CN116751769B - Pc-CL protein of Caesalpinia aphelenchoides, coding gene and application thereof - Google Patents

Abstract

The invention discloses a Pc-CL protein of a coffee brachysenchyma, a coding gene and application thereof, and belongs to the technical field of biology. The invention provides a coffee brachysporum esophageal gland expression gene Pc-CL, which has an expression level in female worms which is obviously higher than that in larvae, male worms and eggs. Through gene silencing, the Pc-CL dsRNA fragment is taken into the coffee short nematode, and the result shows that after the Pc-CL gene is silenced through dsRNA treatment, the fertility of the coffee short nematode is reduced by 42.8% compared with a control EGFP DSRNA, and the infectivity and pathogenicity of the nematode on host corn are also obviously reduced compared with the control EGFP DSRNA, so that the Pc-CL gene can be used as a target gene for preventing and controlling the coffee short nematode.

Description

Pc-CL protein of Pratylenchus coffee nematode, encoding gene and application thereof

Technical Field

The invention relates to the field of biotechnology, and in particular to a Pc-CL protein of Pratylenchus coffee nematode, a coding gene and an application thereof.

Background technique

Pratylenchus Filipjev (1936) is prone to cause root tissue rot and necrosis of crops, so it is also called root rot nematode. The geographical distribution of this nematode is very wide, posing a huge threat to agricultural production safety. As one of the most serious short-bodied nematodes, the coffee nematode (P.coffeae) can not only harm food crops such as corn, wheat, and sorghum, but also economic crops and ornamental flowers such as yam, soybean, sesame, tobacco, and lily. Coffee nematodes mainly use their stylets to damage the cortical tissue of the host's underground roots, causing root rot and necrosis. In addition, fungi and bacteria will invade the parts damaged by the stylets of the nematodes, causing the phenomenon of complex infection of diseases, aggravating the occurrence of diseases and seriously affecting the healthy growth of crops. Due to the rapid intraspecific variation and strong adaptability of coffee nematodes, prevention and control has become a global problem. Identifying and studying the pathogenic genes and their functions of coffee nematodes is the latest breakthrough point for screening safe and effective control targets.

Plant parasitic nematodes need to overcome the host's defense system when infecting the host. The secretory proteins of the esophageal gland can help nematodes manipulate and regulate the defense response of the host plant, inhibit plant disease resistance, and play an important role in the establishment and maintenance of nematode feeding sites. Such proteins are called effector proteins. With the rapid development of modern molecular biology technology and the new generation of high-throughput sequencing technology, common technical means such as nematode genome, transcriptome and proteome have been continuously optimized, and a large amount of data related to effector proteins of plant parasitic nematodes have been mined and published. This precise and rapid technology has accelerated the enrichment of the database of parasitic nematode effector proteins, providing a good opportunity for functional research on effector proteins and nematode evolution. Cysteine proteases play key functions in the physiological and biochemical processes of apoptosis, parasitic infection, immune escape, tissue invasion, etc. in the life activities of various nematodes and animal parasites. In recent years, more and more studies have been conducted on cathepsins on plant parasitic nematodes, and some L-type cathepsins (CL) have been cloned and studied in fixed parasitic root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.). However, the function of CL in migratory parasitic nematodes has not been reported.

Summary of the invention

The purpose of the present invention is to provide a coffee nematode Pc-CL protein, an encoding gene and an application thereof, so as to solve the problems existing in the above-mentioned prior art.

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

The present invention provides a Pc-CL protein, the amino acid sequence of the protein is shown in SEQ ID NO:1.

The present invention also provides a DNA molecule encoding the Pc-CL protein.

Preferably, the nucleotide sequence is as shown in SEQ ID NO:2.

The present invention also provides the use of the DNA molecule as a target gene in preventing and controlling coffee Pratylenchus coffee nematode.

The present invention also provides an in situ hybridization probe for the DNA molecule, the nucleotide sequence of which is shown in SEQ ID NO:3.

The present invention also provides dsRNA of the DNA molecule, and its nucleotide sequence is shown in SEQ ID NO:4.

The present invention also provides a product for controlling coffee nematodes, which comprises the in situ hybridization probe, the dsRNA or a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the DNA molecule.

Preferably, the recombinant expression vector comprises a binary Agrobacterium vector, a viral vector, a bacterial expression vector or a yeast expression vector.

The present invention also provides a method for preventing and controlling coffee Pratylenchus coffeeii, which utilizes the product to silence the DNA molecule.

The present invention studies the function of cathepsin Pc-CL in the infection and pathogenicity of Pratylenchus nematodes by ISH technology, qRT-PCR, in vitro RNAi technology and Agrobacterium-mediated methods.

qPCR can detect the entire PCR process in real time through fluorescent signals, and has the characteristics of sensitivity, accuracy and speed. In situ hybridization, as an emerging tissue localization technology that combines histochemistry, molecular biology and cell biology, has gradually become the most commonly used method in the functional research of plant parasitic nematodes. RNAi is another important means of studying the functional genes of plant parasitic nematodes. The dsRNA in vitro immersion method has been widely used for gene silencing. Its advantages are low operating cost and simplicity. As a difficult problem for the prevention and control of most crops in the world, the coffee nematode has been screened and studied. New effector proteins of the coffee nematode have been screened and studied, and their biological functions in the infection and pathogenesis of the coffee nematode and their molecular mechanisms of interaction with the host have been clarified, opening up new paths for the discovery of anti-parasitic strategies.

The present invention discloses the following technical effects:

The present invention provides a coffee nematode esophageal gland expression gene Pc-CL, the expression level of which in female nematodes is significantly higher than that in larvae, male nematodes and eggs (P<0.05); subcellular localization results show that the Pc-CL protein is localized in the cytoplasm and nucleus of Nicotiana benthamiana leaves. The present invention uses gene silencing to allow coffee nematodes to ingest Pc-CL dsRNA fragments, and measures the effect of Pc-CL gene silencing on the reproduction of coffee nematodes and the infectivity and pathogenicity to host corn. The experimental results show that after the Pc-CL gene is silenced by dsRNA treatment, the reproductive capacity of coffee nematodes is reduced by 42.8% compared with the control eGFP dsRNA, and the infectivity and pathogenicity of the nematodes to the host corn are also significantly reduced compared with the control eGFP dsRNA (P<0.05), indicating that the Pc-CL gene can be used as a target gene in the prevention and control of coffee nematodes.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

Figure 1 shows the tissue localization of the Pc-CL gene in the example of Pratylenchus coffeei, A: no hybridization signal of the sense chain probe (negative control); B and C: hybridization signals of the antisense chain probe are shown in the esophageal gland cells of Pratylenchus coffeei; S: stylet; mb: middle esophageal bulb; eg: esophageal gland;

Figure 2 is a quantitative analysis of the expression of the Pc-CL gene in four different stages of the coffee nematode in the embodiment; the detection was performed by the qRT-PCR method, the relative expression analysis method was the 2- ^△△Ct method, the internal reference gene was eGFP, the RQ of the female nematode was 1, female: female; juvenile: larva; egg: egg; male: male;

FIG3 is the subcellular localization result of the Pc-CL protein in the embodiment, and the Pc-CL protein is localized in the cytoplasm of tobacco leaf cells; A is the superposition result; B is the result observed under GFP green fluorescence; C is the result observed under bright field;

Figure 4 is the silencing efficiency test result of the Pc-CL gene in the embodiment; CK, untreated nematodes; G12, G24, G36 and G48: nematodes treated with eGFP dsRNA for 12, 24, 36 and 48 hours, respectively; R12, R24, R36 and R48: nematodes treated with Pc-CL dsRNA for 12, 24, 36 and 48 hours, respectively;

FIG5 is a statistical result of the number of nematodes inoculated with carrot callus tissues 60 days after different treatments in the embodiment; CK, untreated nematodes; G36: nematodes treated with eGFP dsRNA for 36 hours; R36: nematodes treated with Pc-CL dsRNA for 36 hours;

Figure 6 shows the results of the pathogenicity test of coffee nematode to corn after 36 hours of Pc-CL dsRNA soaking treatment in the embodiment; (a) and (b): 30d aboveground symptoms and 60d root symptoms, respectively, A represents CK without worms; B represents nematode inoculation treatment after 36h of Pc-CL dsRNA treatment; C represents nematode inoculation treatment after 36h of eGFP dsRNA treatment; D represents nematode inoculation treatment after 36h of clean water treatment; (c): measurement of various physiological indicators, including plant height, aboveground fresh weight, root fresh weight, and rhizosphere worm quantity;

Figure 7 shows the results of measuring the infectivity of coffee nematodes to corn after immersion treatment with Pc-CL dsRNA for 36 hours in the example; (a): root worm count 72 hours after inoculation of host corn roots; (b): root staining 72 hours after inoculation of host corn roots; CK, untreated nematodes; G36: nematodes treated with eGFP dsRNA for 36 hours; R36: nematodes treated with Pc-CL dsRNA for 36 hours.

Detailed ways

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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

Example 1 Discovery of Pc-CL protein and Pc-CL gene

1. Collect coffee nematodes, extract total RNA and reverse transcribe to obtain cDNA.

2. Using cDNA as template and Pc-CL-f and Pc-CL-r as primers, PCR amplification was performed. The sequence is as follows:

Upstream primer: Pc-CL-f: 5′-TAATCCAAGATGTCGATGGC-3′;

Downstream primer: Pc-CL-r: 5′-CAAAGCAGTTTTTCAGGCC-3′.

The amplification system was 2×Taq PCR Mix, 12.5 μL; cDNA first-strand template, 1 μL; forward primer, 1 μL; reverse primer, 1 μL; ddH ₂ O was added to 25 μL.

PCR amplification program: preliminary denaturation at 94°C for 2 min, (denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 90 s) 35 cycles; final extension at 72°C for 8 min.

3. Connect the One step ZTOPO-Blunt/TA zero background rapid cloning kit vector to the PCR amplification product in step 2, transform it into DH5α Escherichia coli competent cells, and sequence it. The sequencing results show that the amplified product has an open reading frame shown in the sequence SEQ ID NO: 2, and also encodes the protein shown in the sequence SEQ ID NO: 1. The SEQ ID NO: 1 in the sequence list is named Pc-CL protein, and its encoding gene is named Pc-CL gene.

Pc-CL protein amino acid sequence (SEQ ID NO: 1):

MSMAKREISLSIEPLLLDQPYKQVVKASSNKNPADDGKTAKAIFLFFRILLTFIIICVTIQLLFRFLPLLLSLIQAAINRFQNYEKQTENFFTNEAPEFVHQFVDFEKRFGRVWSGEKERWQRFKVFEKNLKEIERLNAEAIKAKRNVTFGINGMTDKSDEEMRQILLPLDHFKKRRLNAKFIRELNPILRESFTARSDEVLEEYPSHFDW RPKGVVTPVKAQGQCGSCWAFAAVATTESAHAVAHRELISLSEQELLDCNLENNACQGGNEDKAFSFIHERGLVSEWEYPYVAHRQNSCRMNEKSENLTTIDVAVFINPDEDSIIDWLLNFGPVNVGIAVPPDMKPYTGGIYHPSDFDCKFRVLGLHALLVVGYGVTDDGVKYWIVKNSWGDTWGQEHGYVNFIRGINACGIEDEPIGILA;

Pc-CL gene sequence (SEQ ID NO: 2):

atgtcgatggcaaaacgcgaaatttctttgtcaattgagccgctactcttggatcaaccctacaagcaagttgtgaaggcatcatcaaacaaaaaccctgctgacgatggaaaaaccgccaaggcaatctttctatttttccgaattttactcacctttataatcatttgcgtgaccattcaacttcttttccgattccttccactactcctctcactcattcaagcggcaatcaatcggttccagaattatgaaaaacaaacggagaatttcttcaccaatgaggcccctgaattcgtccaccaatttgtggattt cgaaaaacgttttggacgtgtttggtctggtgaaaaagagcgttggcaacgtttcaaagtgttcgagaagaatttgaaggaaattgaacgcctgaatgccgaagcaattaaagcaaaaagaaatgtgacttttggaatcaatggtatgaccgataaatcggatgaagaaatgagacagattctattgcctttggatcatttcaagaaacgacgactaaatgcgaaattcatcagagaactaaatccaattttgagagagtcgttcactgcacgaagcgatgaagtgttggaagaatatccaagtcattttgattggcg accgaagggggttgtgacaccggtgaaggcacagggtcaatgcggttcatgctgggcatttgctgctgtggcgacaacagaatctgcgcatgctgttgctcaccgcgaattgataagtctttctgaacaggaactcttagattgcaatttggagaacaacgcttgccagggaggaaatgaggacaaagcatttagttttatccatgaaagaggcctcgtttcggagtgggaatacccatatgtggcacatcgtcagaattcatgtcgaatgaatgagaagagcgaaaatctgacaacaattgacgttgctgtcttca tcaaccccgacgaggactcaataatcgattggctactcaactttggtcccgtgaatgttggtattgccgtccctcctgatatgaagccttacaccggcggaatctatcatccatctgattttgattgcaaattcagagttctcggacttcatgcacttttggttgtcggctatggcgttacggatgacggcgtaaaatattggatcgtcaaaaacagttggggcgacacatggggccaagaacacggctatgtgaatttcattcgaggaatcaacgcctgtggaattgaagatgaaccgattggaatattggcctga.

Example 2 Analysis of tissue localization of Pc-CL gene

1. Use the Pc-CL gene cloning vector as a template to amplify the target sequence by conventional PCR. The primers are as follows:

ISH-T7F: 5′-GGATCCTAATACGACTCACTATAGGGAACAACGCTTGCCAGG GAG-3′;

ISH-R: 5′-GACGGCAATACCAACATTCAC-3′;

ISH-F1:5′-AACAACGCTTGCCAGGGAG-3′;

ISH-T7R1: 5′-GGATCCTAATACGACTCACTATAGGGGACGGCAATACCAACA TTCAC-3′.

Using ISH-T7F/ISH-R and ISH-F1/ISH-T7R1 as primers, the forward and reverse strand DNA templates required for in vitro transcription of RNA probes were synthesized respectively. The specific steps were referred to the instruction manual of KOD FX DNA polymerase (TOYOBO) to prepare the reaction system for PCR reaction.

The PCR reaction conditions were as follows: 94°C pre-denaturation for 2 min, (98°C denaturation for 10 s, 58.2°C annealing for 30 s, 68°C extension for 90 s) 35 cycles; 72°C final extension for 10 min. PCR products were detected and recovered.

The Pc-CL gene in situ hybridization probe sequence is as follows (SEQ ID NO: 3):

aacaacgcttgccagggaggaaatgaggacaaagcatttagttttatccatgaaagaggcctcgtttcggagtgggaataccc atatgtggcacatcgtcagaattcatgtcgaatgaatgagaagagcgaaaatctgacaacaattgacgttgctgtcttcatcaaccccga cgaggactcaataatcgattggctactcaactttggtcccgtgaatgttggtattgccgtc.

2. Prepare the positive and negative strand RNA probe reaction system of Pc-CL gene according to the instruction of DIG RNA labeling kit (Roche), and incubate the reaction system in a 37℃ metal bath for 2h; then add 2mL of DNaseI, and incubate in the metal bath for 15min; finally, add 2μL of 0.2M EDTA (pH=8.0) to terminate the reaction. In situ hybridization was performed according to the instruction. The results are shown in Figure 1. The results show that the reverse strand probe has hybridization signals, and the hybridization signals are located in the esophageal gland of coffee nematode, indicating that the Pc-CL gene is a gene specifically expressed in esophageal gland cells.

Example 3 Analysis of the expression level of Pc-CL gene in different insect stages of Pratylenchus coffee nematode

RNA from female, male, larvae and eggs of P. coffee nematode was extracted and cDNA was obtained by reverse transcription. Primer5.0 software was used to design Pc-CL gene specific primers.

Upstream primer qpcR-f1: 5′-GAGAACAACGCTTGCCAG-3′;

Downstream primer: qpcR-r1: 5′-ATTGAGTCCTCGTCGGGGT-3′.

Real time PCR relative quantitative technology was used to detect the expression of Pc-CL gene in different insect stages, with 18s gene as the internal reference gene, and the upstream and downstream primers were:

18S-F: 5′-AGTGACGAGAAATAACGAGACC-3′;

18S-R: 5′-CCAGACTTGCCGCTCTCATA-3′.

Real-time PCR was performed on a fluorescent quantitative PCR instrument using TB Green Premix Ex Tap Kit (Takara). The expression differences of sample genes were relatively quantitatively analyzed using the ^2-△△Ct method (△Ct = average Ct value of target gene - average Ct value of reference gene). The experiment was repeated three times.

Reaction system (12μL): SYBR Green PCR Mix, 6μL; PCR Forward Primer (10μM), 0.5μL; PCR Reverse Primer (10μM), 0.5μL; cDNA template, 1μL; RNase-free water, 4μL.

Reaction program: 95°C, 2 min; (95°C, 15 s; Tm, 30 s; 72°C, 30 s) 40 cycles; 95°C, 15 s; 60°C, 60 s; 95°C, 30 s; 60°C, 15 s.

The results are shown in Figure 2. The expression level of the Pc-CL gene in female insects was significantly higher than that in the other three insect stages (P<0.05). It is speculated that the Pc-CL gene plays a major role in infection in female coffee nematodes.

Example 4 Subcellular localization of Pc-CL protein

1. Using Pc-CL gene cDNA as template, design Pc-CL full-length primers with restriction endonuclease AhdⅠ cutting site to amplify the target sequence by conventional PCR. The specific sequences of the primers are as follows:

PGWC-Pc-CL-F: 5′-AGCAGGCTTTGACTTTAGGTC-TAATCCAAGATGTCGA TGGC-3′;

PGWC-Pc-CL-R: 5′-TGGGTCTAGAGACTTTAGGTC-CAAAGCAGTTTTTCAG GCC-3′.

The PCR reaction system was: 2×Phanta Max Buffer, 12.5 μL; dNTP Mix (10 Mm each), 0.5 μL; upstream primer, 1 μL; downstream primer, 1 μL; Phanta Max Super-Fidelity DNA Polymerase, 0.5 μL; cDNA template, 1 μL; ddH ₂ O, made up to 25 μL.

PCR reaction conditions: pre-denaturation at 95°C, 3 min; denaturation at 95°C, 15 s; annealing at 58°C, 15 s; extension at 72°C, 1 min, for a total of 35 cycles; final extension at 72°C, 5 min; storage at 4°C.

After the reaction, the PCR product was electrophoresed with 1% agarose gel to detect the target band, and the amplified fragment was recovered, connected, transformed and sequenced.

2. Use restriction endonuclease AhdⅠ to digest the PGWC empty vector. The digestion reaction system is as follows: PGWC empty vector plasmid: 2μg, AhdⅠ: 2μL, 10×Fast Digest buffer: 2μL, ddH ₂ O supplemented to 20μL. Incubate at 37℃ in a metal bath for about 3h, and purify and recover by agarose gel electrophoresis.

3. Refer to the instructions of the Novogene Recombination Kit to seamlessly clone the target fragment and PGWC empty vector through reaction. The connection system is as follows: PGWC empty vector after enzyme digestion: 2μL, target fragment: 3μL, 5×CEⅡBuffer: 2μL, ExnaseⅡ: 1μL, ddH ₂ O to 10μL. Connect the reaction system at 37℃ for 30min and then introduce it into DH5α competent cells, transform, save the bacterial solution with correct sequencing verification results, extract the plasmid to obtain the recombinant vector of PGWC-Pc-CL.

4. Use LR recombinase to recombinantly replace the target protein gene sequence fragment in the entry vector PGWC-Pc-CL with the expression vector pEarleyGate104 to obtain the recombinant expression vector pEarleyGate104-Pc-CL. Use the freeze-thaw method to transform the recombinant plasmid into Agrobacterium GV3101 competent cells, take an appropriate amount of shaking culture solution and apply it on the LB plate containing the corresponding antibiotics, and culture it at 28°C for about 48 hours.

5. Pick the monoclonal Agrobacterium colony that is positive by PCR and place it in 5mL LB liquid medium containing the corresponding antibiotics, shake and culture at 220rpm (28℃) for 16-20h; centrifuge at 8000rpm for 2min at room temperature, discard the supernatant, resuspend in a buffer (containing 10mM _MgCl2 +10mM MES+200μM As in sterile water) to make _OD600 0.6-1.0; after induction at room temperature for 2-4 hours, mix the Agrobacterium solution containing the recombinant plasmid, and carefully infiltrate and inject it into the lower epidermis of Nicotiana benthamiana leaves (the 7th and 8th leaves) with a syringe. Make sections after 48h, and observe its fluorescence expression under a confocal microscope. The implementation results are shown in Figure 3. The Pc-CL protein is located in the cytoplasm of Nicotiana benthamiana leaves.

Example 5: Verification of the application of Pc-CL gene as a target in anti-nematode by in vitro RNAi silencing

1. The present invention silences the Pc-CL gene of coffee nematode by synthesizing dsRNA through in vitro RNAi amplification, and uses the exogenous gene eGFP as a control. The specific primers for amplifying the sense chain and the antisense chain are as follows:

Pc-CL-T7S:5′-GGATCCTAATACGACTCACTATAGGGCCTTCCACTACTCC TCTCAC-3′

Pc-CL-A:5′-CCAACACTTCATCGCTTC-3′

Pc-CL anti-S:5′-CCTTCCACTACTCCTCTCAC-3′

Pc-CL anti-T7A:5′-GGATCCTAATACGACTCACTATAGGGCCAACACTTCATC GCTTC-3′

eGFP-T7S: 5′-GGATCCTAATACGACTCACTATAGGG CAGTGCTTCAGCCG CTACC-3′

eGFP-positive-A:5′-AGTTCACCTTGATGCCGTTCTT-3′

eGFP anti-S: 5′-CAGTGCTTCAGCCGCTACC-3′

eGFP anti-T7A: 5′-GGATCCTAATACGACTCACTATAGGG AGTTCACCTTGATGC CGTTCTT-3′

The dsRNA sequence of the Pc-CL gene is as follows (SEQ ID NO: 4):

ccttccactactcctctcactcattcaagcggcaatcaatcggttccagaattatgaaaaacaaacggagaatttcttcaccaatgaggcccctgaattcgtccaccaatttgtggatttcgaaaaacgtttttggacgtgtttggtctggtgaaaaagagcgttggcaacgtttcaaagtgttcgagaagaatttgaaggaaattgaacgcctgaatgccgaagcaattaaagcaaaaagaaatgtgacttttggaatcaatggtatgaccgataaatcggatgaagaaatgagacagattctattgcctttggatcatttcaagaaacgacgactaaatgcgaaattcatcagagaactaaatccaattttgagagagtcgttcactgcacgaagcgatgaagtgttgg.

2. Wash out the coffee nematodes with better activity from the carrot callus, rinse with DEPC water 2-3 times, and treat the coffee nematodes with the corresponding Pc-CL dsRNA and eGFP dsRNA (2000ng/μL) at 100rpm/min for different time periods. The time periods were set to 12h, 24h, 36h and 48h. At the same time, each treatment was carried out three biological replicates.

(I) Total RNA of nematodes treated differently at different time periods was extracted, and the reverse transcribed cDNA was used as a qRT-PCR template to detect the silencing efficiency of the Pc-CL gene;

(II) 30 female coffee worms with good activity and treated differently were selected and inoculated on the prepared carrot callus. After 60 days of dark culture at 25°C, the reproduction of nematodes was isolated and counted. The experiment was repeated three times. Nematodes soaked in eGFP dsRNA (2.0 mg/mL) for the same time were used as controls, and nematodes treated with clean water were used as blank controls.

(III) A pot experiment was conducted by inoculating 1,000 nematodes into corn seedlings (grown for about 20 days) after being treated with immersion silencing (Pc-CL dsRNA immersion treatment for 36 hours). Corn plants inoculated with nematodes treated with eGFP dsRNA for 36 hours and water were used as controls to detect whether silencing of the Pc-CL gene affects the pathogenicity of coffee nematodes to the host. After growing in the greenhouse for 60 days, the plant height, aboveground fresh weight and root weight of corn with different treatments were measured, and the number of nematodes in the rhizosphere of the plants was isolated and counted. The experiment was set up with three biological replicates. See Figures 4, 5, and 6;

(IV) To further detect the effect of silencing the Pc-CL gene of coffee nematode on the infectivity of corn, 1000 coffee nematodes with different treatments were inoculated into corn roots with uniform growth for 72 h, and the number of nematodes in the roots was stained and counted.

As shown in Figure 7, Pc-CL dsRNA soaking treatment can effectively inhibit the expression of the target gene, and the silencing efficiency of the target gene is the highest after soaking treatment for 36 hours; after soaking treatment with Pc-CL dsRNA for 36 hours, the reproductive capacity of coffee nematodes and their pathogenicity and infectivity to host corn were significantly reduced compared with the control treatment group (P<0.05). This shows that the Pc-CL gene plays an important role in the reproduction and pathogenicity of coffee nematodes and can be used as a target gene for plant anti-nematode engineering.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (8)

1. A Pc-CL protein, characterized in that the protein has an amino acid sequence as set forth in SEQ ID NO: 1.

2. A DNA molecule encoding the Pc-CL protein of claim 1, characterized in that it has the nucleotide sequence set forth in SEQ ID NO: 2.

3. Use of a DNA molecule according to claim 2 as target gene for controlling aphelenchus coffee nematodes, wherein the expression of the target gene is reduced.

4. The in situ hybridization probe of the DNA molecule according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO: 3.

5. The dsRNA of claim 2 for a DNA molecule having a nucleotide sequence as set forth in SEQ ID NO: 4.

6. A product for controlling the nematode of the short body of coffee, characterized in that it comprises the dsRNA of claim 5 or a recombinant expression vector, an overexpression vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette comprising the DNA molecule of claim 2.

7. The product of claim 6, wherein the recombinant expression vector comprises a binary agrobacterium vector, a viral vector, a bacterial expression vector, or a yeast expression vector.

8. A method of controlling a praecox elegans, wherein the DNA molecule of claim 2 is silenced with the dsRNA of claim 5.