CN114317549A

CN114317549A - Application of muscarinic C-type acetylcholine receptor in prevention and treatment of migratory locust

Info

Publication number: CN114317549A
Application number: CN202210018672.XA
Authority: CN
Inventors: 曾保娟; 周树堂; 郑洪远; 杨洁冰; 贠佳琦; 程冰静
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2022-01-08
Filing date: 2022-01-08
Publication date: 2022-04-12
Anticipated expiration: 2042-01-08
Also published as: CN114317549B

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a muscarinic C-type acetylcholine receptor (mAChR-C) in prevention and treatment of locusta migratoria. The method of in vitro injection and transgenic crop feeding shows that after the expression of mAChR-C gene of the migratory locust larvae is silenced, the phenomena of a large amount of death and eclosion and metaplasia are generated, the population growth and migratory flight harm of migratory locust are effectively inhibited, and the inhibition of mAChR-C gene expression can achieve the good effect of controlling migratory locust. Cultivation of Stable expressionmAChR‑CThe transgenic crop of double-stranded RNA provides a new method for field control of locusta migratoria.

Description

Application of muscarinic C-type acetylcholine receptor in prevention and treatment of migratory locust

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a muscarinic C-type acetylcholine receptor in prevention and treatment of migratory locust.

Background

Locusta migratoria (Locusa migratoria) is a migratory omnivorous pest, prefers to eat grain crops such as wheat, corn and the like, and is an important agricultural pest in China and even in the world. The locusta migratoria is easy to burst and cause disasters, and is closely related to the long-distance migratory flight capacity and high reproductive capacity of the locusta migratoria. At present, the prevention and control of pests are mainly based on a chemical pesticide prevention and control strategy, but the implementation and the use of pesticides not only easily cause the improvement of the drug resistance of the pests and the increase of the prevention and control cost, but also cause pesticide residues, pollute the ecological environment and harm the health of human beings. Therefore, the search for efficient, specific and sustainable green migratory locust control methods to replace chemical pesticides is urgent.

RNA interference (RNAi) is a phenomenon of sequence-specific post-transcriptional gene silencing initiated by small-molecule double-stranded RNA (dsRNA), has the characteristics of high efficiency, specificity and the like, and is gradually becoming a potential pest control technology at present. The technical key of RNA interference for pest control is to obtain dsRNA with high interference efficiency aiming at a target gene of a pest through screening, and the more important technical implementation difficulty is how to effectively transfer the dsRNA into the body of the pest in the field so as to achieve the application effects of gene silencing and pest control. In the past, RNAi mediated pest control is usually realized by methods such as injection, mixed feed feeding and the like to realize gene silencing, but the methods have poor application in field pest control. The first report of using transgenic plants to express pest gene dsRNA to control cotton bollworms appeared in 2007. An effective solution is provided for the field management of the pests by a mode of expressing the target gene dsRNA of the pests through transgenic plants.

G Protein Coupled Receptor (GPCR) is a seven-transmembrane protein superfamily, mediates extracellular signal conduction into cells, and plays an important role in regulation and control of the physiological process of insect organisms. Through previous work, the inventors identified a gene encoding a GPCR protein in migratory locusts. The alignment result of sequence analysis shows that the GPCR is homologous with the identified C-type muscarinic acetylcholine receptor (mAChR-C) protein of the drosophila muscarinic acetylcholine receptor. However, no specific role of mAChR-C in insect growth has been reported.

Disclosure of Invention

The invention discovers that after the expression of mAChR-C is interfered by an RNA technology, a large number of phenomena of death and eclosion of metaplasia migratoria larvae occur; in addition, the invention successfully cultivates a corn plant stably expressing double-stranded RNA of mAChR-C by adopting a transgenic crop method, and feeding experiments show that after migratory locust larvae eat transgenic plants, the expression quantity of target gene mAChR-C is reduced by 79 percent, so that the migratory locust larvae die and have eclosion and metaplasia, the lethality and the metaplasia rate are up to 73.9 percent in total, and the population quantity increase and migratory flight diffusion of migratory locust can be effectively inhibited. The invention provides a new method for controlling locusta migratoria, and has a great application prospect.

The invention adopts the following technical scheme:

according to the gene sequence of the migratory locust mAChR-C, synthetic double-stranded RNA (dsmAChR-C) is designed, the dsmAChR-C is injected into five-instar larvae within 12 hours of the migratory locust molting, and meanwhile, the five-instar larvae within 12 hours of the migratory locust molting, which are injected with green fluorescent protein double-stranded RNA (dsGFP), are used as a control group. Wherein, the sequence of the migratory locust mAChR-C gene is shown as SEQ ID NO. 1, the amino acid sequence coded by the migratory locust mAChR-C gene is shown as SEQ ID NO. 2, and the DNA template sequences for synthesizing dsmAChR-C and dsGFP are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4.

The results show that when the mAChR-C interference efficiency is 82%, the death rate of migratory locust larvae is 53.1%, the wing aberration rate after eclosion is 18.6%, the total mortality rate and wing aberration rate is 71.7%, the survival rate of normal eclosion is 28.3%, and the normal survival rate is obviously lower than that of the dsGFP control group (100%).

In order to analyze the function of mAChR-C in the growth and development process of migratory locust larvae and the molecular mechanism of larval death and eclosion of the metaprotea after silencing mAChR-C expression, qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is carried out to detect the gene expression conditions related to chitin synthesis and degradation in an mAChR-C interference group and a GFP control group after interfering the mAChR-C gene expression. The results show that mAChR-C interferes with the significant decrease of mRNA levels of chitin degradation related genes chitin 5-1(CHT5-1), chitin 5-2(CHT5-2), chitin 10(CHT10), and chitin synthesis related genes chitin synthesis 1(CHS1), UDP-N-acetylglucosamine phosphorylase 2(UAP 2). The results show that mAChR-C participates in regulating and controlling the expression of chitin metabolism related genes, and further influences the metabolism of epidermal chitin in the growth and development process of larvae. Five-instar larval cuticles on day 5 after dsRNA treatment in mAChR-C interference group and GFP control group were further selected and stained for tissue section to observe cuticle morphology. Chitin staining results show that the old epidermal thickness of the larvae of the dsmAChR-C treated group is remarkably increased and the new epidermal thickness is remarkably reduced compared with the control group, and show that the old epidermal shedding and the new epidermal formation are inhibited after the mAChR-C expression is silenced. In conclusion, after the RNAi technology inhibits the expression of mAChR-C, the normal growth of the epidermis is affected, and the normal growth of the larval locust is hindered, so that death and eclosion of the wings are caused.

In order to verify the application prospect of RNAi technology-mediated mAChR-C expression silencing in crop pest control. A template which is shown in SEQ ID NO. 3 and used for synthesizing dsmAChR-C is integrated into a maize immature embryo genome through an agrobacterium transformation method to obtain the transgenic maize stably expressing mAChR-C double-stranded RNA. Feeding five-instar locust larvae on leaves of transgenic corn, wherein feeding experiment results show that the survival rate of individuals eating wild corn leaves is 97.2%, the instar period is 6.2 days, and all wings of eclosion adult corns normally extend; and compared with a wild corn control group, the expression level of the individual mAChR-C eating the dsmAChR-C transgenic corn leaf is reduced by 79.0%, the mortality rate is 53.2%, the instar period of the non-dead larva is remarkably prolonged to 7.2 days, the wing aberration rate of the eclosion adult is 20.7%, the total mortality rate and the wing aberration rate are 73.9%, the survival rate of the normal molting eclosion is 26.1%, and the survival rate is remarkably lower than that of the dsGFP control group (97.2%).

The invention has the beneficial effects that:

the methods of in vitro injection, transgenic crop feeding and the like prove that after the expression of mAChR-C genes of migratory locust larvae is silenced, the phenomena of massive death and eclosion of the larvae occur, the population growth and migratory flight harm of migratory locusts are effectively inhibited, and the inhibition of mAChR-C expression can achieve the good effect of controlling migratory locusts. The cultivation of transgenic crops stably expressing mAChR-C double-stranded RNA provides a new method for field control of locusta migratoria.

Drawings

FIG. 1 shows the gene interference efficiency after double-stranded RNA injection in epidermal tissue. Denotes P < 0.001.

FIG. 2 is a graph of the effect of interfering mAChR-C expression on larval survival and post-emergence wing type. (A) Interfering the survival rate of the migratory locust larvae after the mAChR-C expression. (B) Phenotypic statistics of larvae after interference with mAChR-C expression. (C) Statistics of five instar stages of larvae after interference of mAChR-C expression. (. indicates P < 0.05;. indicates P < 0.01).

FIG. 3 is a graph of the effect of dsmAChR-C injection on chitin metabolism genes and epidermal chitin. (A) Expression levels of genes involved in chitin degradation and synthesis following dsmAChR-C injection. (B) Larvae body walls stained after dsmAChR-C injection. Labeling the nucleus with Propidium Iodide (PI); chitin was stained with Fluorescent Brightener 28 (FB 28). The length of the scale is 50 μm. During normal molting, the old epidermis separates from the epithelial cell layer, sloughs off and is replaced by new epidermis. (C) Index of epidermal thickness after dsmAChR-C injection. Denotes P < 0.01; denotes P < 0.001.

FIG. 4 is a PCR identification of positive transgenic maize plants. Note: and (5) Maker: DL 1500; mAChR-C represents a synthetic template that has been integrated into the maize genome capable of stably expressing dsmAChR-C.

FIG. 5 is a graph of the effect of feeding transgenic maize on larval survival and post-emergence wing shape. (A) Interference efficiency of mAChR-C gene in larval epidermis after feeding transgenic corn. (B) Survival rate of larvae after feeding transgenic corn. (C) And (5) performing phenotype statistics on migratory locusts fed with transgenic corn. (D) And (5) counting the five-instar period of the larvae after the transgenic corn is fed. (represents P < 0.01; represents P < 0.001).

Detailed Description

The present invention will be described in more detail with reference to the following embodiments for understanding the technical solutions of the present invention, but the present invention is not limited to the scope of the present invention.

1. Obtaining of full-length coding sequence of locust-podded poisonous mushroom type C-type acetylcholine receptor gene

Based on a migratory locust transcriptome database (http://159.226.67.243/), a bioinformatics method is adopted to search the sequence of a migratory locust mushroom type C acetylcholine receptor (mAChR-C) gene to obtain a partial sequence of the mAChR-C gene. RACE primers required by the cDNA terminal rapid cloning technology are designed aiming at the partial sequence by using primer premier6.0 software and synthesized by Beijing Liuhe Huada Gene science and technology Limited.

The primer sequences for amplifying the 5 'end and the 3' end of the mAChR-C gene by the cDNA terminal rapid cloning technology are as follows: mAChR-C-5' RACE (SEQ ID NO: 5): 5'-ACCCAAGACCAAAAGTACGACCTGGA-3', respectively; mAChR-C-3' RACE (SEQ ID NO: 6): 5'-TGATAGTCTCAGTCGGCTGGATCCTTTC-3' are provided.

Selecting healthy migratory locust adults (population of the social migratory locust east Asia), and extracting epidermal total RNA by using a Trizol method. The total RNA mentioned above was reverse transcribed to synthesize the first cDNA according to the instructions of the FastKing cDNA first strand synthesis kit of Tiangen. Using this cDNA as a template, the upstream and downstream primers shown by the sequences SEQ ID NO 5 and SEQ ID NO 6 were combined, and the cDNA ends were cloned by the rapid cloning technique (according to Clontech Co., Ltd.)

RACE 5 '/3' kit instruction step) to obtain mAChR-C gene 5 'end and 3' end sequences, and sending the fragments to Beijing Liuhe Hua Dagenescience and technology Limited company for sequencing (all the subsequent primer synthesis and sequencing are completed in Beijing Liuhe HuaDagenescience and technology Limited company). Splicing and comparing a migratory locust transcriptome database sequence with a PCR amplified sequence by using BioEdit software to obtain an mAChR-C full-length Open Reading Frame (ORF), wherein the full length of the ORF sequence is 1005bp and is shown as SEQ ID NO. 1; the ORF sequence encodes 334 amino acids, as shown in SEQ ID NO. 2.

2. Migratory locust mAChR-C double-stranded RNA acquisition

Based on the obtained ORF sequence of mAChR-C shown in SEQ ID NO. 1, primer premier6.0 software is adopted to design primers of a DNA template for synthesizing dsRNA, and the sequences are SEQ ID NO. 7 and SEQ ID NO. 8 respectively. Then sent to Beijing Liuhe Hua Dagenescience and technology Limited company for synthesis.

Primers for amplification of mAChR-C dsRNA template:

SEQ ID NO:7：5’-GTGCTGCCACAAGCCTATATC-3’；

SEQ ID NO:8：5’-ACCAACAGATGGAGAATGAACC-3’。

using cDNA synthesized by reverse transcription in the step 1 as a template, and performing PCR amplification by using upstream and downstream primers shown as SEQ ID NO. 7 and SEQ ID NO. 8 under the PCR reaction conditions of (1) pre-denaturation at 95 ℃ for 3 min; (2) at 95 ℃ for 30 s; at 58 ℃ for 30 s; 35 cycles of 72 ℃ for 40 s; (3) extension was 72 ℃ for 5 min. And (3) connecting the single product obtained by PCR amplification into a pGEM-T (Tiangen) vector to obtain a recombinant plasmid pGEM-T-mAChR-C. The sequencing result shows that the size of the PCR amplification product is 202bp, and the nucleotide sequence is shown as SEQ ID NO. 3.

The recombinant plasmid pGEM-T-mAChR-C obtained above is taken as a template, a paired primer dsmAChR-C-T7-F/dsmAChR-C-T7-R with a T7 promoter sequence is used for carrying out PCR amplification to obtain a DNA template for synthesizing double-stranded RNA (namely a sequence shown as SEQ ID NO:3 with a T7 promoter at two ends, and underlined is marked as a T7 promoter sequence), and the PCR reaction condition is that(1) Pre-denaturation at 95 deg.C for 3 min; (2) at 95 ℃ for 30 s; at 58 ℃ for 30 s; 35 cycles of 72 ℃ for 40 s; (3) extension was 72 ℃ for 5 min. Use of

The SV Gel and PCR Clean-Up System (Promega) kit purifies the target fragment. And measuring the concentration of the NanoDrop 2000, and determining that the concentration of the NanoDrop 2000 is 125-1000 ng/mu l.

dsmAChR-C-T7-F：

5’-TAATACGACTCACTATAGGGTGCTGCCACAAGCCTATATC-3’；

dsmAChR-C-T7-R：

5’-TAATACGACTCACTATAGGACCAACAGATGGAGAATGAACC-3’。

Using the DNA with T7 promoter sequence at both ends as template, and performing DNA sequencing according to T7RiboMAX^TMExpress RNAi System (Promega) kit for the in vitro transcription Synthesis of double-stranded RNA. The concentration was measured using a NanoDrop 2000, placed at-20 ℃ until use.

3. Migratory locust mAChR-C double-stranded RNA interference migratory locust larva

3.1 double-stranded RNA injection of migratory locust

Five-instar larvae within 12 hours after molting are selected as experimental materials, and are randomly divided into a dsmAChR-C treatment group and a dsGFP control group (because green fluorescent protein GFP genes do not exist in locusta migratoria, the green fluorescent protein GFP genes can be used as negative control), and each group is repeated for 3 times at 28-30 times. Mu.g of dsmAChR-C was injected into the body from the third abdominal segment of the larvae using a 10. mu.L format microsyringe, while the larvae injected with the same dose of dsGFP were used as a control. Feeding locusta migratoria after injection in a well ventilated metal cage (25cm × 25cm × 25cm), feeding fresh wheat seedling and wheat bran twice a day at 30 + -2 deg.C and with a photoperiod of 14L: 10D. Mortality of larvae was observed and counted daily after injection, and wing teratogenesis and five-year developmental history were counted after eclosion.

3.2 locusta migratoria mAChR-C double-stranded RNA interference efficiency detection

48 hours after dsRNA injection, larvae were randomly picked at 8 heads in the dsmAChR-C treated group and dsGFP control group, respectively, to test the mAChR-C double-stranded RNA interference efficiency. Extracting driedCarrying out reverse transcription on the total RNA of epidermal tissues of the larvae of migratory locust in a perturbation group and a control group to synthesize first-strand cDNA, carrying out qRT-PCR (quantitative reverse transcription-polymerase chain reaction) on a LightCycler 96(Roche) by using qRT-PCR upstream and downstream primers of mAChR-C and qRT-PCR upstream and downstream primers of beta-actin according to the specification method of a Tiangen SuperReal fluorescent quantitative premix reagent enhanced version (SYBR Green) kit (PF205), and respectively detecting the expression quantities of a target gene (mAChR-C) and an internal reference gene (beta-actin), wherein the PCR reaction condition is (1) pre-denaturation at 95 ℃ for 10 min; (2)95 ℃ for 10 s; at 58 ℃ for 30 s; 40 cycles of 72 ℃ for 30 s; (3) the dissolution curve analysis is carried out for 60s at 95 ℃; 30s at 65 ℃; 95 ℃ for 30 s. Then according to 2^-ΔΔCtThe method calculates the relative expression quantity of mAChR-C in the larval epidermis of the treatment group and the control group, and analyzes the gene silencing efficiency. The results are shown in FIG. 1, and the mAChR-C expression of the dsmAChR-C treated group is reduced by 82% compared with the dsGFP control group, indicating that mAChR-C gene expression is effectively silenced.

qRT-PCR primers:

mAChR-C-qRT-F：5’-CAGAGAAAGTGATATGGGCAACAA-3’；

mAChR-C-qRT-R：5’-GAACTGGCAGGAAGTATAACAAGT-3’。

β-actin-qRT-F：5’-AATTACCATTGGTAACGAGCGATT-3’；

β-actin-qRT-R：5’-TGCTTCCATACCCAGGAATGA-3’。

3.3 interference with mAChR-C expression leads to larval mortality and eclosion of the hind wings

The phenotype of the migratory locust larvae is observed every day immediately after double-stranded RNA injection until all experimental larvae eclosion is completed. The results show that: the mortality rate for the dsmAChR-C interfering group was 53.1%, the post-eclosion wing aberration rate was 18.6%, the rate of normal molting was 28.3%, significantly lower than the survival rate (100.0%) for the normal molting of the dsGFP control group, as shown in fig. 2A and 2B. In addition, the five instar developmental history of the larvae in the dsmAChR-C treated group was significantly longer than the control group, as shown in fig. 2C.

Mechanism of action of mAChR-C in grasshopper emergence

In order to analyze the role of mAChR-C in the growth and development process of migratory locust larvae and the molecular mechanism of lethality and wing deformity after mAChR-C expression is silenced, after interfering mAChR-C expression is implemented, the expression condition of genes related to chitin synthesis and degradation is detected through qRT-PCR, and the change of the epidermal morphology of an interference group (dsmAChR-C) and a control group (dsGFP) is further observed through tissue section staining.

4.1 expression of genes involved in chitin metabolism

The expression conditions of 5 genes related to chitin metabolism after mAChR-C gene silencing are detected by qRT-PCR by using interference group (dsmAChR-C) and control group (dsGFP) samples obtained in 3.1 after dsRNA injection for 48 hours.

qRT-PCR primers:

gene	Forward primer (5 '-3')	Reverse primer (5 '-3')
			CHS1	CTTGAGCCAATTGGTTTGGT	TGAGTTCTGTGGATGCAAGG
UAP2	GTACCTAAATGCTCATGGTGTGGAT	GTCCACCTGGCAAACAACTCCT
			CHT5-1	CATCAAAGCGAAGGGCTACGGC	AGATTAGTGCGTCCTTCGGGCCA
CHT5-2	CAAGGATTATGTGGAGAACC	TCCACAGTGTTTGTTTTCTTTGATT
			CHT10	GCAATTGGTGGTTGGAATGAT	GGTCTAGTCCTTCAAATCCATACTTTTC
β-actin	AATTACCATTGGTAACGAGCGATT	TGCTTCCATACCCAGGATGA

As shown in FIG. 3, the transcript levels of Chitin synthase 1(Chitin synthase 1, CHS1), UDP-N-acetylglucosamine pyrophosphorylase 2(UDP-N-acetylglucosamine phosphorilase, UAP2), chitinase5-1 (Chitin 5-1, CHT5-1), chitinase5-2 (Chitin 5-2, CHT5-2) and chitinase 10(Chitin 10, CHT10) were significantly lower than those of the control group. The transcript level of genes CHS1 and UAP2 related to chitin synthesis is respectively reduced by 76.0 percent and 82.0 percent, and the transcript level of genes CHT5-1, CHT5-2 and CHT10 related to chitin degradation is respectively reduced by 58.0 percent, 93.6 percent and 86.1 percent. The result shows that mAChR-C participates in regulating and controlling the expression of chitin metabolism related genes, and further influences the metabolism of epidermal chitin in the growth and development process of larvae.

4.2 histological Observation of epidermal changes

In order to further detect the effect of mAChR-C on epidermal chitin, the present example utilizes the tissue section staining method to perform microscopic observation on the epidermis of migratory locust sections 3-4 of the interfering group and the control group at day 5 after injection of dsRNA, Propidium Iodide (PI) stains the cell nucleus (red), and Fluorescent Brightener 28(Fluorescent Brightener 28, FB 28) stains chitin (blue). The results are shown in fig. 3B and 3C, where the old cuticle thickness was significantly higher in the cuticle layer of the larvae of the interfering group than in the larvae of the control group, while the new cuticle thickness was significantly lower in the larvae of the interfering group than in the control group. Through measurement and statistics, the average thickness index of the new epidermis of the interference group is reduced by 95.0 percent compared with that of the control group, while the average thickness index of the old epidermis of the interference group is obviously increased by 2.5 times compared with that of the control group, and the old epidermis decomposition and new epidermis synthesis abnormality is presumed to be the reasons for locust migratory death, metaplasia and developmental retardation.

Application of mAChR-C double-stranded RNA transgenic corn in locust control

5.1 construction of transformation vectors

Primers containing BamHI and SpeI sites were designed, and pGEM-T-mAChR-C plasmid was used as template for PCR amplification to recover and purify the PCR product (i.e., SEQ ID NO:3 containing BamHI and SpeI upstream and downstream, respectively). A gene fragment of dsmAChR-C synthetic template (SEQ ID NO:3) was ligated to the pWMB006 vector at both BamHI and SpeI multiple cloning sites in forward and reverse directions, respectively, by a double digestion and infusion reaction. After the connection is successful, transforming escherichia coli, screening positive clones, and obtaining the positive clone with correct sequencing, namely the pWMB006-mAChR-C recombinant plasmid.

5.2 Agrobacterium mediated method for constructing transgenic maize stably expressing mAChR-C double-stranded RNA

And (3) carrying out corn transgenic operation by using an agrobacterium transformation method. The experimental flow of corn genetic transformation is as follows:

(1) the pWMB006-mAChR-C recombinant plasmid is transferred into agrobacterium EHA105 by an electric shock method and identified by PCR.

(2) Using a corn variety KN5855 and taking freshly stripped corn embryos of about 1mm as a material, putting the stripped corn embryos into a 2mL plastic centrifuge tube containing 1.8mL of agrobacterium suspension, and treating about 150 immature embryos in 30 minutes; the Agrobacterium suspension was aspirated, the corn embryos left in the tube, and then 1.0mL of Agrobacterium suspension was added and allowed to stand for 5 minutes.

(3) The young embryos in the centrifuge tube are suspended and poured onto a co-culture medium, and the surplus agrobacterium liquid on the surface is sucked by a liquid transfer device and is cultured for 3 days in the dark at the temperature of 23 ℃.

(4) After co-cultivation, the young embryos were transferred to a resting medium, cultured in the dark at 28 ℃ for 6 days, placed on a screening medium containing bialaphos, and screened for two weeks, after which the screening medium was replaced with a new one for 2 weeks.

(5) Transferring the resistant callus to a differentiation culture medium, and culturing for 3 weeks at 25 ℃ and 5000lx under illumination; transferring the differentiated plantlets to a rooting culture medium, and culturing at 25 ℃ and 5000lx by illumination until the plantlets are rooted; and (3) transferring the plantlets into a small pot for growth, transplanting the plantlets into a greenhouse after a certain growth stage, and harvesting progeny seeds after 3-4 months. The seed is the transgenic corn seed for stably expressing the mAChR-C double-stranded RNA.

(6) Extracting T0 generation corn leaf genome DNA, obtaining positive plants after PCR detection, and taking the positive plants to carry out the next feeding experiment as shown in figure 4.

5.3 detection of Effect of stably expressing mAChR-C double-stranded RNA on controlling locusta migratoria by transgenic maize

Feeding transgenic corn leaves stably expressing mAChR-C double-stranded RNA, and observing and analyzing the influence on the growth and development of fifth-instar locusta migratoria.

(1) And selecting five-instar larvae within 12 hours after molting, repeating for 3 times, wherein 17-20 larvae in each group are obtained. Leaves were cut from plants identified as positive, and larvae were fed after careful cleaning, while larvae fed with wild type maize leaves were used as a control.

(2) Feeding proper amount of corn leaves according to the number of the larvae, and keeping the corn leaves sufficient.

(3) After feeding for 48 hours, randomly picking 5 larvae in a treatment group (feeding transgenic corns) and a control group (feeding wild corns), respectively, collecting epidermal tissues, and detecting the expression quantity of mAChR-C (namely detecting gene interference efficiency) in the epidermal tissues of locusta migratoria after feeding the transgenic corns by qRT-PCR. The detection method is the same as 3.2.

(4) From the day of feeding, phenotypes of larval death, developmental catastrophe, and post-eclosion malformation were recorded for the transgenic corn feeding group and the wild-type corn feeding group by daily observation.

The results show that: the expression level of mAChR-C of the larvae eating the transgenic corn leaves is reduced by 79 percent, as shown in figure 5A; individuals eating wild type corn leaves have a survival rate of 97.2%; the mortality rate of the transgenic corn larvae fed with the transgenic corn is 53.2 percent, the wing aberration rate after eclosion is 20.7 percent, the total mortality rate and the wing aberration rate is 73.9 percent, and the survival rate of normal molting eclosion is 26.1 percent, as shown in figures 5B and 5C; the larvae instar of transgenic corn leaf feeding was significantly longer than that of wild corn control group, and the larvae instar of transgenic corn feeding was 7.2 days, significantly longer than that of wild corn feeding (6.2 days), as shown in fig. 5D.

The above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications in the structure, features and principles described in the present invention should be included in the claims of the present invention.

SEQUENCE LISTING

<110> university of Henan

Application of <120> C-type acetylcholine receptor in prevention and treatment of migratory locust

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Ser Ser Ser Ile Tyr Asn Leu Ile Ala Ile Ala Thr Asp Arg Tyr Ile

115 120 125

Ser Ile Val Tyr Pro Leu His Tyr Ser Thr Tyr Met Thr Ala Arg Lys

130 135 140

Ala Leu Leu Ile Val Ser Val Gly Trp Ile Leu Ser Ala Ile Val Ser

145 150 155 160

Thr Met Pro Ser Tyr Trp Asn Lys Tyr Ser Ala Asp Lys Ala Cys Leu

165 170 175

Val Asp Asn Val Leu Pro Gln Ala Tyr Ile Ile Gly Ile Ile Thr Pro

180 185 190

Ala Phe Val Leu Val Leu Ile Ala Met Leu Val Leu Tyr Trp Arg Ile

195 200 205

Trp Lys Glu Ala Ser Glu Gln Ala Arg Arg Leu Arg Asn Val Thr Asn

210 215 220

Ser His Ser Asp Trp Lys Ser Val Gln Val Val Leu Leu Val Leu Gly

225 230 235 240

Ser Phe Ser Ile Cys Trp Leu Pro Tyr Phe Thr Val Ile Cys Cys Arg

245 250 255

Val Ala Gly Ala Lys Ser Tyr Thr Ser Phe Met Val Tyr Lys Ala Ala

260 265 270

Phe Ser Met Gly Met Ala Asn Ser Cys Ile Asn Pro Leu Ile Tyr Ala

275 280 285

Trp Lys Asn Thr Glu Phe Lys Asn Ala Phe Arg Asn Ile Leu His Cys

290 295 300

Lys Ser Pro Asn Arg Ala Asp His Leu Leu Glu Thr Ile Thr Thr His

305 310 315 320

Thr Glu Leu His Thr Ile Asp Ser Ser Cys Gly Gly Phe Asn

325 330

<210> 3

<211> 202

<212> DNA

<213> Artificial sequence

<400> 3

gtgctgccac aagcctatat cataggtatc atcacaccag catttgttct ggtgctgata 60

gccatgttgg tactttactg gcgaatatgg aaggaagctt cagaacaagc cagaagactt 120

cgaaatgtta caaactctca ctcagattgg aaatctgtcc aggtcgtact tttggtcttg 180

ggttcattct ccatctgttg gt 202

<210> 4

<211> 260

<212> DNA

<213> Artificial sequence

<400> 4

cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa cttgcagacc attatcaaca 60

aaatactcca attggcgatg gccctgtcct tttaccagat aaccattacc tgtccacaca 120

atctaccctt tccaaagatc ccaacgaaaa gagagatcac atgatctatt ttgagtttgt 180

aacagctgct gcgattacac atggcatgga tgaattatac aaataaatgt atagacttca 240

agttgacact aacgtgtccg 260

<210> 5

<211> 26

<212> DNA

<213> Artificial sequence

<400> 5

acccaagacc aaaagtacga cctgga 26

<210> 6

<211> 28

<212> DNA

<213> Artificial sequence

<400> 6

tgatagtctc agtcggctgg atcctttc 28

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<400> 7

gtgctgccac aagcctatat c 21

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<400> 8

accaacagat ggagaatgaa cc 22

Claims

1. The application of muscarinic C-type acetylcholine receptor gene in controlling migratory locust is characterized by silencing muscarinic C-type acetylcholine receptor gene in migratory locust larva to realize migratory locust controlmAChR-CThe sequence of (A) is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the C-acetylcholine receptor gene of the muscarinic type in vivo is silenced by RNA interference technology in young locusta migratoria.

3. The application of double-stranded RNA synthesized based on muscarinic C-type acetylcholine receptor gene in controlling migratory locust is characterized in that the sequence of a DNA template for synthesizing the double-stranded RNA is shown as SEQ ID NO. 3.

4. The use according to claim 3, wherein the locusta migratoria larvae are injected with double-stranded RNA in vitro to achieve locusta migratoria control.

5. The use according to claim 3, wherein the locusta migratoria larvae eats a transgenic crop to realize locusta migratoria control, wherein the transgenic crop is a crop with a foreign sequence shown in SEQ ID NO. 3.

6. Use according to claim 5, characterized in that the crop is maize.

7. The application of double-stranded RNA synthesized based on muscarinic C-type acetylcholine receptor genes in inhibiting synthesis of new and old epidermis of migratory locust and chitin metabolism is characterized in that a sequence of a DNA template for synthesizing the double-stranded RNA is shown as SEQ ID NO. 3.

8. Use of double-stranded RNA synthesized based on muscarinic C-type acetylcholine receptor gene for inhibiting expression of chitin metabolism-related geneCHS1、UAP2、CHT5-1、CHT5-2、CHT5-10。