CN111944824A - Tachykinin receptor gene of fall webworm, dsRNA and application in preventing and treating fall webworm - Google Patents
Tachykinin receptor gene of fall webworm, dsRNA and application in preventing and treating fall webworm Download PDFInfo
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Abstract
The invention discloses a tachykinin receptor gene of fall webworm, dsRNA and application thereof in preventing and treating the fall webworm. The nucleotide sequence of the tachykinin receptor gene is shown as SEQ ID No.1, and the coding sequence is protein of an amino acid sequence shown as SEQ ID No. 2. The nucleotide sequence of the tachykinin receptor gene dsRNA is shown as SEQ ID No. 3. The application of the tachykinin receptor gene dsRNA of the fall webworm in preventing or preparing fall webworm products. The invention can reduce the food intake of the hyphantria cunea larvae, obviously reduce the hunger resistance of the larvae, weaken the vitality of the hyphantria cunea, and finally achieve the effect of prevention and treatment.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a Tachykinin receptor gene (Tachykinin receptor) of fall webworm, dsRNA (double-stranded ribonucleic acid) and application of the dsRNA in control of the fall webworm.
Background
Hyphantria cunea (Drury) of the genus Lepidoptera (Lepidotera) Arctidae (Arctidae), native North America (Morris, 1963). In 1979, the plant was introduced into China from Dandong City of Liaoning province, and the host included 175 plants of 108 genus of 49 families (Yang loyalty and Zhang Yongan, 2007). The pest has the advantages of high propagation speed, strong fertility, more harm to host plants, large food intake and frequent outbreak and disaster formation, and becomes a worldwide quarantine pest. At present, the control is mainly performed by chemical pesticides such as chlorbenzuron III (vermouth, 2000), 3.6% nicotine and matrine microcapsule suspending agent (Zhangwei, etc., 2013) and triflumuron (Qilu, etc., 2009), but the problem of '3R' caused by long-term use cannot be ignored. In recent years, biological methods have been favored for the control of fall webworms (Zhao Haiyan et al, 2012; Guojunhui et al, 2003), such as: yang loyalty et al (2005) control the hyphantria cunea through the natural enemy insect Chouiia cunea Yang, the parasitic rate is as high as 92.67%; zhao Hai Yan et al (2012) adopt HcNPV (4.5X 10)6PIBs/mL) for preventing and controlling 2-3-year-old hyphantria cunea larvae of fraxinus chinensis forest, and the prevention effect is as high as 90.41%. Because the hyphantria cunea nuclear polyhedrosis virus HcNPV is concerned for its specificity to host and safety to natural enemy insects, it has the disadvantages of narrow insecticidal spectrum, slow insecticidal speed, low action activity, etc. In view of these problems, it is necessary to find new targets of pesticides for this species and then develop new insect pesticides, and with the rapid development of biotechnology, the control of pest damage by RNAi technology has become the focus and hot point of research for plant protection workers, and the technology has been applied in pest control in agriculture and forestry. On one hand, the pest target gene dsRNA is transferred into a plant body by a genetic engineering means, when pests eat the transgenic plant, gene silencing in the pest body is triggered to lose functions, and the eating capacity of the pests is reduced; based on RNAi technologyThe method has the characteristics of safety to human and livestock, specificity to target prevention and control objects, safety to non-target organisms, harmless insect resistance and the like, and solves the problem of '3R' caused by long-term application of chemical pesticides.
Neuropeptides are important molecules for cell signaling and are involved in the regulation and coordination of physiological processes in invertebrates (Broeck, 2001; Grimmelikhuijnzen and Hauser, 2012). It is through activating its specific G protein-coupled receptor (GPCR) (Altstein and Nassel,2010), GPCR transmits extracellular signals into the cell, which in turn has important regulatory effects on the development, growth, reproduction, metabolism and behavior of insects, and due to its basic functions in various life stages, the multiple functions of insect neuropeptides make them the main potential target for the development of novel insect control agents based on interfering with their endogenous activities (Altstein, 2001). Because of these important physiological functions of the insect neuropeptide family and receptor genes, insect neuropeptides are one of the hot spots in recent studies on insect physiology and insect neurology. At present, no report is found about the prevention and control of the major quarantine pest hyphantria cunea by inhibiting the transcription level of the gene of the insect neuropeptide receptor family.
Disclosure of Invention
Based on the defects, the invention provides the Tachykinin receptor gene (Tachykinin receptor) of the fall webworm, dsRNA (double-stranded ribonucleic acid) and application in the fall webworm control based on the gene, so that the defects of the prior art are overcome, the feed intake of the fall webworm larva can be influenced, the hunger resistance of the larva is reduced, the vitality of the fall webworm is weakened, and the control effect is finally achieved.
The purpose of the invention is realized by the following technical scheme: a tachykinin acceptor of fall webworm has the nucleotide sequence shown in SEQ ID No.1 and codes the protein with the amino acid sequence shown in SEQ ID No. 2.
Another object of the invention is: provides a tachykinin receptor gene dsRNA of fall webworm, the nucleotide sequence is shown in SEQ ID No. 3.
Another object of the invention is: provides an application of a tachykinin receptor gene dsRNA of the fall webworm in preventing or preparing products for preventing and controlling fall webworm larvae.
The application is characterized in that the feed intake of the hyphantria cunea larvae is reduced.
The application is characterized in that the injection dosage of the dsRNA of the tachykinin receptor gene is 2 mu g.
The dsRNA is used for preventing and controlling fall webworm larvae and comprises the active ingredient of the dsRNA.
The dsRNA is mainly applied to control of the fall webworm, and is mainly characterized in that the ability of the fall webworm to resist hunger is regulated, the food intake of the fall webworm larvae is obviously reduced, and the survival rate of the larvae under the hunger adversity is obviously reduced. The dsRNA is injected into the bodies of the 4 th instar larvae of the fall webworms in a micro-injection way, and the result shows that: compared with a control group dsRNAEGFP treatment group, the feeding intake of the tachykinin receptor gene silencing fall-back webworm larvae is obviously reduced, and the survival rate of the tachykinin receptor gene silencing treatment group under starvation treatment is obviously reduced. RNAi technology can efficiently and specifically silence the mRNA expression of the tachykinin receptor gene in the fall webworm.
The invention has the advantages and beneficial effects that: the invention discloses a full-length nucleic acid sequence of a tachykinin receptor of a fall webworm for the first time at home and abroad, dsRNA is injected into a body cavity of a 4 th instar larva of the fall webworm, a target gene tachykinin receptor of a gypsy moth is efficiently silenced, the fall webworm larva can be reduced in a proper amount, the survival rate of the larva is obviously reduced due to hunger, the survival of the fall webworm on hunger adversity is influenced, and the control effect is finally achieved. The experimental results show that: compared with the control of EGFP gene dsRNA injection, the American white moth larva has stronger silencing effect after injecting the tachykinin receptor gene dsRNA, so that feeding of the American white moth larva is inhibited. Therefore, the invention provides a method for preventing and treating the major quarantine pest fall webworm and the lepidoptera pest with similar structural domains by utilizing the silencing technology of the tachykinin receptor, and provides a new thought and technology for green prevention and treatment of forestry pests.
Drawings
FIG. 1 is an electrophoresis diagram of the tachykinin receptor gene of the fall webworm;
FIG. 2 shows the expression level of the tachykinin receptor gene of 4 th instar larvae of hyphantria cunea after dsRNA injection;
FIG. 3 is a graph showing the effect of feeding by 4 th instar larvae of Spodoptera cunea injected with tachykinin receptor dsRNA;
FIG. 4 is a graph of ghrelin survival rate of Douglas fir (Spodoptera cunea) injected with tachykinin receptor dsRNA.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.
Example 1: full-length cloning of tachykinin receptor gene of fall webworm
The nucleotide sequence 2379bp of the tachykinin receptor gene of the fall webworm, 1230bp of an open reading frame, 409 coded amino acids, 47.29kDa of molecular weight and 9.31 of theoretical isoelectric point PI.
Extracting total RNA of fall webworm, and using reverse transcription kit PrimeScriptTMThe first strand cDNA was synthesized using the RT reagent Kit (TaKaRa), and primers (forward primer: 5'-ATGAGGATGCTTGACGACCTGG-3'; reverse primer: 5'-TCATGCAGAACTCACCAGAGTC-3') were designed on both sides of the gene coding region sequence based on the hyphantria cunea larva transcriptome sequence using the first strand cDNA as a template.
Reaction System 5 XPrimeScript buffer 2. mu. L, PrimeScriptRT Enzyme Mix I0.5μL、Oligo d(T)primer(50μM)0.5μL、Random 6mers(100μM)0.5μL、Total RNA 0.5μg RNase Free ddH2O make up to 10. mu.L. The PCR amplification procedure was as follows: 3min at 94 ℃; 30s at 94 ℃, 30s at 60 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min. The PCR product was detected by electrophoresis on a 1% agarose gel and a 1230bp band was recovered using a gel recovery kit. The recovered bands were ligated with pMD18-T vector overnight and the ligation products were transformed into DH 5. alpha. competent cells. The positive clone bacterial liquid detected by the PCR of the bacterial liquid is sent to Beijing Hua Dazhou biotechnology limited company for sequencing to verify the reading frame.
Example 2: synthesis of American white moth tachykinin receptor gene dsRNA
According to the full length of the tachykinin receptor gene of the fall webworm cloned in the example 1, a forward primer (5'-GTACAACGTCTGTTTCATGGT-3') and a reverse primer (5'-GTGCTGGTACCATTCTTCC-3') of the dsRNA of the tachykinin receptor gene are designed and synthesized, a sequence with the fragment length of 492bp is obtained through amplification, and the dsRNA of the tachykinin receptor gene is obtained through an in-vitro dsRNA synthesis kit.
Specifically, the synthesis process is to add a T7 promoter sequence of about 20bp to the 5' end of each specific primer, and EGFP is used as a control group. Amplifying a target band by a PCR method, wherein the reaction program is 94 ℃ for 3 min; 30s at 94 ℃, 30s at 60 ℃, 1min at 72 ℃ and 35 cycles; at 72 ℃ for 7min, synthesizing dsRNA (refer to MEGAscript RNAi Kit instruction) as a template after the amplified product is detected and confirmed by electrophoresis, detecting the concentration of the dsRNA in an ultraviolet spectrophotometer, and taking 0.5 mu L of the dsRNA to detect and confirm in 1% agarose gel electrophoresis, and storing at-80 ℃ for later use.
Example 3: detection of Gene silencing Effect of tachykinin receptor of fall webworm
The dsRNA (2. mu.g) of the tachykinin receptor gene and EGFP gene synthesized in example 2 was microinjected into the Bombycis mori 4-instar larvae, the live 4-instar larvae were selected at 48 and 72h, respectively, total RNA was extracted using RNeasy Mini animal tissue Total RNA extraction kit (Qiagen), PrimeScript was usedTMSynthesizing a cDNA first chain by using an RT kit (TaKaRa), and detecting the expression quantity of the injected tachykinin receptor gene by using the cDNA first chain as a template and using fluorescent quantitative RT-PCR. The expression level of the tachykinin receptor gene of 4 th instar larvae of the hyphantria cunea after dsRNA injection is shown in figure 2. The result shows that the dsRNA of the exogenous control gene EGFP injected into the larva of the fall webworm can influence the expression of the tachykinin receptor gene. Compared with the control EGFP gene, the silencing effect of the tachykinin receptor gene is higher than that of the control EGFP gene. After 48h of injection, compared with the dsRNAEGFP treatment group, the expression level of the dsRNA tachykinin receptor in the dsRNA tachykinin receptor treatment group is reduced by 72.53%, the silencing effect of the dsRNA tachykinin receptor is reduced along with the increase of time, the silencing effect still exists after 72h of treatment, and the silencing efficiency is 36.38% (P is P)<0.05, the reference genes were RPL13 and EF1 α, and the primers are shown in table 1).
TABLE 1 primers of interest in RNAi interference
The method of cloning the tachykinin receptor of the fall webworm described in the above examples is a method commonly employed in the art. The methods for extracting total RNA of fall webworm larva, synthesizing first strand cDNA, synthesizing dsRNA, real-time fluorescence quantitative RT-PCR and the like which are described in the above embodiments are all mature technologies in the field, and the kits Qiagen and PrimeScriptTMRT, MEGAscript RNAi Kit, SYBR Green Real-time PCR Master Mix (Toyobo), etc. are all available from manufacturing vendors.
Example 4: effect of dsRNA silencing of tachykinin receptor genes on feeding by hyphantria cunea larvae
The in vitro synthesized dsRNA of example 2 was microinjected into the larvae of the fall webworm at age 4, while the effect of feed intake by the larvae of the fall webworm was recorded. More specifically, 2 μ g of dsRNA tachykinin receptor and dsRNAEGFP were injected into the starvation-treated fall webworm on the first day of 4 th instar larvae, 30 larvae were treated in each group, and the larvae were weighed in one group for ten, each treatment was repeated 3 times, and fresh weight of feed was weighed on time per day.
As can be seen from fig. 3: compared with a control group dsRNAEGFP, the experimental group injected with the dsRNA by a trace amount has a remarkable difference in food intake after being fed. Wherein, the micro-injection tachykinin receptor gene dsRNA treatment group shows that the food intake of 4-instar larvae of the fall webworms on day 1 fed by artificial feed is remarkably reduced (P <0.05), and is reduced by 64.54 percent (P <0.05) compared with the EGFP injection control group; on day 2 of feeding, the dsRNA tachykinin receptor panel fed 0.31g, only 60.98% of the control dsRNAEGFP (P < 0.05).
Example 5: effect of dsRNA silencing of tachykinin receptor genes on hunger resistance of hyphantria cunea larvae
The in vitro synthesized dsRNA of example 2 was microinjected into the adult 4 th instar larvae of fall webworm and placed in the absence of any feed (using sterile cotton balls to supplement the water source) while the death of the fall webworm larvae was recorded.
Compared with the control group dsRNAEGFP, the dsRNA tachykinin receptor treatment group starvation condition shortens the median lifetime and obviously reduces the survival rate. The mortality rate of the dsRNA tachykinin receptor treated group is 46.67 percent and is significantly higher than that of the control dsRNAEGFP (30.00 percent) after the starvation treatment is 96 hours, the mortality rate of the dsRNA tachykinin receptor treated group is 93.33 percent after the starvation treatment is 120 hours, and the mortality rate of the dsRNA tachykinin receptor treated group is only 63.33 percent (P <0.05, figure 4) of the control dsRNAEGFP
In the embodiment, a molecular biology approach is utilized to interfere with a tachykinin receptor of a potential defense gene of the fall webworm, so that the survival capability of insects is reduced, the application of the fall webworm in the fall webworm prevention and control has a wide prospect, a theoretical basis is provided for the prevention and control of other lepidoptera pests, the tachykinin receptor of the fall webworm is effectively silenced, the feeding of the fall webworm larva is influenced, the survival rate of the fall webworm larva under starvation stress is reduced, and a new idea and a new material are provided for the prevention and control of the fall webworm.
Sequence listing
<110> northeast university of forestry
<120> application of tachykinin receptor gene and dsRNA of fall webworm in preventing and treating fall webworm
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Claims (6)
1. A tachykinin receptor gene of fall webworm is characterized in that the nucleotide sequence is shown as SEQ ID No.1, and the coding protein is shown as the amino acid sequence of SEQ ID No. 2.
2. The hypha moth tachykinin receptor gene dsRNA of claim 1, which has a nucleotide sequence shown in SEQ ID No. 3.
3. The use of the hypha cunea moth tachykinin receptor gene dsRNA of claim 2 in preventing or preparing products for preventing and treating hypha cunea moth larvae.
4. Use according to claim 3, characterized by a product for reducing food intake by larvae of fall webworm.
5. The use according to claim 3 or 4, wherein the injected dose of the tachykinin receptor gene dsRNA is 2 μ g.
6. The dsRNA for preventing and treating hyphantria cunea larvae contains the dsRNA as claimed in claim 2 as an active ingredient.
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| CN113774065B (en) * | 2021-06-21 | 2023-11-10 | 南京市园林和林业科学研究院(南京市园林绿化指导站) | Fluorescent quantitative internal reference gene for different adults of fall webworm, primer and application thereof |
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| CN113481202A (en) * | 2021-08-17 | 2021-10-08 | 中国林业科学研究院森林生态环境与保护研究所 | Spodoptera cunea Rop gene dsRNA as well as bacterial expression solution and application thereof |
| CN113430200B (en) * | 2021-08-17 | 2022-05-27 | 中国林业科学研究院森林生态环境与保护研究所 | Hypha cunea Yang vATPase A gene dsRNA, bacterial expression liquid thereof and application |
| CN113481202B (en) * | 2021-08-17 | 2023-05-16 | 中国林业科学研究院森林生态环境与保护研究所 | Fall webworm Rop gene dsRNA, bacterial expression liquid and application thereof |
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