CN110184274B - E75 gene and application of dsRNA thereof in pest control - Google Patents
E75 gene and application of dsRNA thereof in pest control Download PDFInfo
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- CN110184274B CN110184274B CN201910464696.6A CN201910464696A CN110184274B CN 110184274 B CN110184274 B CN 110184274B CN 201910464696 A CN201910464696 A CN 201910464696A CN 110184274 B CN110184274 B CN 110184274B
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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Abstract
The invention provides an insect E75 gene cDNA full-length sequence and application of dsRNA thereof in pest control. Specifically, a bioinformatics method is used for obtaining a sequence of SEQ ID NO: 1, full-length cDNA of E75 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
Technical Field
The invention relates to the field of biotechnology and agricultural pest control, in particular to a migratory locust E75 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, and the use of a large amount of chemical insecticides not only easily causes the insects to generate drug resistance, but also brings serious pollution to the ecological environment and also forms a good effect on the health of human beingsThe research and development of green novel pesticides have important significance for the locust control 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 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.
E75 is one of key transcription factors in insect ecdysone signal transduction pathways, and by adopting an RNA interference technology, the subject group firstly proves that 100% of lethality occurs after injection of E75 gene dsRNA to 4-year-old nymphs. Therefore, the E75 gene can be used as a high lethal target gene for RNAi-based pest control technology.
Disclosure of Invention
The invention aims to provide a full-length cDNA sequence of an insect E75 gene and application of dsRNA thereof in killing locusta migratoria.
The invention provides a migratory locust E75 gene cDNA full-length sequence, 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 2238 bp, and the nucleotide sequence is SEQ ID NO: 1.
the invention provides application of migratory locust E75 gene dsRNA in pest control, which is characterized in that based on migratory locust E75 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 was injected into the body cavity of a 4-year migratory locust via a microinjector. The results show that: after dsRNA injection, mRNA expression of the migratory locust E75 gene is remarkably reduced, and the migratory locust has growth and development resistance and causes 100% death.
Drawings
FIG. 1: effect on E75 gene mRNA expression after injection of E75 gene dsRNA into 4-year migratory locusts. Beta-actin is an internal reference gene. WhereinP<0.05。
FIG. 2: influence of dsRNA injected with E75 gene on growth and development of 4-year migratory locust nymphs. dsRNA locusta migratoria injected with E75 gene has growth and development resistance and leads to 100% death. A: the left side is control injected with GFP and the right side is control injected with SEQ ID NO: 2 synthetic dsRNA, i.e. dsLmE75。
Detailed Description
The first embodiment is as follows: full-length cDNA sequence of migratory locust E75 gene and obtaining of gene fragment
Searching a migratory locust E75 gene cDNA sequence by adopting a bioinformatics method based on a transcriptome database of the migratory locust to obtain a migratory locust E75 gene (LmE75) The cDNA full-length sequence is designed by primer premier 5.0 software, and the upstream and downstream primers are used for verifying the cDNA full-length sequence and are sent to Shanghai Weijie Jie biology GmbH for synthesis. Selecting 4 heads of 4-year migratory locust nymphs which are healthy in growth and have the same size as each half of the migratory locust, quickly dissecting the body wall of the migratory locust under a body microscope, and freezing the migratory locust in liquid nitrogen. Extracting RNA according to TaKaRa RNAisso Plus kit, Reverse transcribing the RNA into first strand cDNA with Reverse transcription M-MLV (RNase H-) as instruction, PCR amplifying with the cDNA as template and designed upstream and downstream primersLmE75The full-length sequence of gene cDNA. 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: migratory locust E75 gene specific dsRNA synthesis
1) Design of migratory locust E75 gene dsRNA primer
Based on the full-length cDNA sequence of the migratory locust E75 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 Ente Weiziji Biometrics Ltd.
2) Synthesis of migratory locust E75 gene dsRNA
Using SEQ ID NO: 3 and SEQ ID NO: 4, PCR was performed using the cDNA template of example 1. 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: migratory locust E75 gene dsRNA lethal migratory locust test
1. Injection of migratory locust E75 gene dsRNA
Selecting 40 th day 1 nymphs with healthy growth, consistent size and half of males and females for total age of 4 to carry out experiment group dsLmE75And (4) injecting the gene. Will synthesize dsLmE75Using a microsyringe, 5 μ L (10 μ g ds)LmE75) Lightly inject into the area between the two and three abdominal segments of the nymph flank. The same amount of GFP dsRNA was injected simultaneously into the control group (40 heads, male and female halves) for phenotype observation. In addition, 9 th 4 th day nymphs were selected for the experimental group dsLmE75Injection of gene, along with the same amount of GFP dsRNA into control group (9 heads), was used for detection of gene silencing efficiency. And (3) placing the locusta migratoria after injection into an artificial climate box for feeding (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 E75 gene silencing detection
Collection of injected dsGFP and ds, respectivelyLmE75After 48 h, total RNA extraction, control and injection ds were performed on the nymph body wallLmE753 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 (LmE75) And housekeeping genes: (β-actin) And calculating the silencing efficiency of the gene. ResultsIndicating that ds was injected compared to the control groupLmE75Group test insectLmE75Gene expression was significantly reduced (figure 1).
3. Phenotypic observation of 4-year-old nymphs after injection of E75 gene dsRNA
4-year-old nymph injection dsLmE75Then, the insects in the control group successfully molt to 5 th day on day 6, and the development state of the insects after molting is good. Injection of ds compared to control groupLmE75The migratory locust dies on the 7 th day, the death rate reaches 100%, and two phenotypes appear, namely 67.5% of migratory locusts die before sloughing without crest line cracking, and 32.5% of migratory locusts do not crack at the crest line but can not successfully complete molting, and finally die, 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
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<400> 4
agctggagga ccagtctgcg ctgctggcgc gcgtcgtgcg cgcgcacctc gacacctgcg 60
agttcacgcg cgacaagatc gcgccgctgc tcgccaacgc gcgcgccagg cccacctaca 120
ccagctgtcc atccactctg gcgtgtccgc tgaaccctag cccgcagccg ttgacgggcc 180
agcaggagct gctacaagac ttctcgaagc gcttctcgcc tgccatccgc ggcgtcgtcg 240
agttcgccaa gcgcatcccc ggcttcgcgc tgctgccgca ggacgaccag gtgacgctgc 300
tcaaggcggg cgtgttcgag gtcctgctgg tgcggctcgc ctgcctcttc gactcggcca 360
ccaacagcat ggtgtgcctg gacgggcaga tgctgaagcg cgagtccatc cacagctcgt 420
ccaacgcgcg cttcctcatg gactccatgt tcgacttcgc 460
<210> 3
<211> 39
<212> DNA
<213> migratory locust (Locusa migratoria)
<400> 3
taatacgact cactataggg agctggagga ccagtctgc 39
<210> 4
<211> 40
<212> DNA
<213> migratory locust (Locusa migratoria)
<400> 4
taatacgact cactataggg gcgaagtcga acatggagtc 40
Claims (6)
1. A migratory locust E75 gene, which is characterized in that the nucleotide sequence is SEQ ID NO: 1.
2. A migratory locust E75 gene fragment is characterized in that the nucleotide sequence is SEQ ID NO: 2.
3. The migratory locust E75 gene fragment of SEQ ID NO: 2 synthetic dsRNA.
4. The use of dsRNA of migratory locust E75 gene according to claim 3 for controlling migratory locust.
5. The use of dsRNA of migratory locust E75 gene in the control of migratory locust as claimed in claim 3, wherein: the dsRNA is prepared into a spraying pesticide, or the dsRNA is prepared into a bait, or the dsRNA is transferred into a locusta migratoria to eat plant bodies.
6. The method for obtaining 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 by PCR amplification the 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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Non-Patent Citations (2)
| Title |
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| Nuclear receptor HR3 controls locust molt by regulating chitin synthesis and degradation genes of Locusta migratoria;Xiaoming Zhao et al.;《Insect Biochemistry and Molecular Biology》;20171104;第1-11页 * |
| 保幼激素调控昆虫生殖滞育的研究进展;高俏等;《环境昆虫学》;20181231;第988-944页 * |
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