Monochamus alternatus hope adenosine triphosphate enzyme gene V-ATPase H and application thereof

Abstract

The invention discloses a monochamus alternatus hope adenosine triphosphate enzyme gene V-ATPase H and application thereof. The monochamus alternatus hope adenosine triphosphate enzyme gene V-ATPase H comprises a V-ATPase H superfamily conserved domain, and the base sequence is shown as SEQ ID NO.1. The monochamus alternatus hope adenosine triphosphate enzyme gene V-ATPase H encodes V-ATPase protein, and the amino acid sequence of the monochamus alternatus hope adenosine triphosphate enzyme gene V-ATPase protein is shown as SEQ ID NO.2. The invention also discloses an application of the triphosadenase gene V-ATPase H of the monochamus alternatus hope as an RNAi target gene in prevention and treatment of the monochamus alternatus hope. The dsRNA is designed and synthesized by taking the monochamus alternatus hope adenosine triphosphate enzyme gene V-ATPase H as a template, then the dsRNA is used for interfering expression of monochamus alternatus hope immune genes Dome, Tube and TAB2, and experimental results show that the dsRNA designed and synthesized by utilizing the monochamus alternatus hope adenosine triphosphate enzyme gene V-ATPase H can effectively inhibit expression of monochamus alternatus hope immune related genes, so that the dsRNA has good development and utilization prospects.

Description

Technical Field

[0001] The invention belongs to the field of forest pest prevention and control, and particularly relates to a pine beetle adenosine triphosphatase gene V-ATPase H and an application thereof. Background Art

[0002] Pine beetle is a dangerous forest pest and an important trunk-boring pest that harms pine trees. The larvae bore into the phloem and xylem of the trunk and branches, destroy and cut off the transporting tissues, affect the transport of water and nutrients, seriously affect the growth of pine trees, and cause large-scale death in pine forests. Pine beetle is widely distributed and has posed a serious threat to the healthy and sustainable development of my country's forestry industry and the construction of ecological civilization. In addition, pine beetle is also the main vector insect of pine wood nematode, an important international quarantine object. When pine beetle supplements nutrition or lays eggs on pine trees, pine wood nematodes are inoculated and invaded into healthy pine trees through the wounds caused by supplementing nutrition and laying eggs. The extremely destructive disease in which pine wood nematodes parasitize in pine trees and cause the trees to die quickly is called pine wood nematode disease. In recent years, the area and location of pine wood nematode disease in my country's pine forests caused by pine beetle damage have shown an increasing trend year by year. Therefore, eliminating or reducing pine wood nematodes in trees and forest stands is one of the breakthroughs in controlling pine wood nematode disease.

[0003] RNA interference (RNAi) is a gene silencing phenomenon induced by double-stranded RNA in molecular biology. Its mechanism is to inhibit gene expression by hindering the translation or transcription of specific genes. RNA interference has been proven to be effective in a variety of agricultural and forestry pests, such as tobacco hornworm, cotton bollworm, corn root leaf beetle, and white ash borer. Due to its specificity and effectiveness, RNA interference has been considered an important specific gene blocking technology and an important research method for agricultural and forestry pest control.

[0004] ATPase is an ATP-dependent proton pump involved in various cellular processes and disease processes. ATPase is widely present in epithelial tissues such as the Malpighian tubules, salivary glands, midgut, and sensory organs of insects. It is particularly noteworthy that ATPase plays an important role in the absorption of amino acids and the regulation of pH in the insect midgut. Studies have shown that oral administration of dsRNA / siRNA targeting ATPase to pests (such as western corn rootworm and whitefly) can lead to significant mortality. Summary of the invention

[0005] In view of the technical problems in the background technology, the purpose of the present invention is to provide adenosine triphosphatase gene V-ATPase H of Monochamus alternatus and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a ATPase gene V-ATPaseH of Monochamus alternatus, comprising a V-ATPase H superfamily conserved region, and the base sequence thereof is shown in SEQ ID NO.1.

[0008] Preferably, the V-ATPase H protein encoded by the Monochamus alternatus ATPase gene V-ATPase H comprises 495 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] The second aspect of the present invention provides the use of the above-mentioned pine beetle adenosine triphosphatase gene V-ATPase H as an RNAi target gene in the prevention and treatment of pine beetle (M. alternatus).

[0010] Preferably, the method for controlling Monochamus alternatus using the ATPase gene V-ATPase HRNA of Monochamus alternatus comprises the following steps:

[0011] S1. Extracting total RNA from Monochamus alternatus larvae and determining the total RNA concentration;

[0012] S2, reverse transcription and synthesis of cDNA from total RNA using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMixkit kit;

[0013] S3, using cDNA as template, amplify the conserved region fragment of the target gene V-ATPase H using a kit;

[0014] S4, designing amplification primers, including a V-ATPase H forward primer, namely, ATPase HF, and a V-ATPase H reverse primer, namely, ATPase HR, and performing a PCR amplification reaction using the amplification primers;

[0015] V-ATPase HF: 5'-GAAGAGAGACCGCAATCAGT-3'

[0016] V-ATPase HR: 5'-AACCGACTGGATGTATTCGT-3'

[0017] S5, using 1% agarose gel to detect the PCR amplification product, cutting out the gel containing the band, recovering the kit, and purifying the PCR product;

[0018] S6. Sequencing to confirm the sequence, and using the purified PCR product as a template to synthesize dsRNA of the ATPase gene V-ATPase H of Monochamus alternatus;

[0019] S7. Mix the hemolymph of the larvae of Monochamus alternatus with dsRNA, incubate at 28°C in the dark for 72 hours to interfere with the hemolymph of the larvae of Monochamus alternatus, and detect the expression levels of the immune genes Dome, Tube and TAB2 of Monochamus alternatus.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a pine beetle adenosine triphosphatase gene V-ATPase H, and its use as an RNAi target gene in the prevention and control of pine beetles. By designing and synthesizing dsRNA using the pine beetle adenosine triphosphatase gene V-ATPase H as a template, and then using the dsRNA to interfere with the expression of the pine beetle immune genes Dome, Tube and TAB2, the purpose of effectively preventing and controlling the occurrence of pine wood nematode disease caused by the pine beetle as a medium is achieved, which is of great significance for the prevention and control of forestry diseases and therefore has good application prospects. The present invention also obtains the CDS sequence of the pine beetle adenosine triphosphatase gene V-ATPase H, which is important for studying the function of the gene in the pine beetle. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, 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.

[0023] Figure 1 This is the expression level result of the ATPase gene of Monochamus alternatus after being treated with dsRNA;

[0024] Figure 2 This is a graph showing the expression levels of immunity-related genes Dome, Tube and TAB2 of Monochamus alternatus after dsRNA treatment. DETAILED DESCRIPTION

[0025] In the following description, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are provided to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0026] Example 1

[0027] 1. Experimental Materials and Methods

[0028] 1.1 Experimental Materials and Reagents

[0029] Total RNA Kit I (Omega, Georgia, USA), Monochamus alternatus larvae, EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Quanshijin, Beijing, China), Multiscan GO microplate spectrophotometer with μDrop ultra-microplate (Thermo Fisher Scientific, Massachusetts, USA), PEGFP-C1 plasmid (Miaoling Biotechnology, Wuhan, Hubei), TransStart-KD-Plus-PCR-SuperMix kit (Quanshijin, Beijing, China), SanPrep column DNA gel recovery kit (Shanghai, China), MEGAscript RNAi kit (Thermo Fisher Scientific, Massachusetts, USA)

[0030] 1.2 Experimental methods

[0031] 1.2.1 Synthesis of dsRNA of the V-ATPase H gene of Monochamus alternatus

[0032] (1) Total RNA was extracted from the larvae of Monochamus alternatus using the total RNA Kit I;

[0033] (2) The integrity of the extracted total RNA samples was checked by 1% agarose gel electrophoresis, and the concentration of total RNA was determined using a MultiscanGO microplate spectrophotometer;

[0034] (3) Reverse transcription and synthesis of cDNA from total RNA using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMixkit kit;

[0035] (4) Using the cDNA prepared above and the enhanced green fluorescent protein plasmid, i.e., PEGFP-C1 plasmid, as templates, the TransStart-KD-Plus-PCR-SuperMix kit was used to amplify the target gene V-ATPase H and the enhanced green fluorescent protein gene EGPF fragment, respectively;

[0036] (5) The corresponding amplification primers were designed using the Primer3web software, specifically V-ATPase H forward primer, i.e., ATPase HF, V-ATPase H reverse primer, i.e., ATPase HR, EGFP forward primer, and EGFP reverse primer (see Table 1);

[0037] Table 1. Primers used for PCR amplification

[0038]

[0039] (6) PCR amplification reaction was carried out in A200 PCR instrument using V-ATPase H forward primer, V-ATPase H reverse primer, EGFP forward primer and EGFP reverse primer. PCR cycle conditions were as follows: 1. Initial denaturation: 94°C for 5 min; 2. Cycling (35 cycles): denaturation: 94°C for 30 sec, annealing: 55°C for 30 sec, extension: 68°C for 1 min; 3. Final extension: 68°C for 10 min; 4. End: incubation at 4°C.

[0040] (7) Detect the PCR amplification product using 1% agarose gel, cut the gel containing the band, and purify the PCR product using the SanPrep column DNA gel recovery kit;

[0041] (8) After sequencing to confirm the sequence, the purified PCR product was used as a template and the MEGAscript RNAi kit was used to synthesize dsRNA of the ATPase gene V-ATPaseH of Monochamus alternatus and the dsRNA of the enhanced green fluorescent protein gene EGFP.

[0042] 1.2.2. RNA interference experiment using dsRNA of V-ATPase H gene of Monochamus alternatus

[0043] 20 μL of hemolymph was taken from the fifth-instar larvae of Monochamus alternatus and placed in an RNase-free centrifuge tube. 1 μL of the above-mentioned synthesized dsRNA of the ATPase gene V-ATPase H of Monochamus alternatus (1000 ng / μL) and the enhanced green fluorescent protein gene EGFP dsRNA (as a control) were added to the hemolymph respectively, and mixed well. The mixture was incubated at 28°C in the dark. Samples were taken after 24h, 48h, and 72h for the next step of RNA extraction. The transcription level of the ATPase gene V-ATPase H of Monochamus alternatus after treatment for different time periods was detected, and the silencing efficiency of dsRNA targeting V-ATPase H was studied. Specifically, the expression level of the knocked-down target gene was detected by fluorescence quantitative detection. The results are shown in Figure 1 .

[0044] Depend on Figure 1The results showed that dsRNA treatment caused the transcription level of the target gene to decrease significantly at 24 hours (t=4.429, df=4, P=0.011) and 48 hours (t=3.683, df=4, P=0.021) after treatment, which was significantly different from the control group; and after 72 hours (t=0.583, df=4, P=0.591), the expression level of the target gene recovered, which was not significantly different from the control group. The above results indicate that the RNAi efficiency induced by exogenous dsRNA of the V-ATPase H gene of Monochamus alternatus is a transient effect. dsRNA significantly reduced the expression of the target gene 24 hours after treatment, but recovered 72 hours after treatment.

[0045] 1.2.3. Determination of the expression levels of the adenosine triphosphatase gene V-ATPase H and immune-related genes (such as Dome, Tube, and TAB2) by qPCR

[0046] (1) Total RNA was extracted from hemolymph samples of Monochamus alternatus larvae using the total RNA Kit I, and cDNA was prepared using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit. All steps were performed according to the instructions.

[0047] (2) Primer3web software was used to design (gene-specific) primers for qPCR amplification of the pine beetle adenosine triphosphatase gene V-ATPase H, glyceraldehyde-3-phosphate dehydrogenase GAPDH, and immune-related genes (such as Dome, Tube, and TAB2). See Table 2.

[0048] (3) According to the protocol provided by the PefectStart-Green-qPCR-SuperMix kit, qPCR amplification was performed on the LineGene4800 fluorescent quantitative PCR detection system. The qPCR amplification program was set as follows: 94°C for 30 s; 94°C for 5 s, 60°C for 30 s, and 40 cycles. The specificity of the SYBR green PCR signal was confirmed by the melting curve from 60°C to 94°C. GAPDH was used as the internal reference gene. -ΔΔCT The relative expression levels of genes were determined by Figure 2 .

[0049] Table 2. Primers used for qPCR amplification

[0050]

[0051] Figure 2The results showed that the present invention can effectively determine the expression level of the adenosine triphosphatase gene V-ATPase H and immune-related genes (such as Dome, Tube and TAB2). At the same time, the method was used to detect the expression level of immune-related genes (such as Dome, Tube and TAB2) 72 hours after dsRNA treatment. It was proved that RNAi knockdown of the adenosine triphosphatase gene V-ATPase H inhibited the expression of Dome (t=3.032, df=4, P=0.031), Tube (t=2.996, df=4, P=0.040) and TAB2 (t=2.145, df=4, P=0.026) ( Figure 2 ). The potential of V-ATPase H as an RNAi target gene for the control of pine beetle (M. alternatus) and other pests was further confirmed.

[0052] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A pine beetle adenosine triphosphatase gene V-ATPase H, characterized in that: It contains a V-ATPase H superfamily conserved region, and its base sequence is shown in SEQ ID NO.

1.

2. The ATPase gene V-ATPase H of Monochamus alternatus according to claim 1, characterized in that: The ATPase gene V-ATPase H of Monochamus alternatus encodes V-ATPase protein, and its amino acid sequence is shown in SEQ ID NO.

2.

3. Use of the pine beetle adenosine triphosphatase gene V-ATPase H described in claim 1 as an RNAi target gene in the prevention and treatment of pine beetles.

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