Pc-VAP gene, protein coded by Pc-VAP gene and application of Pc-VAP gene in regulation and control of infection ability of pratylenchus coffee

Abstract

The invention discloses a Pc-VAP gene, a protein coded by the Pc-VAP gene and application of the Pc-VAP gene to regulation and control of infection capacity of pratylenchus coffee, and belongs to the technical field of biology. The Pc-VAP is located in esophageal glands, intestinal tracts, gonads and eggs of Pratylenchus coffee. RT-qPCR results show that the Pc-VAP is expressed in different insect states of the pratylenchus coffee, and the expression quantity of female pratylenchus coffee is the highest. After the Pc-VAP is silenced by the in vitro RNAi, the expression quantity of the Pc-VAP gene in the nematode body is obviously reduced (Plt; 0.05), the infectivity of the nematodes is obviously reduced; after the Pc-VAP gene silencing is mediated by the VIGS, the pathogenicity of the pratylenchus coffee to the host is obviously reduced. The results show that the Pc-VAP gene can be used as a target gene and has important application potential in prevention and control of pratylenchus coffee.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Pc-VAP gene, a protein encoded by the Pc-VAP gene, and applications thereof in regulating the infectivity of Pratylenchus coffee nematode. Background Art

[0002] The root rot nematode (Pratylenchus Filipjev, 1936), also known as the short-bodied nematode, can infect and harm a variety of crops and is one of the three most destructive plant pathogenic nematodes worldwide. There are more than 100 valid species of root rot nematodes, and 11 species of short-bodied nematodes, such as the coffee nematode, the corn nematode, and the sunny nematode, are recognized as plant pathogenic nematodes with more serious damage. The coffee nematode seriously damages a variety of food crops and cash crops such as wheat, corn, soybeans, coffee, citrus, tomatoes and tobacco. The short-bodied nematode is a type of migratory, obligate endoparasitic plant nematode. During the invasion and feeding process, it will destroy root cells and produce necrotic spots, thereby promoting the infection of other soil-borne pathogens and causing damage to plant roots. The root rot nematode disease caused by the coffee nematode has become a major disease affecting agricultural production safety. At present, there is still a lack of effective targets and methods for the prevention and control of this nematode.

[0003] Plant-parasitic nematodes secrete effector proteins through their stylets, whose core function is to regulate the physiological processes of their host plants to establish a parasitic relationship. These effector proteins can target the plant immune system by inhibiting key signaling pathways involved in pathogen-associated pattern-triggered immunity (PTI) and effector protein-triggered immunity (ETI). As key molecular tools for the interaction between nematodes and their host plants, plant-parasitic nematode effector proteins have recently demonstrated significant potential for application in agricultural biotechnology. Venom allergen-like proteins (VAPs) are a class of key effector proteins secreted by plant-parasitic nematodes, named after their structural similarity to allergen proteins found in animal venom. These proteins play multiple roles in the pathogenesis of plant pathogenic nematodes, ranging from immunosuppression to regulation of host cell metabolism. However, research on VAP proteins in root-rot nematodes has been limited. Summary of the Invention

[0004] The purpose of the present invention is to provide a Pc-VAP gene, the protein encoded by it and its application in regulating the infectivity of coffee Pratylenchus coffee nematode, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a Pc-VAP gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] The second technical solution of the present invention is that the amino acid sequence of the protein encoded by the Pc-VAP gene is shown in SEQ ID NO.2.

[0008] The third technical solution of the present invention is the use of the Pc-VAP gene or the protein in regulating the infectivity of coffee nematode.

[0009] A fourth technical solution of the present invention is the use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the Pc-VAP gene in reducing the infectivity of coffee nematode.

[0010] The fifth technical solution of the present invention is a dsRNA of the Pc-VAP gene, the nucleotide sequence of which is shown in SEQ ID NO.20.

[0011] A sixth technical solution of the present invention is a product for reducing the infectivity of coffee nematodes, comprising the dsRNA or a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the Pc-VAP gene.

[0012] The seventh technical solution of the present invention is a method for controlling coffee nematode, which uses the dsRNA or the product to silence or knock out the Pc-VAP gene.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The present invention provides the amino acid sequence of the Pc-VAP protein of the coffee nematode and the nucleotide sequence of its encoding gene. The Pc-VAP protein encodes 206 amino acids, and its encoding gene comprises 621 amino acids. In situ hybridization of the Pc-VAP antisense probe revealed hybridization signals in the esophageal gland, intestine, gonads, and eggs, while the sense probe showed no hybridization signals, indicating that Pc-VAP is localized in these three areas. RT-qPCR results showed that Pc-VAP is expressed in all stages of the coffee nematode, with the highest expression in females. In vitro RNAi silencing of Pc-VAP significantly reduced Pc-VAP gene expression in the nematodes (P<0.05), significantly reducing the nematode's infectivity. VIGS-mediated silencing of the Pc-VAP gene significantly reduced the virulence of the coffee nematode to its host. This suggests that the Pc-VAP gene has significant potential as a target gene for the control of coffee nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 In situ hybridization shows the tissue localization of Pc-VAP, the gene encoding the Pc-VAP protein, in the coffeae nematode. A and E: No signal was detected after hybridization of the sense probe with C. coffeae nematodes. B, C, D, and F: Hybridization of the antisense probe with C. coffeae nematodes revealed hybridization signals in the esophageal gland, intestine, gonad, and egg. mb: mid-esophageal bulb; eg: esophageal gland; i: intestine; v: vulva; g: gonad. egg: egg. Scale bars: 100 μm (A); 20 μm (B)-(F).

[0017] Figure 2 To detect the expression of Pc-VAP gene in different stages of coffee nematode, 2 -△△Ct The difference in expression levels among different insect stages was calculated by the method; **: P < 0.01.

[0018] Figure 3 The subcellular localization of Pc-VAP protein in Nicotiana benthamiana leaf cells; Merge represents the overlapping results; GFP represents the results observed under GFP green fluorescence; TRIRC represents the results observed under TRIRC red fluorescence; Bright represents the results observed under bright field; scale bar = 20 μm.

[0019] Figure 4 RT-qPCR was used to detect the expression level of Pc-VAP gene in coffee nematodes in different dsRNA treatment groups.

[0020] Figure 5 The number of coffee nematodes in the maize roots was counted 72 hours after inoculation with different dsRNA treatment groups; CK represents the sterile water treatment group; G36 represents the eGFP dsRNA treatment group for 36 hours; R36 represents the Pc-VAP dsRNA treatment group for 36 hours; *: P < 0.05; **: P < 0.01; ns: indicates no statistically significant difference.

[0021] Figure 6The phenotypes of corn plants after TRV-induced Pc-VAP gene silencing; A is the aboveground morphology of corn in different treatment groups, and B is the leaf morphology of corn in different treatment groups; WT represents wild-type corn plants; pTRV2-emty represents plants in the empty vector treatment group; pTRV2-Pc-VAP represents plants in the Pc-VAP silencing treatment group; and pTRV2-ZmPDS represents plants in the ZmPDS positive control treatment group.

[0022] Figure 7 Figure 3 shows the growth parameters and rhizosphere insect loads of corn plants in different treatment groups after TRV-induced Pc-VAP gene silencing; A is plant height, B is plant fresh weight, C is root fresh weight, and D is the total insect load in rhizosphere soil; CK represents healthy corn plants that have not been inoculated with insects; pTRV2-emty represents plants in the empty vector treatment group; pTRV2-Pc-VAP represents plants in the Pc-VAP silencing treatment group; and WT represents wild-type plants inoculated with nematodes. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0029] An embodiment of the present invention provides a Pc-VAP gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0030] The embodiment of the present invention also provides a protein encoded by the Pc-VAP gene, whose amino acid sequence is shown in SEQ ID NO.2.

[0031] The embodiments of the present invention also provide the use of the Pc-VAP gene or the protein in regulating the infectivity of Pratylenchus coffee nematode.

[0032] In some specific embodiments, silencing or knocking out the Pc-VAP gene or downregulating the level of the protein it encodes reduces the infectivity of the coffee nematode.

[0033] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium comprising the Pc-VAP gene in reducing the infectivity of the coffee nematode.

[0034] The present invention also provides a dsRNA of the Pc-VAP gene, the nucleotide sequence of which is shown in SEQ ID NO.20.

[0035] Soaking coffee nematodes with the above-mentioned dsRNA and incubating them in the dark at room temperature with shaking for 36 hours can silence the expression of the Pc-VAP gene.

[0036] An embodiment of the present invention also provides a product for reducing the infectivity of coffee nematodes, comprising the dsRNA or a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the Pc-VAP gene.

[0037] An embodiment of the present invention also provides a method for controlling Pratylenchus coffee nematode, using the dsRNA or the product to silence or knock out the Pc-VAP gene.

[0038] The present invention demonstrated that in situ hybridization of the Pc-VAP antisense probe revealed hybridization signals in the esophageal gland, intestine, gonads, and eggs, while no hybridization signals were observed with the sense probe, indicating that Pc-VAP is located in the esophageal gland, intestine, gonads, and eggs of the coffee nematode. RT-qPCR results showed that Pc-VAP was expressed in all stages of the coffee nematode, with the highest expression level in females. In vitro RNAi silencing of Pc-VAP significantly reduced the expression of the Pc-VAP gene in the nematodes (P < 0.05), significantly reducing the nematode's infectivity. VIGS-mediated silencing of the Pc-VAP gene significantly reduced the pathogenicity of the coffee nematode to its host. These results indicate that the Pc-VAP gene has important potential as a target gene for the prevention and treatment of coffee nematodes.

[0039] Example 1

[0040] Obtaining the Pc-VAP gene and Pc-VAP protein sequences

[0041] The Pc-VAP gene of the K1 population of Pratylenchus coffee nematodes was obtained by cloning and sequencing (the K1 population of nematodes was collected by our laboratory from the roots of corn plants in Pingdingshan, Henan Province and stored in the Plant Nematode Laboratory of Henan Agricultural University.) contains 621 nucleotides, and its sequence is as follows (SEQ ID NO. 1):

[0042] SEQ ID NO.1: ATGCATTTTTCGTTTATTTTAACCGTTTTCTTCTTCATCATTCCCTTCAAAAATGTGGCCATTGGCATTCTGACGGATGCAGAACGAAAATCGGCACTGGACAATCACAATTTGAAGCGATCCCAATTGGCCAATGGAATGAGTGCCAATAAAACGGGGACAATGCCGACGGGAAAAAACATTTGGACAATGCTCTATGACACAGCACAGGAAGCTTCAGTGCAATCTTGGCTTGATAAATGCACTTTCAACCATTCGGGCAATTATGAGAATTTGTATTGGATTGGCATTCAGCAAACAAAAGTAGCCGCTTTGAAATCCGCCATCGATTTGTGGTGGTCCGAATTGGTAAATGTTGGGGCACCGGCGGATTTGGTGCTTCCCTCAATTAATTGGCAACCAATTGGCCATTGGTCCGCGATGGCATGGGCCACAACAAACAAAGTTGGCTGCGGAGTGGCATATTGCATGCGAAATGGAGTGAATTACACATATGCGGGGTGCCAATATAGACCAACTGGCAATTCTTTCGGCCAAAAAATCTATGAATCGGGTGCCGTTTGCAGCGGATGCCCGGTCGTCAATGGCCAACTTCAGTGTCAATATGGATTATGCATATAA; <0,000,100><0,000,101>The encoded Pc-VAP protein contains 206 amino acids, and the sequence is as follows (SEQ ID NO.2): <0,000,102><0,000,103>SEQ ID NO.2: MHFSFILTVFFFIIPFKNVAIGILTDAERKSALDNHNLKRSQLANGMSANKTGTMPTGKNIWTMLYDTAQEASVQSWLDKCTFNHSGNYENLYWIGIQQTKVAALKSAIDLWWSELVNVGAPADLVLPSINWQPIGHWSAMAWATTNKVGCGVAYCMRNGVNYTYAGCQYRPTGNSFGQKIYESGAVCSGCPVVNGQLQCQYGLCI. <0,000,104><0,000,105>Example 2 <0,000,106><0,000,107>Detection of the mRNA Tissue Localization of the Pc-VAP Gene in Pratylenchus coffeae Using In Situ Hybridization

[0047] 1. Extraction of total RNA and cDNA synthesis of P. coffeae

[0048] The propagated culture of Pratylenchus coffeae was collected in sterile water and transferred to a 1.5 mL centrifuge tube. The tube was centrifuged at 6000 rpm at room temperature, and the supernatant was discarded. After quick freezing in liquid nitrogen, the tube was ground three times with a sterile pointed glass rod until the nematodes were completely crushed. 1 mL of Trizol reagent was added, and the tube was allowed to stand at room temperature for 5 minutes. 200 μL of chloroform was then added, and the tube was shaken vigorously for 15 seconds. The tube was allowed to stand at room temperature for another 5 minutes. After centrifugation at 12000 rpm at 4°C for 20 minutes, 600 μL of the supernatant was transferred to a new 1.5 mL centrifuge tube. 600 μL of isopropanol was added in equal proportions, mixed by inversion, and allowed to stand at room temperature for 10 minutes. After centrifugation at 12000 rpm at 4°C for 10 minutes, the supernatant was discarded, and 1 mL of 75% ethanol was added, mixed by inversion, to wash the pellet. After air drying, the pellet was dissolved in 40 μL of RNase-free ddH2O to obtain total RNA. Take 2 μL of the sample and perform agarose gel electrophoresis to check RNA quality. Determine the total RNA concentration using a UV spectrophotometer. Prepare the reverse transcription system on ice according to the instructions for the Yisheng in vitro reverse transcription kit. Mix thoroughly and perform reverse transcription in a PCR instrument. Upon completion of the reaction, the caffeine nematode cDNA is obtained.

[0049] 2. Using the coffee nematode cDNA as a template, design Pc-VAP-specific primers VAP-T7S1 / VAP-A1 and VAP-S1 / VAP-T7A1 and eGFP-specific primers. The primer sequences are as follows:

[0050] VAP-T7S1 (SEQ ID NO.3): 5′-GGATCCTAATACGACTCACTATAGGGCGGCACTGGACAATCACAATT-3′;

[0051] VAP-A1 (SEQ ID NO.4): 5′-ACCAATTCGGACCACCACAA-3′;

[0052] VAP-S1 (SEQ ID NO.5): 5′-CGGCACTGGACAATCACAATT-3′;

[0053] VAP-T7A1 (SEQ ID NO. 6): 5′-GGATCCTAATACGACTCACTATAGGGACCAATTCGGACCACCACAA-3′.

[0054] The above primers were used to amplify the transcription template of the Pc-VAP in situ hybridization probe by PCR, which was recovered and used for future use. The reaction system was prepared according to the instructions of the Roche DIG RNA labeling kit. The mixture was incubated in a metal bath at 37°C for 2 h. After adding 2 μL of DNase I, the mixture was incubated at 37°C for 15 min. The reaction was terminated by adding 2 μL of 0.2 M EDTA (pH = 8.0). The DIG-labeled sense and antisense strand probes were synthesized. The sequence of the antisense strand probe is as follows (SEQ ID NO. 7):

[0055] SEQ ID NO.7: CGGCACTGGACAATCACAATTTGAAGCGATCCCAATTGGCCA ATGGAATGAGTGCCAATAAAACGGGGACAATGCCGACGGGAAAAAACATTTGGACAATGCTCTATGACACAGCACAGGAAGCTTCAGTGCAATCTTGGCTTGATAAATGCACTTTCAACCATTCGGGCAATTATGAGAATTTGTATTGGATTGGCATTCAGCAAACAAAAGTAGCCGCTTTGAAATCCGCCATCGATTTGTGGTGGTCCGAATTGGT.

[0056] 3. Perform in situ hybridization

[0057] In situ hybridization was performed according to the instructions. Figure 1 Obvious hybridization signals were observed in the esophageal gland, intestine, gonads and eggs of adult coffee nematodes using the Pc-VAP antisense chain probe. These results indicate that Pc-VAP is expressed in the esophageal gland, intestine, gonads and eggs of adult coffee nematodes.

[0058] Example 3

[0059] RT-qPCR detection of the expression level of Pc-VAP in different stages of P. coffeae

[0060] 1. Design Pc-VAP-specific primers qPC-VAP-F / R for RT-qPCR, using the nematode 18S gene as the internal reference gene. The primer sequences are as follows:

[0061] qPC-VAP-F (SEQ ID NO.8): 5'-GATTTGGTGCTTCCCTCA-3';

[0062] qPC-VAP-R (SEQ ID NO.9): 5'-CAAAGCAGTT TTTCAGGCC-3';

[0063] 18S-F (SEQ ID NO. 10): 5'-AGTGACGAGAAATAACGAGACC-3';

[0064] 18S-R (SEQ ID NO. 11): 5'-CCAGACTTGCCGCTCTCATA-3'.

[0065] 2. RT-qPCR detection

[0066] About 100 eggs, larvae, females and males of Coffea caesaria were separated from carrot culture medium, frozen in liquid nitrogen and ground with glass rods to extract total RNA from different insect stages, and cDNA was synthesized according to the instructions of Yisheng in vitro reverse transcription kit.

[0067] qRT-PCR was performed using cDNA of different stages of Pratylenchus coffee nematodes as templates, qPC-VAP-F / qPC-VAP-R and 18S-F / R as primers, and according to the instructions of 2×HQ SYBR qPCR mix of Beijing Zhuangmeng International Biological Company. -△△Ct The expression differences among different insect stages were calculated.

[0068] Results Figure 2 The Pc-VAP gene was expressed in females, males, larvae and eggs of P. coffee nematode, with the highest expression level in females and the lowest expression level in males, which was significantly lower than that in the other three stages (P<0.05).

[0069] Example 4

[0070] Subcellular localization of Pc-VAP protein in Nicotiana benthamiana

[0071] 1. Construction of plant expression vector

[0072] Using the coffee nematode cDNA as a template, the ORF fragment of Pc-VAP was amplified using specific primers pGWC-VAP-F / R. The primer sequences are as follows:

[0073] pGWC-VAP-F (SEQ ID NO. 12): 5'-AGCAGGCTTTGACTTTAGGTCATGCATTTTTCGTTTATTTTAACCG-3';

[0074] pGWC-VAP-R (SEQ ID NO. 13): 5'-TGGGTCTAGAGACTTTAGGTCTTATATGCACAATCCATATTGACATTG-3'.

[0075] After the amplified product was purified and recovered, the fragment was cloned into the PGWC vector digested with AndI using the Novozymes one-step cloning kit to construct the PGWC-Pc-VAP vector. The specific steps are as follows:

[0076] (1) Prepare the following reaction system on ice: 3 μL of PGWC vector after enzyme digestion; 1 μL of Pc-VAP ORF fragment; 2 μL of 5×CE II buffer; 1 μL of Exnase II; and 10 μL of RNase-free water.

[0077] (2) After the above system is prepared, place it in a 37℃ metal bath for 30 minutes, let it stand on ice for 2 minutes, and transform the ligation product into DH5α competent cells. Resuspend the transformed E. coli and spread it on a solid LB plate with chloramphenicol resistance, and culture it at 37℃ overnight. After a single clone grows on the plate, pick the single clone with a toothpick and transfer it to LB liquid medium with chloramphenicol resistance. Shake the culture at 37℃ and 220rpm for 3-5 hours. After the culture liquid becomes turbid, perform PCR detection on the culture liquid and send it to the company for sequencing. The culture liquid with correct sequencing is expanded and cultured, and the PGWC-Pc-VAP plasmid is extracted using the Novozymes plasmid extraction kit according to the instructions. The Pc-VAP ORF fragment is replaced with the pEarleyGate 104 (N-terminus eGFP) vector through the Gateway reaction using LR cloning enzyme to generate the eGFP-Pc-VAP expression vector peg104-Pc-VAP. The peg104-Pc-VAP vector was transformed into competent E. coli DH5α, and the plasmid was extracted after PCR detection using primers 35s F / R. The sequences of the 35s F / R primers are as follows:

[0078] 35S F (SEQ ID NO. 14): 5'-GACCACTTTGTACAAGAAAGCTGGGTC-3';

[0079] 35S R (SEQ ID NO. 15): 5'-CAATCCCACTATCCTTCGCAAGACCCT-3'.

[0080] 2. Transformation of plant expression vector into Agrobacterium

[0081] Transform the peg104-Pc-VAP vector into competent Agrobacterium tumefaciens GV3101 according to the instructions for the competent Agrobacterium GV3101. After PCR identification, the transformed Agrobacterium culture was mixed with an equal volume of 60% glycerol and stored in a -80°C freezer.

[0082] 3. Agrobacterium infection of Nicotiana benthamiana and detection

[0083] After Agrobacterium propagation, the OD 600 The induction temperature was 1.0, and the cells were cultured in the dark at 28°C for 3 hours. Tobacco seedlings grown to the 4-leaf stage were selected and infiltrated with transgenic Nicotiana benthamiana using the Agrobacterium infiltration method. 48 hours after inoculation, images were obtained using a laser confocal microscope under excitation wavelengths of 488 nm for GFP and 561 nm for mCherry. The results are shown in Figure 2. Figure 3 As shown, the fluorescence signal of Pc-VAP protein is localized in the cell membrane and cytoplasm of Nicotiana benthamiana.

[0084] Example 5

[0085] Silencing of the Pc-VAP gene in Pratylenchus coffeae and detection of its silencing efficiency

[0086] 1. Synthesis of Pc-VAP dsRNA

[0087] PCR amplification was performed using T7 RNA polymerase using the forward and antisense primers VAP-T7S1 / VAP-A1 and VAP-S1 / VAP-T7A1, respectively, containing the T7 promoter, and using P. coffeae cDNA as a template. After purification, the forward and antisense strands of ssRNA were synthesized, respectively, and mixed in equal proportions and annealed to form dsRNA. After detection, the dsRNA was stored at -80°C until use. As a control, non-endogenous eGFP dsRNA (225 bp) was synthesized and purified using primers eGFP-T7-F1 / R1 and eGFP-F1 / T7-R1. The primer sequences are as follows:

[0088] eGFP-T7-F1 (SEQ ID NO. 16): 5′-GGATCCTAATACGACTCACTATAGGGCAGTGCTTCAGCCGCTACC-3′;

[0089] eGFP-R1 (SEQ ID NO. 17): 5′-AGTTCACCTTGATGCCGTTCTT-3′;

[0090] eGFP-F1 (SEQ ID NO. 18): 5′-CAGTGCTTCAGCCGCTACC-3′;

[0091] eGFP-T7-R1 (SEQ ID NO. 19): 5′-GGATCCTAATACGACTCACTATAGGGAGTTCACCTTGATGCCGTTCTT-3′.

[0092] The Pc-VAP dsRNA sequence is as follows (SEQ ID NO. 20):

[0093] SEQ ID NO.20: GGATCCTAATACGACTCACTATAGGGCGGCACTGGACAATCA CAATTTGAAGCGATCCCAATTGGCCAATGGAATGAGTGCCAATAAAACGGGGACAATGCCGACGGGAAAAAACATTTGGACAATGCTCTATGACACAGCACAGGAAGCTTCAGTGCAATCTTGGCTTGATAAATGCACTTTCAACCATTCGGGCAATTATGAGAATTTGTATTGGATTGGCATTCAGCAAACAAAAGTAGCCGCTTTGAAATCCGCCATCGATTTGTGGTGGTCCGAATTGGT.

[0094] 2. dsRNA introduction into coffee nematode

[0095] Mixed-stage P. coffeae nematodes were washed three times with DEPC-treated water and then added to a solution soaked with Pc-VAP dsRNA (2.0 mg / mL). The mixture was incubated in a dark shaker (100 rpm) at 25°C for 12, 24, 36, and 48 hours. Nematodes soaked in eGFP dsRNA (2.0 mg / mL) served as a control, while nematodes treated with water served as a blank control (CK).

[0096] 3. Pc-VAP gene silencing efficiency detection

[0097] Total RNA was extracted from coffee nematodes in different treatment groups and reverse transcribed into cDNA. The expression level of Pc-VAP gene was detected by qRT-PCR and its silencing efficiency was calculated. Figure 4 The silencing efficiency of Pc-VAP was the highest when Pc-VAPdsRNA was soaked in coffee nematodes for 36 hours. The expression levels of Pc-VAP decreased by 81% and 68% compared with the control group CK treated with pure water and the control group treated with eGFP dsRNA for 36 hours, respectively. The differences were significant (P<0.05), indicating that soaking coffee nematodes with Pc-VAPdsRNA for 36 hours can significantly inhibit the expression of the target gene.

[0098] Example 6

[0099] Detection of the effect of Pc-VAP gene silencing on the infectivity of Pratylenchus coffee nematode

[0100] 1. Corn sowing and inoculation with coffee nematode

[0101] Place plump corn seeds in a sterile glass Petri dish and soak them in 75% ethanol for 10 minutes. Discard the alcohol solution, rinse three times with sterile water, and then rehydrate with an appropriate amount of sterile water. Cover the dish and incubate in a 25°C biochemical incubator in the dark until corn sprouts approximately 5 cm in length emerge. Inoculate the roots of germinated corn with Pc-VAP dsRNA-treated Pratylenchus coffeae nematodes for 36 hours. Place the seeds in a flask, support them with vermiculite, and add an appropriate amount of water. Incubate at 25°C in a greenhouse for 72 hours.

[0102] 2. Corn root staining and coffee nematode abundance statistics

[0103] Corn inoculated with Pratylenchus coffee nematodes for 72 hours was gently removed from the flask. The roots were washed with clean water and placed in a beaker. An appropriate amount of 1% sodium hypochlorite solution was added and stirred continuously with a glass rod. After 5 minutes, the root tissue was rinsed with running water for 1 minute, dried with absorbent paper, and placed in the flask. Acid fuchsin solution was added and heated to a boil in a microwave oven. After cooling, the root tissue was rinsed with running water. The rinsed root tissue was placed in acidic glycerol and heated to a boil for decolorization. The decolorized root tissue was prepared on a slide, and the stained nematodes in the root system were counted and photographed under a microscope.

[0104] The results are as follows Figure 5 As shown, the Pc-VAP dsRNA silencing group (R36) had 121 coffee nematodes in the maize root system, significantly lower (P<0.05) than the 312 in the water treatment group (CK) and the 298 in the eGFP dsRNA treatment group (G36). There was no significant difference between the latter two control treatments (P>0.05). These results indicate that silencing the Pc-VAP gene significantly reduces the infectivity of coffee nematodes to their hosts, and that Pc-VAP is a key gene required for coffee nematodes to infect their hosts.

[0105] Example 7

[0106] Detection of the effect of Pc-VAP gene silencing on the pathogenicity of Pratylenchus coffee nematode

[0107] 1. Construction of recombinant vector

[0108] Using the coffee nematode cDNA as a template, the specific primers TRV2-VAP-F / R were used to amplify the specific fragment of Pc-VAP. The primer sequences are as follows:

[0109] TRV2-VAP-F (SEQ ID NO. 21): 5'-GTGAGTAAGGTTACCGAATTCCGGCACTGGACAATCACAATT-3';

[0110] TRV2-VAP-R (SEQ ID NO. 22): 5'-CGTGAGCTCGGTACCGGATCCACCAATTCGGACCACCACAA-3'.

[0111] After the amplified product was purified and recovered, it was cloned into the linearized pTRV2 vector after digestion with EcoRI and BamHI using the ClonExpress II One Step Cloning Kit to construct the pTRV2-Pc-VAP recombinant vector.

[0112] 2. Transformation of plant expression vector into Agrobacterium

[0113] The successfully constructed pTRV2-Pc-VAP vector, pTRV2-PDS vector and pTRV2 empty vector were transformed into Agrobacterium tumefaciens GV3101, respectively. Then, the above three Agrobacteria were expanded and mixed with Agrobacterium containing pTRV1 vector in equal proportions to prepare pTRV1::pTRV2, pTRV1::pTRV2-Pc-VAP and pTRV1::pTRV2-PDS Agrobacterium mixtures.

[0114] 3. Agrobacterium corn seeds and testing

[0115] Corn seeds were infiltrated with the three Agrobacterium-mediated inoculation mixtures described above. The maize PDS (phytoene desaturase) gene was used as a positive control, and the empty pTRV2 vector was used as a negative control. Twenty corn plants were used in each treatment group. After 14 days, the positive control plants were observed for typical photobleaching symptoms. Successful expression of the viral vectors in maize was then confirmed using the TRV2-specific primers TRV2-JC-F1 / R1 and TRV2-JC-F2 / R2. The primer sequences are as follows:

[0116] TRV2-JC-F1 (SEQ ID NO.23): 5'-AAAGATGGACATTGTTACTCAAGG-3';

[0117] TRV2-JC-R1 (SEQ ID NO. 24): 5'-ATTACAAAAGACTTACCGATCAAT-3'.

[0118] TRV2-JC-F2 (SEQ ID NO.25): 5'-CCAATGGAATGAGTGCCA-3';

[0119] TRV2-JC-R2 (SEQ ID NO. 26): 5'-ACTTTCAACCATTCGGGC-3'.

[0120] The results are as follows Figure 6 As shown, maize exhibited significant phenotypic changes. The positive control (pTRV2-ZmPDS) showed typical photobleaching, while wild-type plants (WT), silenced plants (pTRV2-Pc-VAP), and negative control plants (pTRV2-empty) showed no significant changes. RT-PCR analysis of RNA extracted from the roots of the differently treated plants successfully detected a 498-bp target band. These results indicate that the target gene is successfully expressed in maize.

[0121] 4. Determination of pathogenicity of P. coffeeensis to hosts in different treatment groups

[0122] Corn seedlings successfully transformed with pTRV2-Pc-VAP and showing consistent growth were selected and inoculated with coffee nematodes, 1000 per pot. After 60 days of cultivation in a greenhouse at 25°C, RNA was extracted from the corn roots. RT-qPCR was used to detect the expression of Pc-VAP in nematodes in different treatment groups. Growth parameters such as plant height, aboveground fresh weight, and root fresh weight were measured, and the number of nematodes in the corn rhizosphere was counted. Data analysis was performed using 2 -△△Ct The experiment was performed in triplicate. pTRV2-emty plants and wild-type control plants (WT) were inoculated with nematodes as controls, and healthy maize seedlings that were not inoculated with P. coffeae served as blank controls (CK).

[0123] Potted plants 60 days after inoculation showed a significant reduction in root infection with the coffee nematode in the pTRV2-emty and wild-type (WT) control groups, with some roots showing distinct brown or dark brown lesions and obvious damage symptoms. Root damage symptoms were significantly reduced in the Pc-VAP silencing group (pTRV2-Pc-VAP) compared to the pTRV2-emty and WT wild-type control groups. Plant height, shoot fresh weight, and root fresh weight of the Pc-VAP silencing group (pTRV2-Pc-VAP) were significantly higher (83.9 cm, 40.1 g, and 14.7 g, respectively) than those in the pTRV2-emty (69.8 cm, 27.7 g, and 10.4 g) and WT wild-type control groups (70.1 cm, 28.1 g, and 10.3 g, respectively) (P < 0.05). The average number of coffee nematodes in the rhizosphere of corn in the pTRV2-Pc-VAP silencing treatment group was 6073, which was significantly lower than that in the pTRV2-emty (10535) and WT wild type control (11007) (P<0.05) ( Figure 7 The above results indicate that TRV-induced silencing of the Pc-VAP gene reduces the pathogenicity of P. coffeei to maize, and Pc-VAP is a key gene in the pathogenicity of P. coffeei.

[0124] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A Pc-VAP gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the Pc-VAP gene according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. Use of the Pc-VAP gene according to claim 1 or the protein according to claim 2 in regulating the infectivity of Pratylenchus coffee nematode.

4. The use according to claim 3, characterized in that Silencing or knocking out the Pc-VAP gene or downregulating the level of its encoded protein can reduce the infectivity of coffee nematode.

5. Use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria comprising the Pc-VAP gene according to claim 1 in reducing the infectivity of Pratylenchus coffee nematode.

6. A dsRNA of a Pc-VAP gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

20.

7. A product for reducing the infectivity of Pratylenchus coffee nematode, characterized in that: A recombinant vector, expression cassette, transgenic cell line or recombinant bacteria comprising the dsRNA of claim 6 or the Pc-VAP gene of claim 1.

8. A method for controlling Pratylenchus coffeei, characterized in that: The Pc-VAP gene according to claim 1 is silenced or knocked out by using the dsRNA according to claim 6 or the product according to claim 7.

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