTAACGGCATGACAGTGGGIIGGGIIGGGIIGNRT1：5′-GTCTCTTGACAATTCACAGGCTG-3′。

NR1：5′-CAGCAATCTGCCAGAGCTAACC-3′；

NR2：5′-TTTATCTGAGACCGCTACGTTTCC-3′。

5.3′outer：5′-GCTGTCAACGATACGCTACGTAAC-3′。

PCR procedure: first round: pre-denatured 60s at 94 ℃, 30s at 94 ℃,70 ^*1 30s (1 represents a 1 ℃ drop per cycle), 60s at 72 ℃,10 cycles; a second wheel: 94 ℃ for 30s,60 ℃ for 30s,72 ℃ for 60s, 25 times of circulation and 72 ℃ for 480s; the PCR product obtained was separated by 1% agarose gel electrophoresis (FIG. 1E), the target DNA was recovered using SanPrep column type DNA gel recovery kit (B518131, division of Biotechnology (Shanghai)) and ligated to T-Vector to construct T-PthLRK, the Vector ligated with the PCR product was transformed into E.coli DH 5. Alpha., and positive clones were selected and sequenced, the sequence size was 404bp。

5' race derived sequence:

GCTGTCAACGATACGCTACGTAACGGCATGACAGTGGGGGGGGGGGGGGGGTAATTTCTGGTCTTGTGGAAGGATTTTTGAGTATCGACTGCGGAGGGAAAACGAACCGAACTGCAGAAAACAATATAATGTGGGTCACTGATGATAATTATATAGACGTGGGTCACAGAGGAGAGATCGGAAACGCTAGTGCTTACGGTTCTTACTTGCACACCTTGCGAGTTTTCCCAAAGCCTCTCAACAAGTCTTGCTATCAGTTGCCTGTGGTCCCTGATGTGCCTTATCTCTTAAGGTTATGGTTTGAAGTTGGAAATTACAGTGGATTCAAACAACTTCCAAGCTTTGCTTTCTCTATTGAGACAGAGGGTATGTTAGCTATGGGAAACGTGACGATCTCAGACTGT(SEQ ID NO:7)

4. sequencing of full-length cDNA sequence of Pinus thunbergii PthLRK1 Gene

The 5 sequences (1017 bp, 1222bp, 1099bp, 375bp and 404 bp) obtained above were spliced, and the total cDNA sequence of the PthLRK1 gene of Pinus koraiensis was deduced to be 3081bp. The ORF Finder (https:// www.ncbi.nlm.nih.gov/orffinder /) is used to search the open reading frame of the spliced full-length cDNA sequence, the ORF region of the black pine PthLRK1 gene is 2679bp, is positioned at 78-2756bp, and codes 892 amino acids.

Sequence 1: full-length cDNA of Pinus nigra PthLRK1 gene

TAATTTCTGGTCTTGTGGAAGGATTTTTGAGTATCGACTGCGGAGGGAAAACGAACCGAACTGCAGAAAACAATATAATGTGGGTCACTGATGATAATTATATAGACGTGGGTCACAGAGGAGAGATCGGAAACGCTAGTGCTTACGGTTCTTACTTGCACACCTTGCGAGTTTTCCCAAAGCCTCTCAACAAGTCTTGCTATCAGTTGCCTGTGGTCCCTGATGTGCCTTATCTCTTAAGGTTATGGTTTGAAGTTGGAAATTACAGTGGATTCAAACAACTTCCAAGCTTTGCTTTCTCTATTGAGACAGAGGGTATGTTAGCTATGGGAAACGTGACGATCTCAGACTGTGACACACCATGTTATGATGAAATAATATTTGTTAGTTCTGGCAGAGTCCTGTACATTTGCTTGATCAGAACCTCGGAGTCTGATGATCCCTTCGTCTCTGCTATCGAGTTGAGAACGCTTCAACAAGGCATGTATGGACTGGCTAAGCCAGGAACAATGTTGATGCTAGTATGGAGATATGATGCTGGTGGAAATATTACCATTAGGTATCCTCAAGACATGTTCGACCGGATCTGGGATTACGAATTGAGAGTAACTTTTGGCAATCATTATCCTGTACAGCCTGTGAATTGTCGAGAGACCATTTCAACCAGCAATACCACAGAACTTCCTCCAAGTGCTGTGATGCAAACAGCGGCGGTTATTATTGACCCGGCACGTGCGTTCGATGTCGGATCGACTAATGGTCATAGATTATTATTGCTGTTATATGTTGCAGAGATCGAGCAGCTAAATATGTCTGAACATAGATCCTTTTATGTGACAATAAATGATGAGAAACGGTCTGAGGCCATTACTTCTCTGAGCGATTATTCTACCCGGGAGCTAAAATTTATATCCAATCCAACAGATGATTTCGATTTTGCCTTGGTCAACACTAGGGATGCAACCAGTGATCCCATAATGAACGCCTTTGAGGCTTACGAAATAATTGACACCCAACCGGCAACATATGCGCAAGATAACAAAGCTCTTGAGGCTATCAAAAGCAGGTTTGGCGTAAAGGATTGGATTTCTGATCCTTGTTTTTTGATCCAGTGGAATGGAATTGTGTGTGAAAGCAGCACTTACCCCATAAGAATTTCGGAAATTGATTTGTCAGGAAAGAATCTTACAGGATTGGTGCCCGACGATATTAGACAGTTGACGGCATTAGTTAAAGTGTCGCTTTACAATAATCATTTGATAGGGCAATTGCCAAATTTGTCCAGTTTAACCATGTTGGAGAGATTGTATCTACAAAACAACAACCTGAGCGGTACTCTTCCGTCCTGGCTATGTGAACTGAAAAACCTAAAAGAATTGAATATAGAGAATAATAATTTCAGCGGTGTAATACCTGTCCAACTTCTCAATGGATCATTGAAATTTAGTTACTGTGGAAACCCCTACTTACCTATGCATAAGGGGAAATGCACCCTGCATACTACAAATAAGAATAAATTGAAGATTATACTCGGAATAACACTAGGCGGAATATTAATCATCGCTTTAGCGCTGATAGTAGCTATTATTATGTATCGCAATAAATTCGGGCGAAAAGAACAGGGTATTGACAGCAGAAGAAGTGGACTGGGAATGGAACCTAGATCTTACTTGTACCAAGACTACTCAATGGTTACCGTGCCAAATTCAACAAAATCCCGTTCCTTCACTCTAGATGAGATGATAGCGGCCACACAAGGCTTTAGCCAGGAGATCGGACGAGGAGGTTTTGGAGCGGTGTTTTTAGGTAAATTGCCCGAAGGAAAATACATAGCCGTCAAATTGCTCTCACAGTTCTCCCAACAAGGGGTTCAAGAATTCTTGAATGAGGTTGATCTTCTTTCCAGAATCCATCATAAGAACTTGGTATCTTTACTGGGATATTGTAACGAATCAAGAGAAATTATGCTTATCTACGACTATATGGCAGAAGGGTCTTTAAGGGATCATCTTTCTGGTCCTAACGCACATCATTCCAAACTGACTTGGAGAGCCAGACTCAAAATAACTTTAGATGCAGCTCAAGGACTGGAATACCTGCACGTTGGTTGCACCCCCAAAATAATTCACAGGGATATCAAGACCGCCAACATCTTGTTAGACAGCGATTTGAATGGAAAACTAGGCGATTTCGGTCTTTCCAAGATGACAACTGATGGGGAGGCTACTCATGTTACCACTGCCGTAAAAGGAACTGCAGGATACCTGGATCCAGAGTATTTCAACACTCAAATGTTGACGGAGAAGAGTGATGTGTACAGTTTTGGAGTGGTTTTGCTGGAGATCATATGTGGTAGACAACCCATAGATCTAAAACTACCTGCAGAAGAACTGAACATCGTTAGATGGGTGAAGCCGTATGTAGTGGAGAATGATAATCCTAGCAGAATATCAGAAATCATTGACAAGAGGTTAGGTGGAGATTACGACATGACATCTATCACTAGTGTTGCCAAAGTAGCGATGAGATGTGTTCACGCTGAACCGTGGTCTAGGCCGAACGTGAGCGAGATAGTGGCCGAGCTGAAAGAGGCTATCAAACATGAGGATCATCGTGCTTCTGTTTCAATTTCAGAGGAAGCTGGTATTCAAAGTAGCGATTTGTCGTCGGGGCCAGTCTCTTTATCTGTGGATTCGACTGCACACGGAGGAATGGAGTGGAGTGACAACTCCAATATTTCTCAATTGGGAAGGTAGGTTGGCACGTTGCTGATATGGATTGAAGGGAAGTTGGCCAAGAAGTCAAACCTACAGGTACTAGTGATGGCATGATTTACAATATGGGTTGATGTCGTGCATTTGACATAATTGTGTCGCACACATCTGAAAAAATTGGTGAAACTTAAACTGTAACCATCTCGTCAAATCTTGGGGTGGTACGTGGATGTATATACACGATCATACATTATGCTGAAACTAATCTTTATTAAATAGGTATTGTGTATTTTAAATCTTCATATCAGTTTTAGAATTTTCCAGGTTAAAATGTTTACAAATTAATAATTTCAAAAAAAAAAAAAAA(SEQ ID NO:1)

Sequence 2: amino acid encoded by black pine PthLRK1 gene ORF

Example 2 bioinformatic analysis of Pinus koraiensis PthLRK1 Gene sequence and protein Structure prediction

The sequencing results of the 5 sequences were spliced to infer the full-length cDNA sequence of the PthLRK1 gene of Pinus koraiensis. And (3) splicing the sequencing results by adopting bioinformatics software to obtain the complete sequence of the PthLRK1 gene of the Pinus koraiensis, which is 3081bp in total. The ORF region of the black pine PthLRK1 gene is predicted to be 2679bp, is positioned at 78-2756bp and encodes 892 amino acids by using ORF finder on-line software.

The result of predicting physicochemical properties by using the ProtParam program (https:// web. Expasy. Org/protParam /) shows that the theoretical molecular mass of the protein encoded by the PthLRK1 gene is about 99.63kDa and the theoretical isoelectric point (pI) is 5.46. The signal peptide results were predicted using the SignalP-5.0 program (https:// services. Healthcare. Dtu. Dk.) and indicated that the protein did not have a signal peptide region.

Analysis of the transmembrane domain of the PthLRK1 gene-encoded protein using the TMHMM-2.0 program (https:// services. Healthcare. Dtu. Dk.) showed that the PthLRK1 gene-encoded protein has a single transmembrane region at amino acids 484-507 (FIG. 2A), extending from extracellular to intracellular (FIG. 2B), which is a transmembrane protein.

The functional domains of the proteins were predicted on the InterPro website using the program (https:// www.ebi.ac.uk/InterPro/result/InterProScan/iprscan5-R20220307-030447-0221-57005514-p2m /) (FIG. 3), which shows that there is a Malcrin domain at amino acid positions 2-309, a membrane-anchored protein on the endoplasmic reticulum; the amino acid position 317-472 contains an LRR (Leucine-rich repeat domain) superfamily domain with disease resistance characteristics; at amino acid positions 562-386 there is a Serine-threonine, tyrosine-Protein kinase (Serine-threonine/tyrosine-Protein kinase) catalytic domain, which overlaps partially with this domain, with the superfamily of Protein kinases (Protein kinase-like domain) located at amino acid positions 543-836 and 560-840. These domains and the cognate superfamily constitute the main functional structure of the PthLRK1 protein, and function together as PthLRK1 genes.

Using BLAST programs, the nucleotide sequence of the PthLRK1 gene from Pinus koraiensis was analyzed for homology to the nucleotide sequence of Pinus koraiensis (P.tabuliformis, KI 711073.1), picea glauca (BT 118800.1), pinus taeda (JQ 019187.1), picea gigantea (Picea sitchensis, BT 123520.1), ji Hudie orchid (Phalaenopsis equestris, XM_ 020737730.1), juglans regia (Carya illinoinensis, XM_ 043109997.1), lactuca sativa (Lactuca sativa, XM_ 023908240.3), and oil palm (Elaeis guineensis, XM_ 029268492.1), and as a result, the highest homology of the gene sequence to the nucleotide sequence of Pinus koraiensis (P.tabuliformis, KI 711073.1) was found to be 89.14%. The phylogenetic tree analysis result shows (figure 4) that the gene and the Chinese pine are on the same branch, the relationship is relatively close, and the phylogenetic tree result and the nucleotide sequence homology analysis result keep relatively high consistency.

Three-dimensional structural modeling is carried out on the black pine PthLRK1 gene coding protein by a Phyre2 program (https:// www.sbg.bio.ic.ac.uk/Phyre2/html/page. Cgi), and the result shows that the protein has clear alpha-helix, beta-sheet and random coil (figure 5).

Example 3 experiments on pine wood nematodes infesting black pine seedlings

Transferring the pine wood nematodes separated and stored in a laboratory onto Botrytis cinerea mycelium, culturing in dark at 25 ℃ for about 7d, flushing the bottle wall with sterile water after the pine wood nematodes climb up the triangular bottle wall, and collecting and preparing into pine wood nematode suspension. Randomly sucking 1 mu L of pine wood nematode suspension, observing and counting under a microscope, repeating 50 times, calculating the quantity of pine wood nematodes per microliter, and finally preparing a suspension containing 25 pine wood nematodes per microliter, wherein the suspension is used for a pine Miao Qinran experiment.

The black pine plants adopt two-year-old black pine seedlings, and the average plant height is about 50 cm. And (3) beveling 1 wound with the length of about 1cm on one side of the main stem which is 10cm away from the black pine basal part by using a sterile blade, fixing a sterilized absorbent cotton ball in the wound, fixing the cotton ball into an inclined funnel shape by using a sealing film, injecting 100 mu L of pine wood nematode suspension into the cotton ball so that the number of the pine wood nematodes inoculated to each black pine is 2500, and finally sealing the wound by using a paraffin film to form a treatment group (BL). And keeping moisture for 24 hours to ensure successful infection of the pine wood nematodes, and simultaneously injecting an equal amount of sterile water according to the method to form a control group (CK), wherein 15 plants of the control group and the treatment group are respectively placed in a greenhouse at 30 ℃ for culture.

1 plant of black pine is randomly extracted from CK and BL groups on the 1 st day, 3 rd day, 5 th day and 7 th day after infection treatment, and is cut into a plurality of sections, and pine wood nematode separation experiments are carried out to judge whether the infection of the pine wood nematodes is successful and whether the pine wood nematodes are transferred. And successfully separating the pine wood nematodes from stems and lateral branches 10cm above the BL black pine infection position on the 5 th day after inoculation, wherein the successful infection of the pine wood nematodes and the transfer occur.

EXAMPLE 4 expression and functional verification of the PthLRK1 Gene of Pinus densiflora in affected Pinus densiflora

5 days, 10 days, 15 days, 20 days after the pine wood nematodes infest the black pine seedlings, and dividingPine needles were taken as samples, using omega companyThe Plant RNA kit is used for extracting total RNA, and the specific operation is as follows:

1) After pine needles are ground by liquid nitrogen, 100mg of plant samples are collected into a centrifuge tube, 500 mu L of RCL Buffer (beta-mercaptoethanol is added in advance) is added, and immediately vortex and mix uniformly;

2) Water bath at 55deg.C for 1-3min, and centrifuging at maximum rotation speed (higher than 14000 Xg) at room temperature for 5min;

3) Sleeving the gDNA Filter column into a 2mL collecting pipe, transferring supernatant to the gDNA Filter column, and centrifuging at room temperature of 14000 Xg for 2min;

4) Adding RCB Buffer with equal volume into the filtrate, sucking up and down, and mixing for 5-10 times;

5) Will beThe RNA Mini binding column was sleeved into a 2mL collection tube and half of the mixture was transferred to +.>Centrifuging 10000 Xg in RNA Mini binding column at room temperature for 1min, and discarding filtrate;

6) Will beThe RNA Mini binding column is sleeved into the same 2mL collecting pipe, and the rest mixed solution is transferred toCentrifuging 10000 Xg in RNA Mini binding column at room temperature for 1min, and discarding filtrate;

7) Will beThe RNA Mini binding column is sleeved into the same 2mL collecting tube, and is +.>RNA Mini binding column 400. Mu.L RWF buffer was addedCentrifuging at room temperature for 1min at r,10000 Xg, and discarding filtrate; />

8) Will beThe RNA Mini binding column is sleeved into the same 2mL collecting tube, and is +.>Adding 500 mu L RNA Wash Buffer II (diluted with absolute ethanol in advance) into RNA Mini binding column, centrifuging at 10000 Xg room temperature for 1min, and discarding filtrate;

9) Repeating the step 8;

10 Will) beThe RNA Mini binding column is sleeved into the same 2mL collecting pipe, and is centrifuged for 2min at 10000 Xg at room temperature, and is spin-dried>RNA Mini binding column matrix;

11 Will) beThe RNA Mini binding column is sleeved into a new 1.5mL centrifuge tube, 80 mu L DEPC water is taken and accurately added into the +.>And (3) placing 2min at normal temperature in the center of the RNA Mini binding column membrane, centrifuging at 10000 Xg at room temperature for 1min, eluting RNA, and preserving at-80 ℃ for later testing.

The extracted RNA needs to detect the integrity of the RNA sample by 1% agarose gel electrophoresis; and detecting the concentration of RNA by a micro-spectrophotometer. After the quality of the RNA is qualified, referring to the instruction of a HiScript III RT SuperMix for qPCR reverse transcription kit of Vazyme company, the cDNA template is obtained through reverse transcription, and the specific reverse transcription steps are as follows:

1) Genomic DNA removal: the following mixtures were placed in RNase-free centrifuge tubes: 4 XgDNA Wiper Mix 4. Mu.L; total RNA 100ng; RNase Free dH ₂ Up to 16. Mu.L. Light weight with pipettorLightly blowing and mixing at 42 ℃ for 2min;

2) Reverse transcription system: the following mixture was placed in an RNase-free centrifuge tube, and gently mixed by pipetting: 5 XHiScript III RT SuperMix 4. Mu.L; 16 mu L of the mixed solution in the last step;

3) Reverse transcription reaction procedure: 37 ℃ for 15min;85 ℃,5s.

To verify the role of PthLRK1 gene in the process of resisting pine wood nematode disease in black pine, two gene-specific primers (41830-Fwd: GACCGGATCTGGGATTACG;41830-Rev: CATCACAGCACTTGGAGGAA) were designed for qRT-PCR analysis, U2af was used as an internal reference gene, and 2 was used ^-ΔΔCT The relative quantitative analysis was performed on the expression level of PthLRK1 gene at various times (5 days, 10 days, 15 days, 20 days) after the black pine-feeling disease, and the results are shown in FIG. 6. In this study, both the treatment group and the control group included 3 biological replicates and 3 technical replicates. The total reaction system was 20. Mu.L: primer F0.4. Mu.L, primer R0.4. Mu.L, SYBR real-time PCR premixture. Mu.L, cDNA 1. Mu.L, ddH ₂ O8.2. Mu.L. The reaction procedure: 95℃for 5min,95℃for 15s,60℃for 30s, cycle number 40.

As shown in FIG. 6, the expression level of PthLRK1 gene in treated black pine seedlings showed a tendency to increase and decrease as compared with control, and reached a peak at 10 days, and was lower in the latter period than in the control. The expression shows that the black pine PthLRK1 gene participates in the black pine disease resistance reaction process in the early stage of black pine infection, and pine wood nematodes continue to reproduce along with the time from day 5 to day 10, and the resistance gene defends against the infection of the pine wood nematodes by improving the expression. In the later stage of the black pine disease, secondary metabolic substances generated in the disease-resistant process of the black pine accumulate to cause toxicity possibly due to serious infection, so that pine wilting is caused, and the PthLRK1 gene expression is reduced.

Therefore, by researching the gene of key enzyme involved in the process of resisting pine wood nematode disease of black pine and cloning and analyzing the gene, a foundation is laid for researching the response mechanism of resisting pine wood nematode disease of black pine and regulating and controlling the molecular level thereof, and the method has important significance for genetic engineering breeding of black pine, prevention and control of pine wood nematode disease and detection and judgment of whether black pine plants are infected with pine wood nematode disease.

The embodiment knows that the PthLRK1 gene expression quantity of the black pine is related to the pine wood nematode disease of the black pine, and can be used for judging whether the black pine is infected by the pine wood nematode or not by detecting the PthLRK1 gene expression quantity in the black pine in the early infection stage of the pine wood nematode so as to make a defense measure in time.

Claims

1. A cDNA sequence for coding the leucine-rich repetitive sequence type receptor protein kinase of black pine features that its nucleotide sequence is shown in SEQ ID No. 1.

2. A cDNA sequence encoding a leucine rich repeat receptor-like protein kinase according to claim 1, wherein the full-length cDNA sequence is 3081bp, contains an open reading frame of 2679bp, encodes 892 amino acids, and the encoded protein has a theoretical molecular weight of 99.63kDa and a theoretical isoelectric point of 5.46.

3. A kind of black pine leucine-rich repetitive sequence type receptor protein kinase is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

4. Use of a cDNA sequence encoding a leucine rich repeat receptor-like protein kinase according to claim 1 for the regulation and control research of the response mechanism of black pine against pine wood nematode stress and its molecular level.