CN109628459B - HR4 gene and application of dsRNA thereof in pest control - Google Patents

Abstract

The invention provides an insect HR4 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 HR4 gene full length cDNA sequence. 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

HR4 gene and application of dsRNA thereof in pest control

Technical Field

The invention relates to the field of biotechnology and agricultural pest control. In particular to an locust migratory HR4 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. The pest control through 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 which are crucial to the growth and development of pests are key links for developing novel pesticides by applying RNAi technology, because not all dsRNA can effectively silence the expression of target genes, nor all dsRNA can kill pests.

HR4 is one of the key transcription factors in insect ecdysone signal transduction pathway, and by means of RNA interference technology, the subject group proves 100% lethality after injecting HR4 gene dsRNA to 4-year-old nymph. Therefore, the HR4 gene can be used as a high lethal target gene in RNAi-based pest control technology.

Disclosure of Invention

The invention aims to provide a full-length cDNA sequence of an insect HR4 gene and application of dsRNA thereof in killing locusta migratoria.

The invention provides a full-length cDNA sequence of migratory locust HR4 gene, 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 1221 bp, and the nucleotide sequence is SEQ ID NO: 1.

the invention provides application of migratory locust HR4 gene dsRNA in pest control, which is characterized in that based on migratory locust HR4 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 dsRNA is injected, mRNA expression of the HR4 gene of the migratory locust is remarkably reduced, and the migratory locust has growth and development resistance and causes 100 percent of death.

Drawings

FIG. 1: effect of dsRNA injected from HR4 gene on HR4 gene mRNA expression after entering into 4-year migratory locust. Beta-actin is an internal reference gene. WhereinP<0.05。

FIG. 2: influence of dsRNA injected with HR4 gene on growth and development of 4-year migratory locust nymphs. dsRNA locusta migratoria injected with HR4 gene appears to have growth retardation and resulted in 100% death. A: the left side is a control injected with water, the right side is a control injected with SEQ ID NO: 2 synthetic dsRNA, i.e. dsLmHR4。

Detailed Description

The first embodiment is as follows: obtaining of full-length cDNA and gene fragment of migratory locust HR4 gene

On the basis of the transcriptome database of migratory locust, adopting a bioinformatics method to search the migratory locust HR4 gene to obtain the migratory locust HR4 gene (LmHR4) 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 4-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 THR4scriptase M-MLV (RNase H-) as instruction, PCR amplification with designed upstream and downstream primersLmHR4The full-length sequence of the gene. The obtained product is purified, cloned and transformed into escherichia coli, and sent to Shanghai Weichai Jie organism Limited company for sequencing, and the sequence of the product is SEQ ID NO: 1.

example two: migratory locust HR4 gene specific dsRNA synthesis

1) Design of dsRNA primer of migratory locust HR4 gene

Based on the full-length cDNA sequence of the migratory locust HR4 gene, primer premier 5.0 software is adopted to design dsRNA primers, 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 dsRNA of migratory locust HR4 gene

The sequence is SEQ ID NO: 1, HR4 gene extraction plasmid as a template, 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, after purification with the FastPeure Gel DNA Extraction Mini Kit (Vazyme) Kit, dsRNA was synthesized by in vitro transcription as indicated by 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: migratory locust HR4 gene dsRNA lethal migratory locust experiment

1. Injection of dsRNA of migratory locust HR4 gene

Selecting 2-day nymphs with healthy growth, consistent size and 60 male and female halves and 5 ages for carrying out experiment group dsLmRanAnd (4) injecting the gene. Will synthesize dsLmRanUsing a microsyringe, 5 μ L (10 μ g ds)LmHR4) 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 HR4 gene silencing detection

Separately collecting the injection water and dsLmHR4Total RNA extraction was performed on nymphs after 48 h, control and injection ds at each time pointLmHR43 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 (LmHR4) And housekeeping genes: (β-actin) And calculating the silencing efficiency of the gene. The results show that ds was injected compared to the control groupLmHR4Group test insectLmHR4Gene expression was significantly reduced (as shown in figure 1).

3. Nymph phenotype observation after HR4 gene dsRNA injection

4-year-old nymph injection dsLmHR4Then, the control group insects successfully molt to adults, and the developmental state of the adults after molting is good. Injection of ds compared to control groupLmHR4Molting of migratory locustThe phenomenon was delayed for 2 days, and then all died, the mortality rate reached 100%, as shown in fig. 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

<110> university of Shanxi

<120> HR4 gene and application of dsRNA thereof in pest control

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Claims (6)

1. A migratory locust HR4 gene, which is characterized in that the nucleotide sequence is SEQ ID NO: 1.

2. A migratory locust HR4 gene fragment, which is characterized in that the nucleotide sequence is SEQ ID NO: 2.

3. The locust migratory HR4 gene fragment of SEQ ID NO: 2 synthetic dsRNA.

4. The use of dsRNA of the migratory locust HR4 gene according to claim 3 for migratory locust control.

5. The use of dsRNA of the migratory locust HR4 gene according to claim 4 for migratory locust control, 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; after purification, the product was transcribed in vitro to synthesize dsRNA as described in Promega T7 RiboMAX Express RNAi System kit.

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