CN110129327B - Migratory locust pore canal formation related geneLmsnarAnd application of dsRNA thereof in locusta migratoria control - Google Patents
Migratory locust pore canal formation related geneLmsnarAnd application of dsRNA thereof in locusta migratoria control Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and provides a locusta migratoria pore channel formation related geneLmsnarAnd the application of dsRNA thereof in locusta migratoria control. Firstly, a bioinformatics method is applied to clone related genes for forming epidermal ducts from locusta migratoriasnarObtaining the polypeptide with the sequence of SEQ ID NO: 1 migratory locust pore formation related gene (Lmsnar) (ii) a Designing a primer, synthesizing dsRNA interfering the gene, and injecting the dsRNA into a locusta migratoria body cavity by an injection method. The results show that:Lmsnarafter silencing, locusts died before, during or after molting (the lethality rates were 3.3%, 6.7% and 18.3%, respectively), and their epidermal permeability was enhanced; its viability was significantly reduced in a dry environment compared to the control. The invention provides a new specific molecular target for pest control based on RNA interference and provides a new approach for pest control.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a locusta migratoria pore channel formation related geneLmsnarAnd the application of dsRNA thereof in controlling locusta migratoria.
Background
Migratory locusts are global agricultural pests, have wide distribution range and strong outbreak, often appear in a social form and have strong migratory flight capability. It mainly takes gramineae as food, once locust disasters occur, the range of the locusts is wide, and the locusts are seriously damaged. In recent years, due to long-term unreasonable chemical prevention and control, not only is the agricultural environment seriously polluted, but also locusta migratoria generates drug resistance to common pesticides, so that a novel molecular target is screened, and the research and development of novel green and environment-friendly pesticides become important social concerns.
RNA interference (RNAi) refers to a phenomenon that cells induce by using endogenous or exogenous double-stranded RNA (dsRNA) to induce and enable homologous mRNA to be efficiently and specifically degraded by specific enzymes so as to cause gene silencing, and belongs to post-transcriptional gene silencing. RNAi research has made a breakthrough in recent years, and is evaluated as one of ten scientific advances in 2001 by the "Science" journal, and listed as the first of ten scientific advances in 2002. RNAi is not only a powerful tool for studying gene function, but also has great potential in pest control. The pest control by RNA interference 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. Therefore, scholars at home and abroad refer to the pesticide as a fourth generation pesticide, and RNAi technology becomes a main research direction for biological control of pests in the future.
The insect body wall covers the whole insect body, and has the effects of maintaining the shape of the insect body, inhibiting the evaporation of water in the body, resisting the invasion of exogenous substances and the like. The insect body wall is mainly composed of the upper epidermis (epicuticle), the pro-epidermis (procuticle) and the dermis cell layer (epidermal cells). The insect epidermis has stronger hydrophobic property and water retention function, and can make the insect body better adapt to relatively dry land environment. The structure and content of the lipid substances of the insect epidermis are main factors for determining whether the insect epidermis can survive in adverse environments such as dry high temperature and the like. The pore canals located in the protoepidermis are the main pathways for the transport of lipid substances from the dermal cells to the upper epidermis. Snar is identified as an extracellular protein in drosophila melanogaster and plays an important role in preventing epidermal dehydration and invasion of exogenous substances. Therefore, it is of great theoretical and practical significance to deeply study the physicochemical properties and biological functions of locusta migratoria.
Disclosure of Invention
The inventionAims to provide a gene related to the formation of locusta migratoria pore canalsLmsnarAnd the application of dsRNA thereof in controlling locusta migratoria.
The invention is realized by the following technical scheme: migratory locust pore channel formation related geneLmsnarThe locusta migratoria tunnel forms related genesLmsnarThe nucleotide sequence of (a) is shown as SEQ ID NO: 1 is shown.
The locusta migratoria tunnel formation related geneLmsnarThe coded amino acid sequence is shown as SEQ ID NO: 2, respectively.
The described migratory locust pore canal can form dsRNA synthesized by related gene, its nucleotide sequence is SEQ ID NO: 3.
the locusta migratoria tunnel formation related geneLmsnarThe specific synthetic method of the synthesized dsRNA comprises the following steps: according to SEQ ID NO: 1 designing an upstream primer SEQ ID NO: 4 and the downstream primer SEQ ID NO: 5, obtaining the nucleotide sequence of SEQ ID NO: 3, which contains a T7 promoter, and the length of the nucleotide sequence is 550 bp; the product was synthesized into dsRNA, ds, by T7 RiboMAX Express RNAi System (Promega) kitsnar。
The locusta migratoria tunnel formation related geneLmsnarThe application of the synthesized dsRNA in controlling locusta migratoria.
The locusta migratoria pore canal formation related gene of the inventionLmsnarThe nucleotide sequence of (a) is SEQ ID NO: 1. the full-length gene sequence is obtained by splicing searched fragments based on a migratory locust transcriptome database through BioEdit software, designing an upstream primer and a downstream primer, and performing PCR amplification and clone sequencing. The nucleotide is 743 bp in length and encodes 177 amino acids.
The locusta migratoria pore canal formation related gene of the inventionLmsnarThe coded amino acid sequence is shown as SEQ ID NO: 2, respectively. The amino acid sequence is relative to SEQ ID NO: 1 is predicted after bioinformatics analysis. Bioinformatics analysis shows thatsnarThe gene codes 177 amino acids, the molecular weight is 19.9KD, and the theoretical isoelectric point is 6.95.
The locusta migratoria pore canal formation related gene of the inventionLmsnarSynthetic dsRNA, based on SEQ ID NO: 1 is set up by primer premier5.0 softwareCalculating the upstream primer SEQ ID NO: 4 and the downstream primer SEQ ID NO: 5, obtaining the nucleotide sequence of SEQ ID NO: 3, which contains a T7 promoter, and the length of the nucleotide sequence is 550 bp. Further, dsRNA (ds) was synthesized using T7 RiboMAX Express RNAi System (Promega) kitsnar）。
SEQ ID NO: 3, application of the synthesized dsRNA in pest control: SEQ ID NO: 3 injecting the synthesized dsRNA into the body cavity of the migratory locust. The results show that: SEQ ID NO: 3, the dsRNA synthesized by the method can specifically silence the canals of the migratory locust to form mRNA expression of related genes, and cause death (the death rates are respectively 3.3%, 6.7% and 18.3%) before, during and after molting of the migratory locust, and the permeability of the epidermis is enhanced; in a dry environment (humidity 15 ± 2%), its viability is significantly reduced.
Drawings
FIG. 1: mRNA silencing condition of related gene formed by locusta migratoria tunnel 24 h after dsRNA injection (left is injection ds)GFPRight is the experimental group injected with dsRNA).EF1-αIs an internal reference gene. Wherein the first and second groups are different from each other,P<0.001。
FIG. 2: effect of dsRNA injection on the growth and development of 4-year migratory locust nymphs (A is injection ds)GFPB is an experimental group injected with dsRNA). Locusts in the experimental group showed the phenotype of death before, during or after molting (the lethality rates were 3.3%, 6.7% and 18.3%, respectively).
FIG. 3: effect of dsRNA injection on epidermal permeability of migratory locust (upper is injection ds)GFPThe control group of (1), the experimental group injected with dsRNA). The epidermis of the experimental locusta migratoria can be stained with eosin.
FIG. 4: viability of dsRNA nymphs injected under dry conditions. Blue: injection dsGFPNymphs (humidity 15 ± 2%). Red: injection dsGFPNymphs (humidity 50 ± 2%). Green: injection dsLmsnarNymphs (humidity 15 ± 2%). Purple: injection dsLmsnarNymphs (humidity 50 ± 2%). The survival rate of locusta migratoria in the dry environment was significantly reduced compared to the control.
Detailed Description
Example 1: sequence of locusta migratoria tunnel formation related gene and obtaining of dsRNA thereof
1. Sequence acquisition of related genes for locusta migratoria tunnel formation
On the basis of migratory locust transcriptome database, a bioinformatics method is adopted to search the sequence of the relevant genes formed by the migratory locust pore canals to obtainLmsnarPartial sequence. Performing Blast analysis on the sequence by using NCBI website, obtaining the coding sequence by combining with locusta migratoria genome sequence, splicing and comparing by using BioEdit software to obtainLmsnarFull open reading frame sequence. An upstream primer and a downstream primer are designed by using primer premier5.0 software and sent to Shanghai biological engineering Co.
Extracting total RNA of 5-instar nymphs of migratory locusts, carrying out reverse transcription on the extracted total RNA into first-strand cDNA by adopting M-MLV reverse transcriptase (TaKaRa company), taking the first-strand cDNA as a template, carrying out PCR amplification by combining designed upstream and downstream primers, purifying a product by using a Gel Extraction Kit (Sigma), connecting a pEASY-T3 vector (Beijing all-gold biotechnology, Inc.) and transforming the product into escherichia coli Trans-T1 competence (Beijing all-gold biotechnology, Inc.), sending the escherichia coli Trans-T1 competence to Shanghai Biotechnology, Inc., carrying out sequencing and extracting plasmids. And obtaining the full-length open reading frame sequence of the related gene formed by the pore channel, wherein the sequence is SEQ ID NO: 1. the ORF sequence was translated using ExPASy website translation tool and the molecular weight and isoelectric point (pI) of the protein were predicted. The sequence is SEQ ID NO: 2. the gene codes 177 amino acids, has a molecular weight of 19.9KD and a theoretical isoelectric point of 6.95.
2. dsRNA synthesis of locusta migratoria tunnel formation related gene
1) dsRNA primer design of locusta migratoria tunnel formation related gene
The sequence of the related gene for the locusta migratoria channel formation obtained based on the research is SEQ ID NO: 1, designing dsRNA primers by using primer premier5.0 software, wherein the sequences are respectively shown as SEQ ID NO: 4 and SEQ ID NO: 5. then sent to Shanghai biological engineering Co., Ltd for synthesis.
2) dsRNA synthesis of migratory locust tunnel formation related gene
Taking the pore channel formation related gene extraction plasmid as a template, wherein the expression sequence of SEQ ID NO: 4 and SEQ ID NO: 5, upstream and downstream primers, and PCR amplification is performed. The amplified PCR product was purified and then transcribed to synthesize dsRNA in vitro according to the instructions of T7 RiboMAX Express RNAi System (Promega) kit. Quantification was performed using NaNoDrop 2000 (Thermo scientific) to achieve a final concentration of 2.5. mu.g/. mu.l. Storing in a low temperature refrigerator at-20 deg.C for use.
Example 2: migratory locust channel formation related gene dsRNA lethal 4-year migratory locust
1. Specificity dsRNA injection of related gene formed by migratory locust pore canal
4 μ l (10 μ g) of SEQ ID NO: 3 dsRNA (ds)snar) The mixture was injected into the 4 th day 1 nymph between the two and three abdominal segments with a 25 μ l microsyringe, and 60 nymphs were injected in total, half each. Simultaneously selecting 60 nymphs of the first day of 4 years old as a control group, and injecting ds with the same volume and concentrationGFPTo the body cavity. And (3) respectively feeding the nymphs 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 40 +/-5%), and feeding the young wheat seedlings and the wheat bran every day.
2. Detection of silencing efficiency of locusta migratoria tunnel formation related gene
Collecting nymphs injected with dsRNA for 24 h, dissecting the nymph epidermis, extracting RNA, reversely transcribing the nymphs into first strand cDNA, and detecting a target gene by Real-time PCR (Lmsnar) And housekeeping genes: (EF1-α) To calculate the silencing efficiency of the target gene. 4 biological replicates were set up, and 3 worms were taken per biological replicate. The results showed that the treatment group was treated after injection of dsRNA as compared with the control groupLmsnarThe expression level of (2) was very significantly reduced (FIG. 1).
3. Observation of the phenotype of 4-instar migratory locust after dsRNA injection
After the 4 th nymph is injected with dsRNA, the nymphs of the control group start to molt after 5 days and all successfully molt to the nymphs of 5 th instar after 6 days, and the development state of the nymphs after molting is good. Injection dssnarThe treated group of (1) had 60 nymphs, of which 2 were dead before molting (mortality rate 3.3%); chest before molting 4 nymphsThe back plate and the head are slightly cracked along the ridge line, the back is arched, the wing buds are opened, and the old epidermis is difficult to be sloughed off and continues to die (the fatality rate is 6.7%); the 11 nymphs died within a few days after molting (mortality rate 18.3%) (fig. 2).
Example 3: aconitum migratorium pore passage formation related gene dsRNA (double-stranded ribonucleic acid) influencing acreage epidermis permeability
After injection of dsRNA into 4-year-old nymphs, the molted nymphs were transferred to 2 ml centrifuge tubes containing 1.5 ml of dye solution (0.5% eosin (W/V, Sigma, red water-soluble dye, molecular mass 691.86 Da)), at 45 ℃ for 30 minutes and washed three times with water. The results show that ds is injectedGFPThe migratory locust of (1) can hardly be colored, whereas injection dsLmsnarThe locusta migratoria epidermis was clearly colored (fig. 3), indicating that the epidermal permeability was changed.
Example 4: the water retention of the locusta migratoria epidermidis influenced by dsRNA (double-stranded ribonucleic acid) of related genes formed by the locusta migratoria tunnel
After the dsRNA is injected into a 4-year-old nymph body cavity, nymphs which can successfully exuviate and survive for 24 hours are taken and are respectively and independently placed in 50ml centrifuge tubes, and a proper amount of dried wheat bran is placed at the bottom of the centrifuge tubes for feeding. The centrifuge tube is covered with a gauze. Then placing the mixture into a drying tower, and placing a proper amount of allochroic silica gel (Shanghai New fire silica gel factory) at the bottom of the drying tower to keep the interior of the drying tower relatively dry. Monitoring with a mijia bluetooth hygrothermograph indicated that the relative humidity dropped to 15% within 1 hour of the drying tower shutdown. The number of surviving individuals was recorded every 1 hour and the survival rate was calculated. The results show that ds is injectedLmsnarThe survival rate of locusta migratoria reaches 19% in 72 hours after drying treatment, while injection dsGFPThe survival rate of the migratory locust can reach more than 90 percent. Show thatLmsnarCan influence the drought resistance of locusta migratoria.
Sequence listing
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<120> locusta migratoria tunnel formation related gene Lmsnar and application of dsRNA thereof in locusta migratoria control
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Claims (3)
1. Migratory locust pore channel formation related geneLmsnarThe method is characterized in that: migratory locust pore canal formation related geneLmsnarThe nucleotide sequence of (a) is as shown in SEQ ID NO: 1 is shown.
2. The locusta migratoria pore channel formation-associated gene according to claim 1LmsnarA synthetic dsRNA characterized by: the specific synthesis method comprises the following steps: according to SEQ ID NO: 1-way pipeDesigning an upstream primer SEQ ID NO: 4 and the downstream primer SEQ ID NO: 5, obtaining the nucleotide sequence of SEQ ID NO: 3, which contains a T7 promoter, and the length of the nucleotide sequence is 550 bp; the product is synthesized into dsRNA, ds, by Promega T7 RiboMAX Express RNAi System kitsnar。
3. The locusta migratoria pore formation associated gene according to claim 2LmsnarThe application of the synthesized dsRNA in controlling locusta migratoria.
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| Identification and expression of cuticular protein genes based on Locusta migratoria transcriptome;Xiaoming Zhao et al.;《Scientific Reports》;20170403;第1-14页 * |
| 飞蝗表皮蛋白基因LmNCP1 的分子特性及功能分析;杨亚亭等;《中国农业科学》;20181231;第1303-1314页 * |
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