CN109504684B

CN109504684B - Application of Ran gene and dsRNA thereof in pest control

Info

Publication number: CN109504684B
Application number: CN201811563907.3A
Authority: CN
Inventors: 刘晓健; 张政; 张学尧; 张建珍; 马恩波
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2021-07-27
Anticipated expiration: 2038-12-20
Also published as: CN109504684A

Abstract

The invention provides an insect Ran gene full-length cDNA sequence and application of dsRNA thereof in pest control. Specifically, a bioinformatics method is used for obtaining a sequence of SEQ ID NO: 1, the full-length cDNA sequence of the Ran gene. According to SEQ ID NO: 1, dsRNA of the gene is designed and synthesized, and the dsRNA can specifically silence a target gene after being injected into a locusta migratoria body cavity, so that the locusta migratoria is prevented from growing and developing and leads to death. Multiple experiments show that the lethality reaches 100%. Due to the specificity and the high-efficiency lethality rate, the invention has important practical significance for pest control and can provide a new approach for pest control.

Description

Application of Ran gene and dsRNA thereof in pest control

Technical Field

The invention relates to the field of biotechnology and agricultural pest control. In particular to an locusta migratoria Ran gene and application of dsRNA thereof in pest control.

Background

Migratory locustLocusta migratoriaIs an intercontinental agricultural pest which is mainly distributed in Asia, Europe, Africa and Australia. The method has the characteristics of outbreak, cluster and migration, and once the outbreak occurs, the method is wide in related range, violent in incoming situation and serious in disaster. At present, chemical insecticides are still used as main means in locust control work, and the use of a large amount of chemical insecticides not only easily causes insects to generate drug resistance, but also brings serious pollution to the ecological environment and threatens the health of human beings, so that the research and development of green novel pesticides have important significance for locust control work in China.

RNA interference (RNAi), a phenomenon of specific post-transcriptional gene silencing caused by double-stranded RNA molecules, has been ascending at the technology front since the acquisition of the nobel prize in 2006. RNAi is not only a powerful tool for studying gene function, but also has great potential in pest control. Pest control by RNAi has the following characteristics: 1) the insecticidal has specificity and no killing effect on non-target organisms; 2) RNA is easy to degrade in nature and has no residue; 3) is nontoxic and harmless to the environment and is relatively safe. Currently, RNAi-based pest control has been internationally referred to as fourth generation pesticides. Screening and identifying genes essential to the growth and development of pests is a key link in the development of novel pesticides by applying RNAi technology, because not all dsRNA can effectively silence the expression of target genes, nor all dsRNA can kill pests

Ran is one of small G protein family members, can switch between two forms of RanGDP and RanGTP, and plays an important role in regulating and controlling nuclear mass transport, microtubule formation, mitotic spindle formation, and nuclear membrane assembly after cell division. By adopting an RNA interference technology, the subject group firstly proves that 100% of lethality occurs after injecting the Ran gene dsRNA of the migratory locust to a nymph of 5 years. Therefore, the Ran gene can be used as a high lethal target gene of an RNAi-based pest control technology.

Disclosure of Invention

The invention aims to provide a full-length cDNA sequence of an insect Ran gene and application of dsRNA thereof in killing locusta migratoria.

The invention provides a full-length cDNA sequence of a Ran gene of migratory locust, the nucleotide sequence of which is SEQ ID NO: 1. the full-length cDNA sequence of the gene is obtained based on migratory locust transcriptome database search, the length of the nucleotide is 648 bp, and the nucleotide sequence is SEQ ID NO: 1.

the invention provides application of migratory locust Ran gene dsRNA in pest control, which is characterized in that based on migratory locust Ran gene cDNA sequence, an upstream primer and a downstream primer containing a T7 promoter are designed through primer premier 5.0 software, and a template with two ends being T7 promoters is obtained through PCR amplification. After purification, dsRNA was synthesized by in vitro transcription as described in T7 RiboMAX Express RNAi System (Promega) kit. The synthesized dsRNA is injected into the locusta migratoria body cavity through a micro-injector. The results show that: after the dsRNA is injected, the mRNA expression of the Ran gene of the migratory locust is obviously reduced, and the migratory locust has growth and development resistance and causes 100 percent of death.

Drawings

FIG. 1: and (3) injecting dsRNA of the Ran gene into a 5-year migratory locust body, and influencing mRNA expression of the Ran gene after 24 h, 48 h and 72 h. Beta-actin is an internal reference gene. WhereinP<0.05，****P<0.001。

FIG. 2: influence of dsRNA injected with Ran gene on growth and development of 5-year migratory locust nymph. dsRNA locusta migratoria injected with Ran gene appears to have growth and development resistance and cause 100% death. A: the left side is water-injected control and the right side is injected SEQ ID NO: 2 synthetic dsRNA, i.e. dsLmRan。

Detailed Description

The first embodiment is as follows: full-length cDNA of migratory locust Ran gene and acquisition of gene fragment

Searching for the Ran gene of migratory locust by bioinformatics method based on transcriptome database of migratory locust to obtain Ran gene of migratory locustLmRan) And (3) designing an upstream primer and a downstream primer by using primer premier 5.0 software to verify the full-length cDNA sequence, and sending the full-length cDNA sequence to Shanghai Weiji microbial limited company for synthesis. Selecting 3 heads of 5-year-old locusta migratoria which is healthy in growth and is consistent in size and sex, quickly dissecting the body wall of the locusta migratoria under a body microscope, and freezing the locusta migratoria in liquid nitrogen. RNA was extracted according to TaKaRa RNAisso Plus kit. Reverse transcription of the RNA into first strand cDNA using Reverse transcription M-MLV (RNase H-) as a template, PCR amplification with designed upstream and downstream primersLmRanThe full-length sequence of the gene. Purifying the obtained product, cloning and transforming the product into escherichia coli, and sending the escherichia coli to Shanghai Weiji fundic organism Limited for sequencing, wherein the sequence of the escherichia coli Weiji fundic organism Weiji is SEQ ID NO: 1.

example two: synthesis of specific dsRNA of Ran gene of migratory locust

1) Design of dsRNA primer of migratory locust Ran gene

Based on the full-length cDNA sequence of the migratory locust Ran gene, a primer premier 5.0 software is adopted to design a dsRNA primer, and the sequences are respectively SEQ ID NO: 3 and SEQ ID NO: 4. the upstream and downstream primers carry the T7 promoter sequence. All the primers were synthesized by Shanghai Weiji fundic organisms, Ltd.

2) Synthesis of migratory locust Ran gene dsRNA

The sequence is SEQ ID NO: 1 as a template, and SEQ ID NO: 3 and SEQ ID NO: 4 as upstream and downstream primers, performing PCR amplification. And (3) carrying out PCR amplification on the PCR product with the sequence of SEQ ID NO: 2, purified by the FastPure Gel DNA Extraction Mini Kit (Vazyme) Kit and then transcribed in vitro to synthesize dsRNA according to the instructions of the T7 RiboMAX ™ Express RNAi System (Promega) Kit. Quantification was performed using NANODROP 2000 (Thermo scientific) to achieve a final concentration of 2. mu.g/. mu.L. Storing in a super low-temperature refrigerator at-80 deg.C for use.

Example three: experiments of DsPRC (migratory locust Ran) gene dsRNA (double-stranded ribonucleic acid) lethal migratory locust

1. Injection of horsefly grasshopper Ran gene dsRNA

Selecting 60-head 5-day nymphs with healthy growth, consistent size and half male and female for carrying out experiment group dsLmRanAnd (4) injecting the gene. Will synthesize dsLmRanUsing a microsyringe, 5 μ L (10 μ g ds)LmRan) Lightly inject into the area between the two and three abdominal segments of the nymph flank. The control group (60 heads, male and female halves) was injected with the same volume of water at the same time. And (3) feeding the locusta migratoria after injection in a constant-temperature biochemical incubator at the temperature of 30 ℃ (the illumination: dark time =14 h: 10 h, the temperature is 30 +/-2 ℃, and the humidity is 60%), and feeding fresh wheat seedlings and wheat bran every day.

2. Migratory locust Ran gene silencing detection

Separately collecting the injection water and dsLmRanTotal RNA extraction was performed on nymphs after 24 h, 48 h and 72 h, control and injection ds at each time pointLmRan3 biological repeats are combined, each biological repeat is tested for 3 worms, the worms are reversely transcribed into first strand cDNA, and a target gene is respectively detected by adopting an RT-qPCR method (LmRan) And housekeeping genes: (β-actin) And calculating the silencing efficiency of the gene. The results show that ds was injected compared to the control groupLmRanGroup test insectLmRanGene expression was significantly reduced (as shown in figure 1).

3. Phenotype observation of 5-year-old nymphs after Ran gene dsRNA injection

5-year-old nymph injection dsLmRanAfter that, the insects in the control group successfully molt to adults within 7 days, and the developmental state of the adults after molting is good. Injection of ds compared to control groupLmRanThe locusta migratoria does not have molting delay phenomenon, all the locusta migratoria die on the 7 th day, and the death rate reaches 100 percent, as shown in figure 2.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

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ttgtggttag ctagaaagct aattggtgac cctaatctgg agtttgttgc tatgcctgct 540

cttgtgcctc ctgaggtgac aatggaccca acatggcaac aacaaataga aaatgatctg 600

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Claims

1. A migratory locust Ran gene is characterized in that the nucleotide sequence is SEQ ID NO: 1.

2. A migratory locust Ran gene fragment is characterized in that the nucleotide sequence is SEQ ID NO: 2.

3. The locust fly Ran gene fragment of SEQ ID NO: 2 synthetic dsRNA.

4. The use of dsRNA of a migratory locust Ran gene as claimed in claim 3 for migratory locust control.

5. The use of dsRNA of a migratory locust Ran gene as claimed in claim 3 for the control of migratory locust, wherein: the dsRNA is prepared into a spray type pesticide, or the dsRNA is prepared into a bait, or the dsRNA is transferred into a migratory locust to ingest plant bodies.

6. The method of obtaining the dsRNA of claim 3, comprising the steps of: according to SEQ ID NO: 1, designing an upstream primer SEQ ID NO: 3 and the downstream primer SEQ ID NO: 4, obtaining the nucleotide sequence of SEQ ID NO: 2, the product of which contains the T7 promoter; the product was purified and transcribed to synthesize dsRNA in vitro as described in T7 RiboMAX Express RNAi System (Promega) kit.