Plant nematode pedigree specific gene and application of dsRNA of plant nematode pedigree specific gene in nematode control

Abstract

The invention discloses 24 plant nematode pedigree specific genes in ditylenchus destructor and application of dsRNA of the 24 plant nematode pedigree specific genes in nematode prevention and control, and belongs to the technical field of gene engineering. CDS nucleotide sequences of the 24 specific genes of the plant nematode lineage are as shown in SEQ ID NO: 1-24, and nucleotide sequences of positive-sense strands of dsRNA of the 24 specific genes of the plant nematode lineage are as shown in SEQ ID NO: 25-48. The parasitic ability of the nematodes to hosts after dsRNA treatment is determined through an in-vitro interference method, the expression levels of the 24 plant nematode pedigree specific genes are all remarkably reduced after the 24 plant nematode pedigree specific genes are treated by respective corresponding dsRNA fragments, and the planting ability and the infection area of the ditylenchus destructor are reduced by 20-59% and 22-72% respectively after the 24 plant nematode pedigree specific genes are inoculated into sweet potatoes for 25 days; results show that the specific genes of the 24 plant nematode pedigree participate in regulation and control of infection and reproduction of nematodes on hosts, and can be used as target genes for development of plant nematode prevention and control drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to 24 plant nematode-specific genes in Ditylenchus destructor and application of dsRNA of the 24 genes in nematode control. BACKGROUND

[0002] Plant nematodes are a kind of pathogenic nematodes that can parasitize plants and cause plant diseases, seriously damaging global agricultural production, and causing direct economic losses of up to hundreds of billions of dollars each year. However, the basic biology of plant nematodes lags behind, which cannot provide sufficient knowledge system and theoretical basis for the development of effective nematode control strategies. Ditylenchus destructor is an important migratory endoparasitic nematode and is considered as a model nematode for studying the basic biology of plant nematodes. D. destructor mainly damages underground reproductive plants such as potato, sweet potato and peanut. After invading the host, D. destructor migrates in the host body while feeding, causing destructive necrosis of plant tissues. In China, D. destructor is one of the most destructive pathogens that restricts the potato and sweet potato planting industry, causing 20%-50% of yield loss each year, and even leading to complete crop failure. In recent years, D. destructor has also been found to parasitize corn in the northeast of China, which is a potential constraint factor for corn production. In addition to crops, D. destructor can also infect medicinal herbs such as angelica, American ginseng and mint, resulting in reduced yield and quality of Chinese medicinal materials.

[0003] Studies have found that plant nematodes (including D. destructor) have strong stress resistance and hidden transmission mode, making it difficult to control. Current chemical control of D. destructor is an effective means, but it has also brought about problems such as destruction of the ecological environment and public safety issues of human and animal health. Resistance breeding and biological pesticides are effective and safe means for controlling the nematode disease, but there are currently very few commercial resistance germplasm resources and biocontrol products available. Non-host crop rotation is the safest control method, but limited arable land resources and economic benefits make rotation restricted. Therefore, there is an urgent need to develop new and safe and effective control technologies. Mining functional genes of plant nematodes, especially plant nematode lineage-specific genes, has considerable application value in developing broad-spectrum nematode control strategies. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application aims to provide a plant nematode lineage-specific gene, specifically 24 plant nematode lineage-specific genes in D. destructor, and also to provide dsRNA designed using the 24 lineage-specific genes as target genes, and application of the above lineage-specific genes and dsRNA in plant nematode control, so as to solve the problem of lack of plant nematode resistance germplasm resources and biocontrol products.

[0005] In order to achieve the above technical purposes, the present inventors, on the basis of the previous research on identification of plant nematode lineage-specific genes, have screened plant nematode lineage-specific genes involved in the processes of individual development, growth and reproduction, and infection and pathogenesis of nematodes by functional verification in D. dipsaci, and used the genes as new targets to prevent and control plant nematodes at the time nodes of growth and development of plant nematodes.

[0006] Specifically, the present inventors have found, through gene cloning and functional analysis, that 24 plant nematode lineage-specific genes in D. dipsaci affect the parasitism of nematodes on hosts, and the 24 genes include 2 collagen genes (Dd_09745, Dd_08440), 1 Innexin gene (Dd_02590), 1 transcription factor (Dd_07141), 1 papain family cysteine protease gene (Dd_09367), 1 anthrone oxygenase gene (Dd_12572), 1 Occludin homology domain-containing protein gene (Dd_02817), 1 Pyrroline-5-carboxylate reductase dimerization domain-containing protein gene (Dd_05767), 1 Macro domain-containing protein gene (Dd_12830), 1 WW domain-containing protein gene (Dd_11417), 1 PAP2 family protein gene (Dd_07143), 2 proteins containing unknown functional domains (Dd_02545, Dd_12125), and 11 proteins without any domain information (Dd_07123, Dd_00317, Dd_05965, Dd_10104, Dd_11278, Dd_05610, Dd_00131, Dd_10653, Dd_01604, Dd_10709, Dd_09195). By interfering the 24 genes in vitro, it was found that the colonization and infection ability of D. dipsaci on hosts was significantly reduced.

[0007] Therefore, a first object of the present application is to provide 24 plant nematode lineage-specific genes in Ditylenchus destructor and the application of the 24 genes as dsRNA target genes in the prevention and treatment of plant nematodes; it should be noted that the 24 plant nematode lineage-specific genes in Ditylenchus destructor are Dd_09745, Dd_08440, Dd_02590, Dd_07141, Dd_09367, Dd_12572, Dd_02817, Dd_05767, Dd_12830, Dd_11417, Dd_07143, Dd_02545, Dd_12125, Dd_07123, Dd_00317, Dd_05965, Dd_10104, Dd_11278, Dd_05610, Dd_00131, Dd_10653, Dd_01604, Dd_10709, and Dd_09195, the CDS nucleotide sequences of which are represented by SEQ ID NO: 1-24, respectively.

[0008] A second object of the present application is to design specific primers using the above-mentioned 24 plant nematode lineage-specific genes in Ditylenchus destructor as templates, synthesize dsRNA of the 24 lineage-specific genes by a T7 in vitro transcription kit, and construct an expression vector containing the dsRNA. Further preferably, the dsRNA consists of a sense strand and an antisense strand which is reverse complementary to the sense strand, and the nucleotide sequence of the sense strand is represented by any one of SEQ ID NO: 25-48. Further preferably, the expression vector is an RNA interference vector. Meanwhile, the second object of the present application also provides the application of the above-mentioned dsRNA or the above-mentioned expression vector in the prevention and treatment of plant nematodes. Further preferably, the plant nematodes include Ditylenchus destructor and root-knot nematodes. Still further preferably, the root-knot nematodes are selected from Meloidogyne incognita and Heterodera glycines.

[0009] A third object of the present application is to provide a pesticide composition for preventing and treating plant nematodes, which comprises the above-mentioned dsRNA or the above-mentioned expression vector.

[0010] A fourth object of the present application is to provide a method for preventing and treating plant nematodes, which comprises silencing at least one of the plant nematode lineage-specific genes in the above-mentioned Ditylenchus destructor using the above-mentioned dsRNA or the above-mentioned expression vector. For example, mixed-age D. destructor is soaked in a treatment solution of dsRNA of each of the 24 plant nematode lineage-specific genes in D. destructor, and incubated at room temperature in the dark with low-speed shaking for 24 hours, so as to inhibit the expression of the 24 plant nematode lineage-specific genes and achieve the effect of inhibiting the parasitism of nematodes on plants. Compared with the prior art, the 24 plant nematode lineage-specific genes in D. destructor provided by the present application have the following beneficial effects: the 24 plant nematode lineage-specific genes obtained by the present application are genes that are reported for the first time in D. destructor, and RNA-seq data shows that these genes have different expression patterns, so that the plant nematodes can be prevented and controlled at the time nodes of growth and development of the nematodes according to the expression patterns of these genes. In addition, the present application determines the parasitic ability of the nematodes on the host after dsRNA treatment by an in vitro interference method, and the expression levels of the 24 plant nematode lineage-specific genes in D. destructor are all significantly decreased after treatment with the respective corresponding dsRNA fragments, and the colonization ability and the infestation area of D. destructor are decreased by 20%-59% and 22%-72%, respectively, after being inoculated into sweet potatoes for 25 days. The results show that the 24 lineage-specific genes are involved in the regulation of the infestation and reproduction of D. destructor on the host, and can be used as target genes for the development of plant nematode control drugs. Finally, the present application has great theoretical guidance and application value for the study of the pathogenic mechanism of plant nematodes and the development of nematode control strategies. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Dd_09745, Dd_08440, Dd_02590, Dd_07141, Dd_09367, Dd_12572, Dd_02817, Dd_05767, Dd_12830, Dd_11417, Dd_07143, Dd_02545, Dd_12125, Dd_07123, Dd_00317, Dd_05965, Dd_10104, Dd_11278, Dd_05610, Dd_00131, Dd_10653, Dd_01604, Dd_10709, and Dd_09195 are cloned from D. destructor by PCR.

[0012] Figure 2 The expression patterns of the 24 plant nematode lineage-specific genes in D. destructor in the life history of the nematodes.

[0013] Figure 3 The changes in the expression levels of the 24 plant nematode lineage-specific genes in D. destructor after treatment with the respective corresponding dsRNA fragments.

[0014] Figure 4 Nematode infestation area (cm2) of sweet potato inoculated with mixed age R. sacchari 25 days after inoculation.

[0015] Figure 5 Nematode infestation area (cm2) of sweet potato inoculated with mixed age R. sacchari 25 days after inoculation. 2 DETAILED DESCRIPTION

[0016] The present application is described in detail below with reference to the attached drawing and examples, but should not be construed as limiting the present application. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0017] Example 1: Cloning of 24 plant nematode lineage-specific genes in R. sacchari

[0018] Extraction of total RNA from R. sacchari: Fresh mixed age nematodes (million heads) were taken into a 1.5 mL centrifuge tube, centrifuged at 2500 g for 3 min, and the supernatant was discarded. 300 μL TransZol Up (TransGen Biotech) was added to the centrifuge tube, and the sample was quickly frozen in liquid nitrogen and ground thoroughly with an electric grinder. 700 μL TransZol Up was added, mixed well, and incubated at room temperature for 5 min. The total RNA of R. sacchari was extracted according to the TransZol Up kit instructions.

[0019] cDNA preparation: The extracted total RNA of R. sacchari was used as a template, and the PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit (Takara) was used to obtain the reverse transcription product cDNA.

[0020] ​Target gene coding sequence amplification: The cDNA obtained above was used as a template, and 24 plant nematode lineage-specific genes in R. decalineata were amplified by PCR using the respective upstream and downstream primers. The amplification system was FastPfu DNA polymerase (TransGen) 1 μL, 5×FastPfu Buffer (TransGen) 10 μL, dNTP mix 4 μL, 10 mM upstream primer / 10 mM downstream primer 1 μL each, cDNA 2 μL, and sterile ultrapure water 21 μL; the total reaction system was 50 μL. The amplification program was 94°C pre-denaturation for 10 min, 32 cycles: 94°C denaturation for 15 s, 52°C annealing for 30 s, 72°C extension for several seconds (according to the size of the gene fragment); 72°C for 10 min.

[0021] Amplification product recovery: The PCR amplification product was separated and identified by 1% agarose gel electrophoresis, and the target band was cut and treated at 60°C with 300 rpm shaking until the gel was completely dissolved. The target band was recovered and purified according to the instructions of the gel recovery kit (Omega), and the gel recovery product was obtained, as shown in Figure 1

[0022] Amplification product sequencing: The above-mentioned recovery product was ligated to the pGBKT7 vector (Clontech), and E. coli DH5α was cultured, and 500 μL of bacterial solution was sent to the company for DNA sequencing. The coding sequence of the target gene was obtained by sequencing, as shown in SEQ ID NO: 1-24.

[0023] The full-length amplification primers of 24 plant nematode lineage-specific genes in R. decalineata are as follows:

[0024] Dd_09745 clone F: CGCCCATGGAGATGCAATACGAAAAGATG

[0025] Dd_09745 clone R: CGCCCCGGGTTAATATCCTGGGGCTGT

[0026] Dd_08440 clone F: ctgatctcagaggaggacctgGAATTCATGAACACGTTCGTCAGT

[0027] Dd_08440 clone R: gttatgctagttatgcggccgGTCGACTCATCTAGCGCGCTCGTC

[0028] ​Dd_02590 clone F: CGCGAATTCATGGCCATGCCCGTTATG

[0029] Dd_02590 clone R: CGCGTCGACCTACCAGGCGTCATTTTC

[0030] Dd_07141 clone F: ctgatctcagaggaggacctgCATATGATGAACGGTAATCCAAGC

[0031] Dd_07141 clone R: gttatgctagttatgcggccgGGATCCTTAAATCACAGGTGGAGG

[0032] Dd_09367 clone F: CGCCATATGATGAAAATTCTTCTCACC

[0033] Dd_09367 clone R: CGCGGATCCTTATGCCTTGACTGCGCC

[0034] Dd_12572 clone F: CGCCATATGATGCCGGCACTTTCTGAA

[0035] Dd_12572 clone R: CGCGGATCCCTAGAACAGAGTTCGTTT

[0036] Dd_02817 clone F: CGCGAATTCATGAATGCAAAGAATGCG

[0037] Dd_02817 clone R: CGCCTGCAGTTACATTACCATCATATC

[0038] Dd_05767 clone F: CGCCATATGATGCTTTGGAGCGGGAAA

[0039] Dd_05767 clone R: CGCCTGCAGTCATTGTGAATTTGCAAA

[0040] Dd_12830 clone F: CGCCATATGATGTATCAACAGACGCCC

[0041] Dd_12830 clone R: CGCGGATCCTTATTCCTTTCTCGGGAT

[0042] Dd_11417 clone F: CGCGAATTCATGTTGAAAGGTCGTAA

[0043] Dd_11417 clone R: CGCCTGCAGTTATGAAGTGATTTGTCG

[0044] Dd_07143 clone F: GCGGAATTCATGCAGGAGCGACAACGA

[0045] Dd_07143 clone R: GCTGGATCCCTATGATTTAATTTGGCT

[0046] Dd_02545 clone F: CGCGAATTCATGGACGAAGCATCTCAAC

[0047] Dd_02545 clone R: CGCCTGCAGTCAAGAATGCTGTCCAAT

[0048] Dd_12125 clone F: ctgatctcagaggaggacctgCATATGATGTGCCACTGGGGCGTG

[0049] Dd_12125 clone R: gttatgctagttatgcggccgGGATCCTCAGTAAAATTTGTCGTT

[0050] Dd_07123 clone F: GCGCATATGATGTCCGTTAAAGAGGAA

[0051] Dd_07123 clone R: GCTGGATCCTCAAAGTGTCAATTGTCG

[0052] Dd_00317 clone F: CGCCATATGATGAGCAAAGTGGCACCA

[0053] Dd_00317 clone R: CGCGGATCCTTAATCATCGAGACTCAT

[0054] Dd_05965 clone F: GCGGAATTCATGAATACCGACGGTGAA

[0055] Dd_05965 clone R: GCTCTGCAGCTAAATCATCTCTAAGTC

[0056] Dd_10104 clone F: CGCGGATCCGTATGGATGAAGACCTGAGC

[0057] Dd_10104 clone R: CGCCTGCAGCTACTTCTTACGCTTTTC

[0058] Dd_11278 clone F: CGCCATATGATGGTGTCTCACATTACA

[0059] Dd_11278 clone R: CGCGGATCCTTATTGAAACCAAAGATA

[0060] Dd_05610 clone F: ctgatctcagaggaggacctgCATATGATGACAGTGGAAAGGCCA

[0061] Dd_05610 clone R: gttatgctagttatgcggccgGGATCCTTAGAGGTGTTGCACAGC

[0062] Dd_00131 clone F: ctgatctcagaggaggacctgCATATGATGCTAAGACAAGAAAGG

[0063] Dd_00131 clone R: gttatgctagttatgcggccgGGATCCTCAAATGTTTGCGCGTAG

[0064] Dd_10653 clone F: CGCGAATTCATGATAGCATTTGCGTGG

[0065] Dd_10653 clone R: CGCGGATCCTTAATGGGCAGATCCCAT

[0066] Dd_01604 clone F: CGCCATATGATGACTATGCTATTGAAC

[0067] Dd_01604 clone R: CGCGGATCCCTATTGGAAGAAGCAGTA

[0068] Dd_10709 clone F: ctgatctcagaggaggacctgCATATGATGACTGCAAGAAGGCTC

[0069] Dd_10709 clone R: gttatgctagttatgcggccgGGATCCTCATACGCTATCATAGAA

[0070] Dd_09195 clone F: CGCGAATTCATGAGTGAACGTAGTTCC

[0071] Dd_09195 clone R: CGCGGATCCTCACTGGTGTCTGGTCTG

[0072] Example 2: Expression patterns of genes specific to 24 plant nematode lineages in *Steep rot nematode*

[0073] Based on our previous RNA-seq data obtained from transcriptome sequencing of *Nematodea canis* at different instars, we used the RPKM value (Reads per kilobase per million mapped reads) to represent the expression level of the target gene at each instar of the nematode. The results are as follows: Figure 2 As shown, the expression levels of genes Dd_02545, Dd_07123, Dd_09745, Dd_08440, Dd_10104, Dd_11417, Dd_12125, and Dd_12830 are highest during the egg stage, and gradually decrease as the nematode develops. The expression levels of genes Dd_01604, Dd_05610, Dd_05965, Dd_07141, Dd_07143, Dd_09195, Dd_10653, and Dd_12572 gradually increase from the egg stage to the J2 stage, peaking in the J2 stage, and then decreasing as the nematode develops further. The expression levels of genes Dd_00131, Dd_00317, Dd_02590, Dd_02817, Dd_05767, Dd_09367, and Dd_10709 gradually decrease from the egg stage to the J2 stage, reaching their lowest level in the J2 stage. Subsequently, their expression levels increase as the nematode grows. These results indicate differences in the expression patterns of these target genes, which may reflect their functions at different stages.

[0074] Example 3: Silencing effect of in vitro RNAi on 24 plant nematode lineage-specific genes in Ditylenchus destructor

[0075] Synthesis of dsRNA: According to the 24 target gene sequences cloned in Example 1, specific primers for RNAi were designed, and a T7 promoter sequence TAATACGACTCACTATAGGG was added at the 5' end of the specific primers. The primer sequences are as follows:

[0076] Dd_09745 RNAi F: TAATACGACTCACTATAGGGCCAATGCTCTACAGTTATGC

[0077] Dd_09745 RNAi R: TAATACGACTCACTATAGGGGGCGTTATCATTCACACC

[0078] Dd_08440 RNAi F: TAATACGACTCACTATAGGGCTGGTAGACCTGGCAATA

[0079] Dd_08440 RNAi R: TAATACGACTCACTATAGGGGGAGTTCTTGTTGAGTATG

[0080] Dd_02590 RNAi F: TAATACGACTCACTATAGGGGGCCATGCCCGTTATGCT

[0081] Dd_02590 RNAi R: TAATACGACTCACTATAGGGGGATTCTTCACTTCGCG

[0082] Dd_07141 RNAi F: TAATACGACTCACTATAGGGTAATCCAAGCAATAAC

[0083] Dd_07141 RNAi R: TAATACGACTCACTATAGGGAGATTGCTGTGTTCCG

[0084] Dd_09367 RNAi F: TAATACGACTCACTATAGGGGGAAACGATGATGAGAAA

[0085] Dd_09367 RNAi R: TAATACGACTCACTATAGGGGGTGCGCATTGGGGCCAT

[0086] Dd_12572 RNAi F: TAATACGACTCACTATAGGGGGCACTTTCTGAACTCCT

[0087] Dd_12572 RNAi R: TAATACGACTCACTATAGGGGGCCAATTTGCAAAAATT

[0088] Dd_02817 RNAi F: TAATACGACTCACTATAGGGGGAATACACAAACCGCTC

[0089] Dd_02817 RNAi R: TAATACGACTCACTATAGGGGGTGTGCCTTCACCATTG

[0090] Dd_05767 RNAi F: TAATACGACTCACTATAGGGGGAGCGGGAAATCCAAGA

[0091] Dd_05767 RNAi R: TAATACGACTCACTATAGGGGGGCATTAGTCTGATCAC

[0092] Dd_12830 RNAi F: TAATACGACTCACTATAGGGGGCGCAGGTGCCACAGAA

[0093] Dd_12830 RNAi R: TAATACGACTCACTATAGGGGATCAATTTCTTTTGTCA

[0094] Dd_11417 RNAi F: TAATACGACTCACTATAGGGGGTCGTAAAAGTCATCAA

[0095] Dd_11417 RNAi R: TAATACGACTCACTATAGGGGGTGGGAGTCTGCGGAAT

[0096] Dd_07143 RNAi F: TAATACGACTCACTATAGGGGGCGTTATGGAGTGCCAT

[0097] Dd_07143 RNAi R: TAATACGACTCACTATAGGGGGACGGAACAGCTGAGTG

[0098] Dd_02545 RNAi F: TAATACGACTCACTATAGGGGGACGAAGCATCTCAACC

[0099] Dd_02545 RNAi R: TAATACGACTCACTATAGGGGGCCAATGTCATCGTAAT

[0100] Dd_12125 RNAi F: TAATACGACTCACTATAGGGGGGGCGTGTACGTTGGCA

[0101] Dd_12125 RNAi R: TAATACGACTCACTATAGGGGGCAGTGCCAAGCTCTAA

[0102] Dd_07123 RNAi F: TAATACGACTCACTATAGGGAAACGACACCGAATAT

[0103] Dd_07123 RNAi R: TAATACGACTCACTATAGGGAGATTTCGTTTGACTT

[0104] Dd_00317 RNAi F: TAATACGACTCACTATAGGGCACCAGAGTTTGTAGA

[0105] Dd_00317 RNAi R: TAATACGACTCACTATAGGGACAAAGGATCATCCTC

[0106] Dd_05965 RNAi F: TAATACGACTCACTATAGGGTGAAAATAGTATTGAA

[0107] Dd_05965 RNAi R: TAATACGACTCACTATAGGGTTCTGCGTCGATACAG

[0108] Dd_10104 RNAi F: TAATACGACTCACTATAGGGATGAAGACCTGAGCAA

[0109] Dd_10104 RNAi R: TAATACGACTCACTATAGGGTTTTCTTCTTGAACT

[0110] Dd_11278 RNAi F: TAATACGACTCACTATAGGGGGTGTCTCACATTACATC

[0111] Dd_11278 RNAi R: TAATACGACTCACTATAGGGGGCTCGATCGCTTCCCAA

[0112] Dd_05610 RNAi F: TAATACGACTCACTATAGGGGGAAAGGCCAAAACATGT

[0113] Dd_05610 RNAi R: TAATACGACTCACTATAGGGGGCCAGAGACGAAAATGG

[0114] Dd_00131 RNAi F: TAATACGACTCACTATAGGGAAATCGTTTGAAGCCG

[0115] Dd_00131 RNAi R: TAATACGACTCACTATAGGGCATTAAGTGCACTAGC

[0116] Dd_10653 RNAi F: TAATACGACTCACTATAGGGATCGCAGTTTTTTGCT

[0117] Dd_10653 RNAi R: TAATACGACTCACTATAGGGCAACAACGTCTTCCTG

[0118] Dd_01604 RNAi F: TAATACGACTCACTATAGGGGACTATGCTATTGAACGCTG

[0119] Dd_01604 RNAi R: TAATACGACTCACTATAGGGCCACCGTGGAGAATCACA

[0120] Dd_10709 RNAi F: TAATACGACTCACTATAGGGGCAAGAAGGCTCAAGCCA

[0121] Dd_10709 RNAi R: TAATACGACTCACTATAGGGGGCGAAACAGTCGAATAC

[0122] Dd_09195 RNAi F: TAATACGACTCACTATAGGGGGGTATGGCGACCGCGTG

[0123] Dd_09195 RNAi R: TAATACGACTCACTATAGGGGGGCACATTGCCATATGC

[0124] The amplified product recovered in Example 1 was used as a template to synthesize dsRNA using a T7 RNAi Transcription Kit (Vazyme) according to the manufacturer's instructions. The reaction system was NTP Mix 8 μL, 10x Transcription Buffer 2 μL, T7 Enzyme Mix 2 μL, template 1 μg, and RNA-free water to 20 μL. The reaction system was incubated at 37°C for 2 h in a PCR instrument. Then the reaction product was added to a double enzyme digestion system to remove residual DNA and single-stranded RNA. The digestion system was reaction product 20 μL, DNase I 1 μL, RNase T1 (10 U / μL) 2 μL, and RNA-free water 17 μL. After mixing, the sample was gently blown to mix evenly, and incubated at 37°C for 30 min to obtain dsRNA. The nucleotide sequence of the sense strand of the dsRNA is shown in SEQ ID NO: 25-48. The target gene dsRNA was identified by 1% agarose gel electrophoresis and detected for concentration at 260 nm, and then stored at -80°C for later use.

[0125] RNAi treatment of D. dipsaci: About 1000 fresh mixed age D. dipsaci were taken and immersed in 300 μL of control group treatment solution (50 mM octopamine, 3 mM spermidine, 0.01% Triton X-100, 1 μg / μL dsGFP) and 300 μL of treatment group treatment solution (50 mM octopamine, 3 mM spermidine, 0.01% Triton X-100, 1 μg / μL dsRNA-target gene), respectively. Incubate at room temperature in the dark with 10 rpm shaking for 24 h.

[0126] Quantitative PCR assay: The total RNA of the control and treatment groups of D. dipsaci described above was extracted and reverse transcribed into cDNA according to the method in Example 1. The actin gene was used as an internal reference gene, and SYBR® Select Master Mix kit (Thermo Fisher) was used to perform qPCR detection on an ABI QuantStudio 5 fluorescent quantitative PCR instrument (Thermo Fisher Scientific). The 2 -ΔΔCtThe expression amount of the target gene was calculated. The reaction system was 2x Premix Ex Taq 5 μL, 10 μM upstream primer / 10 μM downstream primer 0.5 μL each, cDNA 2 μL, sterile ultrapure water 2 μL; the total reaction system was 10 μL. The reaction program was 95°C pre-denaturation for 10 min, 40 cycles: 95°C denaturation for 15 s, 60°C annealing for 30 s, 72°C extension for 30 s. The sequence of the fluorescence quantitative PCR primer was as follows:

[0127] Dd_09745 qPCR F: GTTCAGTCTTCGCTCAGT

[0128] Dd_09745 qPCR R: CATATCCTCCTTGTCGTAGAA

[0129] Dd_08440 qPCR F: CATACTCAACAAGAACTCCAAT

[0130] Dd_08440 qPCR R: GTCTCTACCAGCCATTCC

[0131] Dd_02590 qPCR F: TACTGCGACTACCACTTC

[0132] Dd_02590 qPCR R: GGATTGAGCCACTTCTTG

[0133] Dd_07141 qPCR F: CTACACATACTCCACCTACC

[0134] Dd_07141 qPCR R: CGCCTATTACCACATCTCT

[0135] Dd_09367 qPCR F: CGAACAGTCCTTCATCCT

[0136] Dd_09367 qPCR R: GAACCAGTTGAGCAAGTC

[0137] Dd_12572 qPCR F: CTTGGCATAATGCCTGTC

[0138] Dd_12572 qPCR R: ACAGAGTTCGTTTTCGTG

[0139] Dd_02817 qPCR F: CTCGTCAGCATCCTCATC

[0140] Dd_02817 qPCR R: CAGCCTCTCAAGTTCCAT

[0141] Dd_05767 qPCR F: CCAACACTGATGACATACTAAG

[0142] Dd_05767 qPCR R: GTCTAAGTCTCGCTTCTGTA

[0143] Dd_12830 qPCR F: ATCTATCCGCCAAGTTGT

[0144] Dd_12830 qPCR R: ATCGTTCGCTGACTATGT

[0145] Dd_11417 qPCR F: AGTCATCCATATCTTCAGTGT

[0146] Dd_11417 qPCR R: TTGTTCGCTTGTTGTTGT

[0147] Dd_07143 qPCR F: ATGTTGAGGACTATGTATGT

[0148] Dd_07143 qPCR R: TGCTTATGGTCACGAACT

[0149] Dd_02545 qPCR F: GGATGGACAGAATGAAGGA

[0150] Dd_02545 qPCR R: CAGCGGAATAGTGGTTGA

[0151] Dd_12125 qPCR F: GGTGGCTAATCGTAATGAC

[0152] Dd_12125 qPCR R: GCTAAGGACACAGATTCAAT

[0153] Dd_07123 qPCR F: GTGTCAATACCTCATCTTCAG

[0154] Dd_07123 qPCR R: CATACTCATCGTCATCATAGC

[0155] Dd_00317 qPCR F: GGTTATATTCTGAGGCTGTGA

[0156] Dd_00317 qPCR R: TAGTTCCGCTGTCTTGTG

[0157] Dd_05965 qPCR F: CCAAAGATGTGGTCGTAACG

[0158] Dd_05965 qPCR R: CACATGCATAGGCCTTCAC

[0159] Dd_10104 qPCR F: CGATAGAGGAGGAAGATGG

[0160] Dd_10104 qPCR R: CGTCACAGTAAGGCAATG

[0161] Dd_11278 qPCR F: CAATAGTCGCTCTGCCGA

[0162] Dd_11278 qPCR R: ATTGAAACCAAAGATAGCG

[0163] Dd_05610 qPCR F: GCTACCTTCAAGTATCTGTTC

[0164] Dd_05610 qPCR R: GCTGGATTCATAGATGGATTAG

[0165] Dd_00131 qPCR F: ATGCTGCTATTACTGGATTG

[0166] Dd_00131 qPCR R: ATTCGTAACCATTCATCACAA

[0167] Dd_10653 qPCR F: GTCGGTGAGGAGGTTATG

[0168] Dd_10653 qPCR R: TAATGGAGATGATTCTGGC

[0169] Dd_01604 qPCR F: GGAACCGCTTTCATTCTCC

[0170] Dd_01604 qPCR R: TGGAAGAAGCAGTACC

[0171] Dd_10709 qPCR F: AATCCACCGAGTCATTACA

[0172] Dd_10709 qPCR R: ACGCTGAGTTGTTCTTCT

[0173] Dd_09195 qPCR F: CTACTGGTCCTGGTCCTA

[0174] Dd_09195 qPCR R: GTTCGCTGAATAATGACTGT

[0175] Results are shown in Table 4. Figure 3 After treatment with dsRNA-target gene, the expression of target gene in R. sacrohaemae was significantly reduced.

[0176] Example 4: In vitro RNAi experiment to verify the function of 24 plant nematode lineage-specific genes in R. sacrohaemae

[0177] About 1000 fresh mixed-age R. sacrohaemae were soaked in 300 μL control group treatment solution (50 mM octopamine, 3 mM spermidine, 0.01% Triton X-100, 1 μg / μL dsGFP) and 300 μL treatment group treatment solution (50 mM octopamine, 3 mM spermidine, 0.01% Triton X-100, 1 μg / μL dsRNA-target gene), respectively. Incubate at room temperature for 24 h with shaking at 10 rpm in the dark. The control group and treatment group nematodes were inoculated into the same sweet potato to avoid interference with the experimental results caused by individual differences in sweet potato, and then the sweet potato was placed in a constant temperature incubator at 72°C and 70% humidity. After 25 days, the number of nematodes and the area of infection in the sweet potato were counted. From the results, it can be seen that the number of R. sacrohaemae and the area of infection in the sweet potato were significantly reduced after treatment with dsRNA-target gene fragments. Figure 4 and Figure 5 It can be seen that after treatment with dsRNA-target gene fragments, the number of R. sacrohaemae and the area of infection in the sweet potato were significantly reduced. The results show that inhibiting the expression of 24 plant nematode lineage-specific genes in R. sacrohaemae can effectively inhibit the parasitism of R. sacrohaemae on sweet potato, and these genes can be used as target genes for the development of nematode control products or technologies.

Claims

1. A plant nematode lineage-specific gene selected from any one of the following: Dd_09745, Dd_08440, Dd_02590, Dd_07141, Dd_09367, Dd_12572, Dd_02817, Dd_05767, Dd_12830, Dd_11417, Dd_07143, Dd_02545, Dd_12125, Dd_07123, Dd_00317, Dd_05965, Dd_10104, Dd_11278, Dd_05610, Dd_00131, Dd_10653, Dd_01604, Dd_10709, Dd_09195, whose CDS nucleotide sequences are shown in SEQ ID NO: 1-24, respectively.

2. Use of a plant nematode lineage-specific gene as a dsRNA target gene in the control of plant nematodes, the gene being selected from at least one of the following: Dd_09745, Dd_08440, Dd_02590, Dd_07141, Dd_09367, Dd_12572, Dd_02817, Dd_05767, Dd_12830, Dd_11417, Dd_07143, Dd_02545, Dd_12125, Dd_07123, Dd_00317, Dd_05965, Dd_10104, Dd_11278, Dd_05610, Dd_00131, Dd_10653, Dd_01604, Dd_10709, Dd_09195.

3. Use according to claim 2, characterized in that, The CDS nucleotide sequences of the genes Dd_09745, Dd_08440, Dd_02590, Dd_07141, Dd_09367, Dd_12572, Dd_02817, Dd_05767, Dd_12830, Dd_11417, Dd_07143, Dd_02545, Dd_12125, Dd_07123, Dd_00317, Dd_05965, Dd_10104, Dd_11278, Dd_05610, Dd_00131, Dd_10653, Dd_01604, Dd_10709, Dd_09195 are shown in SEQ ID NO: 1-24, respectively.

4. A dsRNA designed using the plant nematode lineage-specific gene of claim 1 as a target gene.

5. The dsRNA of claim 4, wherein, The dsRNA consists of a sense strand and an antisense strand complementary thereto, and the nucleotide sequence of the sense strand is shown in any one of SEQ ID NO: 25-48.

6. An expression vector containing the dsRNA of claim 5, which is an RNA interference vector.

7. Use of the dsRNA of claim 4 or 5 or the expression vector of claim 6 in the control of plant nematodes.

8. Use according to claim 7, characterized in that, The plant nematodes include Ditylenchus destructor and Meloidogyne hapla.

9. A pesticidal composition for controlling plant nematodes, characterized by, The pesticidal composition comprises the dsRNA of claim 4 or 5, or comprises the expression vector of claim 6.

10. A method of controlling nematodes on plants, comprising applying to the plants a composition comprising a compound of Formula (I) or a salt thereof. The method comprises silencing at least one plant nematode lineage-specific gene of claim 1 using the dsRNA of claim 4 or 5 or the expression vector of claim 6.

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