CN115678903B - Sogatella furcifera Ago1 gene, method for synthesizing dsRNA and application thereof - Google Patents
Sogatella furcifera Ago1 gene, method for synthesizing dsRNA and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a sogatella furcifera Ago1 gene, a method for synthesizing dsRNA and application thereof in RNAi-mediated pest control, wherein the sogatella furcifera Ago1 gene is prepared by adopting an RNA interference technology, and a higher lethal phenomenon occurs after Bai Beifei sogatella furcifera Ago1 gene dsRNA is injected to 4-year-old nymphs, so that the sogatella furcifera Ago1 gene can be used as a novel control target in pest control.
Description
Technical Field
The invention relates to the field of microbial technology, in particular to a sogatella furcifera Ago1 gene, a method for synthesizing dsRNA and application thereof in RNAi-mediated pest control.
Background
Bai Beifei lice Sogatalla furcifera (Horvth) belongs to the family Hemiptera, the plant hopper of the order Hemiptera, delphacidae, is one of the serious pests in rice production, and mainly uses nymphs and adults to pierce the juice of rice stems, so that nutrients are lost; and simultaneously, the viruses such as southern rice black-streaked dwarf virus (Southern rice black-streaked dwarf virus, SRBSDV) and the like are transmitted. As a migratory pest, the sogatella furcifera is easy to explode under the condition of suitable environment, and serious economic loss is brought to rice production. The control of Bai Beifei lice still mainly depends on chemical pesticides at present, however, the long-term unreasonable and excessive use of chemical pesticides leads to the generation of drug resistance of the sogatella furcifera, and natural enemies are killed and the environment is polluted. Therefore, research and development of the novel environment-friendly pesticide have great significance for the prevention and control of the sogatella furcifera.
RNA interference (RNAi) is a phenomenon in which small molecule dsRNA causes specific degradation of mRNA sequences of a target gene, resulting in silencing or down-regulation of the expression level of the target gene. RNAi has become an important tool for studying insect gene function, and the technology has become a new pest control means in the field of plant protection: the gene segment in the insect body is utilized, and the aim of preventing and controlling pests is achieved by inhibiting the transcription level of genes which are vital to the growth and development of the pests through RNAi. Therefore, the application of RNAi technology to develop novel pesticides has important significance for pest control.
Proteins in the insect RNAi pathway are mainly Drosha protein, dicer protein, argonaute protein, and dsRNA binding domain protein (double-stranded RNA binding domain protein), wherein Argonaute protein serves as a core element of RNA-induced silencing complex (RNA-induced silencingcomplex, RISC). Two members of the Ago protein subclass are Ago1 and Ago2, respectively, wherein Ago comprises a DUF1785 domain, a PAZ domain, and a PIWI domain. Ago1, an important component of the miRNA pathway in insects, can specifically recruit mirnas to participate in the silencing of mRNA, thereby regulating gene expression.
Functional researches of interfering Ago1 gene in aspects of growth and development, reproduction, resistance, immunity and the like of insects prove that the gene is closely related to various growth and development processes of insects. However, no related report is found in the current research on the sogatella furcifera Ago1 gene, and no report is found on RNAi-mediated pest control of the sogatella furcifera Ago1 gene.
Disclosure of Invention
In order to solve the problems, the primary object of the invention is to provide a sogatella furcifera Ago1 gene, a method for synthesizing dsRNA and an application thereof in RNAi mediated pest control, wherein the sogatella furcifera Ago1 gene is prepared by adopting an RNA interference technology, and a higher lethal phenomenon occurs after Bai Beifei th of the dsRNA of the sogatella furcifera Ago1 gene to 4 th of nymphs is injected, so that the sogatella furcifera 1 gene can be used as a novel control target in pest control.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a sogatella furcifera Ago1 gene comprising SEQ ID NO:1, the nucleotide sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3, and a target gene sequence shown in 3.
The SEQ ID NO:1 is:
atggcagttc cttgttctga tatgcagctt ataaggaggg aaccacaaac agtgtccatg cttggaaaaa cctatggatg tggacagtgt tcagctcctc cgcctggagc gttgggagca gctccggggc cagtggctcc agctggtggg ccagtagccg gcatgcctcc gggagcgatg ggcctacttc cgccacagca gccccaccag caacctccgc agcctcccga tcttcccatg ttcaactgcc ccagaaggcc gaaccttggc agagaaggtc gaccaatcgt tttgcgtgcc aatcattttc aaatcacgat gccccgcggg ttcgttcatc attatgacat aaacattcag ccggacaagt gtcctcgcaa agtcaaccgt gagatcattg aaactatggt gacagcttac agtaaaatat ttggaaactt gaagccagtc ttcgatggcc gcagcaactt gtacacccga gatcccctgc caattggcaa tgatcgcatg gagcttgagg tgacattgcc gggcgaaggc aaggatcgtg tgttcagggt ggcgatcaag tggctggcgc aggtgtcgct gttcgcgctg gaggaggcgc tggaaggccg tacccgacag atcccctacg acgccatcct ggcactcgac gtcgtcatga ggcacttgcc ctcgatgacc tacacccctg tcggccgctc cttcttctcc tcacccgacg gctactatca tccgctcggc ggcggcagag aggtgtggtt cgggttccat caatcagtga ggccgtcaca gtggaagatg atgttgaaca ttgacgtatc tgcgacggcg ttttacaaag cgcagcctgt gatagagttc atgtgcgaag tgctcgacat tcgcgacata aacgaccagc gcaagccatt gactgattcc cagcgcgtca aatttaccaa agaaatcaaa ggcttgaaaa ttgagattac tcactgtgga actatgagac gcaagtaccg tgtttgcaac gtcacgcgcc ggcctgccca gatgcagtcg tttcctctgc agctggaaaa tggacaaacc gtcgagtgta cagtggccaa gtacttcttg gacaagtaca aaatgaaact gagatacccc caccttcctt gtcttcaggt cggacaagag cataagcata cttatcttcc cctagaggtg tgcaacatag tggccgggca gcggtgcatc aagaagctga ccgacatgca gacgtcgacg atgatcaagg cgacagcgcg ttcggcgccg gaccgcgagc gtgagatcaa caacctggtg cggcgcgccg acttcaacaa cgatgcctac gtgcaggagt tcggcctcac catctccaac aatatgatgg aggtgcgcgg tcgcgtcctc cccccgccca agctacagta cggcggacgc gtcagctctc tctccggaca gcaggaattt cagggctgca atgtgctgca gacgaaacag caagcgatgc ccaatcaggg tgtgtgggac atgcgtggca agcagttctt cactggcgtc gagattcgag tctgggcgat cgcctgcttt gctccacaac gcactgtgcg tgaagatgcg ctcagaaact tcactcaaca attgcagaaa atcagtaacg atgctggcat gccaataatc ggccaacctt gtttttgcaa gtacgctact ggccctgacc aagtggagcc gatgttccgg tacctgaaaa attctttcca ggctctacaa ttggtcgtag ttgttcttcc aggaaaaact ccagtatatg ctgaagtgaa gcgagtgggc gatacagtgc tgggaatggc gacccagtgt gtgcaggcca aaaatgtcaa caagacttcc ccacagaccc tttccaatct ttgtctaaag atcaatgtca agcttggtgg aatcaacagc atacttgttc ctagcattag acccaaggtg ttcaatgagc cggtgatatt cctgggtgcc gacgtgacgc acccgccggc cggcgacaac aagaagccgt cgattgcggc agtggtcggg tcgatggacg cgcatcctag ccggtatgca gccactgtgc gcgtgcaaca gcatcgccag gagatcattc aggagctgag ctccatggtc agagaacttc ttatcatgtt ctacaaaagc actggaggct acaaaccaca tcgtattatt ctatacagag atggtgtatc cgagggacag ttccttcacg ttctacagca tgagctgact gctatcagag aagcgtgtat caaacttgaa ggagactata agcctggaat cacattcatt gttgttcaga agcgacatca cacaaggctg ttctgtgcag acaagaagga acagtctgga aaatcaggca acattccagc aggcacaacg gttgacgtag gcatcacaca tccaactgaa tttgatttct acctttgcag tcatcaaggc attcaaggta cgagcagacc gagtcactac cacgtgctgt gggacgacaa ccactttgac tcagacgagc tgcagtgcct gacctaccag ctgtgccaca cctatgtgcg ctgcactaga tccgtctcca ttccggcgcc cgcctactac gcgcacctcg tcgccttccg cgcccgctat catctcgtcg agaaagagca tgacagtcat ggtgattgtt tcagtggcga gggctcgcac cagagcggct gcagcgagga ccgcacgcca ggagccatgg cccgcgccat cactgtccac gccgacacca aaaaggtcat gtactttgct tag。
the SEQ ID NO:2 is:
mavpcsdmql irrepqtvsm lgktygcgqc sapppgalga apgpvapagg pvagmppgam gllppqqphq qppqppdlpm fncprrpnlg regrpivlra nhfqitmprg fvhhydiniq pdkcprkvnr eiietmvtay skifgnlkpv fdgrsnlytr dplpigndrm elevtlpgeg kdrvfrvaik wlaqvslfal eealegrtrq ipydailald vvmrhlpsmt ytpvgrsffs spdgyyhplg ggrevwfgfh qsvrpsqwkm mlnidvsata fykaqpvief mcevldirdi ndqrkpltds qrvkftkeik glkieithcg tmrrkyrvcn vtrrpaqmqs fplqlengqt vectvakyfl dkykmklryp hlpclqvgqe hkhtylplev cnivagqrci kkltdmqtst mikatarsap drereinnlv rradfnnday vqefgltisn nmmevrgrvl pppklqyggr vsslsgqqef qgcnvlqtkq qampnqgvwd mrgkqfftgv eirvwaiacf apqrtvreda lrnftqqlqk isndagmpii gqpcfckyat gpdqvepmfr ylknsfqalq lvvvvlpgkt pvyaevkrvg dtvlgmatqc vqaknvnkts pqtlsnlclk invklggins ilvpsirpkv fnepviflga dvthppagdn kkpsiaavvg smdahpsrya atvrvqqhrq eiiqelssmv rellimfyks tggykphrii lyrdgvsegq flhvlqhelt aireacikle gdykpgitfi vvqkrhhtrl fcadkkeqsg ksgnipagtt vdvgithpte fdfylcshqg iqgtsrpshy hvlwddnhfd sdelqcltyq lchtyvrctr svsipapayy ahlvafrary hlvekehdsh gdcfsgegsh qsgcsedrtp gamaraitvh adtkkvmyfa*。
the SEQ ID NO:3 is:
tggtcgtagt tgttcttcca ggaaaaactc cagtatatgc tgaagtgaag cgagtgggcg atacagtgct gggaatggcg acccagtgtg tgcaggccaa aaatgtcaac aagacttccc cacagaccct ttccaatctt tgtctaaaga tcaatgtcaa gcttggtgga atcaacagca tacttgttcc tagcattaga cccaaggtgt tcaatgagcc ggtgatattc ctgggtgccg acgtgacgca cccgccggcc ggcgacaaca agaagccgtc gattgcggca gtggtcgggt cgatggacgc gcatcctagc cggtatgcag ccactgtgcg cgtgcaacag catcgccagg agatcatt。
a method for synthesizing dsRNA in vitro by adopting the Bai Beifei lupulus Ago1 gene fragment, which is based on the nucleotide sequence SEQ ID NO:1 design of dsRNA upstream and downstream primers carrying T7 promoter by Primer Premier 6.0 software, the sequences of the upstream and downstream primers are SEQ ID NO:4 and SEQ ID NO:5, amplifying by PCR to obtain a PCR product; purifying the obtained PCR product, and cloning and converting the PCR product into escherichia coli; amplifying and culturing by using bacterial liquid with correct sequencing, and extracting plasmids containing target gene fragments; and (3) re-PCR amplification of the primer with the T7 promoter by taking the extracted plasmid as a template, and in-vitro transcription synthesis of dsRNA by taking a high-concentration product recovered by the gel as the template and referring to a kit instruction book of a trans Aid T7 High Yield Transcription (Thermo) after the recovery and purification of the PCR product gel.
The SEQ ID NO:4 is: taatacgact cactataggg tggtcgtagt tgttcttcc.
The SEQ ID NO:5 is: taatacgact cactataggg aatgatctcc tggcgatg.
The sequencing is correct, and the nucleotide sequence in the DNA fragment is shown as SEQ ID NO:3.
an application of the sogatella furcifera Ago1 gene in RNAi-mediated pest control, wherein the in-vitro synthesized dsRNA comprises a substance with a destructive effect on the sogatella furcifera molting process. In RNAi mediation, in-vitro synthesized dsRNA is transferred into a 4-year-old nymph of the sogatella furcifera in a microinjection mode; after the target gene is specifically silenced, the molting process of the sogatella furcifera is destroyed, and death is finally caused. The injection volume of the dsRNA is 0.1 mu l, the injection concentration is 100 ng/mu l, and the injection site is the junction of the forechest and the midchest of the 4-year nymphs of the sogatella furcifera.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses the sogatella furcifera to contain SEQ ID NO: the 3 gene is a target gene and enters the body of the sogatella furcifera in a dsRNA microinjection mode, the phenotype is observed after injection, the death number is counted, and as a result, the experimental group has obvious phenotypes such as abnormal molting, malformation of wings and the like, the death rate after 10d is disturbed, and the death rate of abnormal molting is 85%. The invention can be used for pest control mediated by RNA interference and can also be used for research and development of novel pesticide target genes.
Drawings
FIG. 1 is a protein domain distribution diagram of the Ago1 gene of the sogatella furcifera of the present invention.
FIG. 2 is a schematic diagram showing the relative expression level of SfAgo1 gene (dsSfAgo 1 is an experimental group and dsGFP is a control group) of the invention after injection of dsRNA48 and 72h of SfAgo1 gene in the flea plant hopper 4-year-old nymphs.
FIG. 3 is a graph showing survival rate of the nymphs of the sogatella furcifera after 1-10d injection of dsRNA of the SfAgo1 gene fragment into the 4-year-old nymphs of sogatella furcifera of the present invention (wherein dsGFP is a control group and SfAgo1 is an experimental group).
Fig. 4 is a control graph of the effect on the ecdysis development of Bai Beifei lice after injection of dsSfAgo1 and dsGFP by the nymphs of the sogatella furcifera 4-year-old nymphs.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The sogatella furcifera Ago1 gene realized by the invention comprises SEQ ID NO:1, the nucleotide sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3, and a target gene sequence shown in 3.
The SEQ ID NO:1 is:
atggcagttc cttgttctga tatgcagctt ataaggaggg aaccacaaac agtgtccatg cttggaaaaa cctatggatg tggacagtgt tcagctcctc cgcctggagc gttgggagca gctccggggc cagtggctcc agctggtggg ccagtagccg gcatgcctcc gggagcgatg ggcctacttc cgccacagca gccccaccag caacctccgc agcctcccga tcttcccatg ttcaactgcc ccagaaggcc gaaccttggc agagaaggtc gaccaatcgt tttgcgtgcc aatcattttc aaatcacgat gccccgcggg ttcgttcatc attatgacat aaacattcag ccggacaagt gtcctcgcaa agtcaaccgt gagatcattg aaactatggt gacagcttac agtaaaatat ttggaaactt gaagccagtc ttcgatggcc gcagcaactt gtacacccga gatcccctgc caattggcaa tgatcgcatg gagcttgagg tgacattgcc gggcgaaggc aaggatcgtg tgttcagggt ggcgatcaag tggctggcgc aggtgtcgct gttcgcgctg gaggaggcgc tggaaggccg tacccgacag atcccctacg acgccatcct ggcactcgac gtcgtcatga ggcacttgcc ctcgatgacc tacacccctg tcggccgctc cttcttctcc tcacccgacg gctactatca tccgctcggc ggcggcagag aggtgtggtt cgggttccat caatcagtga ggccgtcaca gtggaagatg atgttgaaca ttgacgtatc tgcgacggcg ttttacaaag cgcagcctgt gatagagttc atgtgcgaag tgctcgacat tcgcgacata aacgaccagc gcaagccatt gactgattcc cagcgcgtca aatttaccaa agaaatcaaa ggcttgaaaa ttgagattac tcactgtgga actatgagac gcaagtaccg tgtttgcaac gtcacgcgcc ggcctgccca gatgcagtcg tttcctctgc agctggaaaa tggacaaacc gtcgagtgta cagtggccaa gtacttcttg gacaagtaca aaatgaaact gagatacccc caccttcctt gtcttcaggt cggacaagag cataagcata cttatcttcc cctagaggtg tgcaacatag tggccgggca gcggtgcatc aagaagctga ccgacatgca gacgtcgacg atgatcaagg cgacagcgcg ttcggcgccg gaccgcgagc gtgagatcaa caacctggtg cggcgcgccg acttcaacaa cgatgcctac gtgcaggagt tcggcctcac catctccaac aatatgatgg aggtgcgcgg tcgcgtcctc cccccgccca agctacagta cggcggacgc gtcagctctc tctccggaca gcaggaattt cagggctgca atgtgctgca gacgaaacag caagcgatgc ccaatcaggg tgtgtgggac atgcgtggca agcagttctt cactggcgtc gagattcgag tctgggcgat cgcctgcttt gctccacaac gcactgtgcg tgaagatgcg ctcagaaact tcactcaaca attgcagaaa atcagtaacg atgctggcat gccaataatc ggccaacctt gtttttgcaa gtacgctact ggccctgacc aagtggagcc gatgttccgg tacctgaaaa attctttcca ggctctacaa ttggtcgtag ttgttcttcc aggaaaaact ccagtatatg ctgaagtgaa gcgagtgggc gatacagtgc tgggaatggc gacccagtgt gtgcaggcca aaaatgtcaa caagacttcc ccacagaccc tttccaatct ttgtctaaag atcaatgtca agcttggtgg aatcaacagc atacttgttc ctagcattag acccaaggtg ttcaatgagc cggtgatatt cctgggtgcc gacgtgacgc acccgccggc cggcgacaac aagaagccgt cgattgcggc agtggtcggg tcgatggacg cgcatcctag ccggtatgca gccactgtgc gcgtgcaaca gcatcgccag gagatcattc aggagctgag ctccatggtc agagaacttc ttatcatgtt ctacaaaagc actggaggct acaaaccaca tcgtattatt ctatacagag atggtgtatc cgagggacag ttccttcacg ttctacagca tgagctgact gctatcagag aagcgtgtat caaacttgaa ggagactata agcctggaat cacattcatt gttgttcaga agcgacatca cacaaggctg ttctgtgcag acaagaagga acagtctgga aaatcaggca acattccagc aggcacaacg gttgacgtag gcatcacaca tccaactgaa tttgatttct acctttgcag tcatcaaggc attcaaggta cgagcagacc gagtcactac cacgtgctgt gggacgacaa ccactttgac tcagacgagc tgcagtgcct gacctaccag ctgtgccaca cctatgtgcg ctgcactaga tccgtctcca ttccggcgcc cgcctactac gcgcacctcg tcgccttccg cgcccgctat catctcgtcg agaaagagca tgacagtcat ggtgattgtt tcagtggcga gggctcgcac cagagcggct gcagcgagga ccgcacgcca ggagccatgg cccgcgccat cactgtccac gccgacacca aaaaggtcat gtactttgct tag。
the SEQ ID NO:2 is:
mavpcsdmql irrepqtvsm lgktygcgqc sapppgalga apgpvapagg pvagmppgam gllppqqphq qppqppdlpm fncprrpnlg regrpivlra nhfqitmprg fvhhydiniq pdkcprkvnr eiietmvtay skifgnlkpv fdgrsnlytr dplpigndrm elevtlpgeg kdrvfrvaik wlaqvslfal eealegrtrq ipydailald vvmrhlpsmt ytpvgrsffs spdgyyhplg ggrevwfgfh qsvrpsqwkm mlnidvsata fykaqpvief mcevldirdi ndqrkpltds qrvkftkeik glkieithcg tmrrkyrvcn vtrrpaqmqs fplqlengqt vectvakyfl dkykmklryp hlpclqvgqe hkhtylplev cnivagqrci kkltdmqtst mikatarsap drereinnlv rradfnnday vqefgltisn nmmevrgrvl pppklqyggr vsslsgqqef qgcnvlqtkq qampnqgvwd mrgkqfftgv eirvwaiacf apqrtvreda lrnftqqlqk isndagmpii gqpcfckyat gpdqvepmfr ylknsfqalq lvvvvlpgkt pvyaevkrvg dtvlgmatqc vqaknvnkts pqtlsnlclk invklggins ilvpsirpkv fnepviflga dvthppagdn kkpsiaavvg smdahpsrya atvrvqqhrq eiiqelssmv rellimfyks tggykphrii lyrdgvsegq flhvlqhelt aireacikle gdykpgitfi vvqkrhhtrl fcadkkeqsg ksgnipagtt vdvgithpte fdfylcshqg iqgtsrpshy hvlwddnhfd sdelqcltyq lchtyvrctr svsipapayy ahlvafrary hlvekehdsh gdcfsgegsh qsgcsedrtp gamaraitvh adtkkvmyfa*。
the SEQ ID NO:3 is:
tggtcgtagt tgttcttcca ggaaaaactc cagtatatgc tgaagtgaag cgagtgggcg atacagtgct gggaatggcg acccagtgtg tgcaggccaa aaatgtcaac aagacttccc cacagaccct ttccaatctt tgtctaaaga tcaatgtcaa gcttggtgga atcaacagca tacttgttcc tagcattaga cccaaggtgt tcaatgagcc ggtgatattc ctgggtgccg acgtgacgca cccgccggcc ggcgacaaca agaagccgtc gattgcggca gtggtcgggt cgatggacgc gcatcctagc cggtatgcag ccactgtgcg cgtgcaacag catcgccagg agatcatt。
a method for synthesizing dsRNA in vitro by adopting the Bai Beifei lupulus Ago1 gene fragment, which is based on the nucleotide sequence SEQ ID NO:1 design of dsRNA upstream and downstream primers carrying T7 promoter by Primer Premier 6.0 software, the sequences of the upstream and downstream primers are SEQ ID NO:4 and SEQ ID NO:5, amplifying by PCR to obtain a PCR product; purifying the obtained PCR product, and cloning and converting the PCR product into escherichia coli; amplifying and culturing by using bacterial liquid with correct sequencing, and extracting plasmids containing target gene fragments; and (3) re-PCR amplification of the primer with the T7 promoter by taking the extracted plasmid as a template, and in-vitro transcription synthesis of dsRNA by taking a high-concentration product recovered by the gel as the template and referring to a kit instruction book of a trans Aid T7 High Yield Transcription (Thermo) after the recovery and purification of the PCR product gel.
The SEQ ID NO:4 is: taatacgact cactataggg tggtcgtagt tgttcttcc.
The SEQ ID NO:5 is: taatacgact cactataggg aatgatctcc tggcgatg.
The following describes the practice of the present invention in connection with examples.
Example 1: bai Beifei lice Ago1 gene acquisition, domain analysis and target gene fragment verification.
1. Bai Beifei acquisition of the Lu Ago1 Gene sequence.
Based on the sogatella furcifera genome and transcriptome database, searching the sogatella furcifera Ago1 gene by genetic ius R9 software, and obtaining the sogatella furcifera Ago1 gene sequence after sequence alignment analysis by NCBI database BLAST: SEQ ID NO:1.
2. bai Beifei lice Ago1 gene bioinformatics analysis.
According to cDNA sequence of sogatella furcifera Ago1 gene, utilizing on-line protein translation software Expasy-TrThe anslate tool translation nucleotide sequence SEQ ID NO:1, obtaining the amino acid sequence shown as SEQ ID NO: 2. The amino acid sequence analysis of the Bai Beifei lice Ago1 gene encoded by the online software ProtParam (https:// web. Expasy. Org/protParam /) shows that the deduced protein molecular formula is C 4681 H 7384 N 1338 O 1333 S 57 . The predicted molecular weight is 105.56kDa and the theoretical isoelectric point is 9.17. The instability factor of this protein was 47.90, which is presumed to be an unstable protein. The conserved domain was predicted using ScanProsite database (https:// prosite. Expasy. Org /), and the protein contained the typical PAZ conserved domain (amino acid positions 285-405) and PIWI conserved domain (amino acid positions 591-893) as shown in FIG. 1.
Example 2: verification and synthesis of dsRNA gene fragments
(1) Based on the nucleotide sequence SEQ ID NO:1, the Primer Premier 6.0 software was used to design the upstream Primer SEQ ID NO:4 and downstream primer SEQ ID NO:5. since green fluorescent protein GFP is not present in insects, GFP gene was selected as a control gene to synthesize dsRNA. The designed specific primers were sent to Beijing qingke biosciences, inc. for synthesis. The specific sequence is as follows:
T7-SfAgo1-F：TAATACGACTCACTATAGGGTGGTCGTAGTTGTTCTTCC SEQ ID NO 4；
T7-SfAgo1-R：TAATACGACTCACTATAGGGAATGATCTCCTGGCGATG SEQ ID NO 5；
T7-GFP-F：TAATACGACTCACTATAGGGGCCAACACTTGTCACTACTT SEQ ID NO 6；
T7-GFP-R：
TAATACGACTCACTATAGGGGGAGTATTTTGTTGATAATGGTCTG SEQ ID NO 7。
(2) Bai Beifei lice nymphs were selected and mixed with adults, placed in 1.5mL RNAse-free centrifuge tubes, and Total RNA from the sogatella furcifera was extracted using the RNA extraction Kit HP Total RNA Kit (Omega Co., U.S.A.), with reference to the Kit instructions, and then reverse transcribed into first strand cDNA using the reverse transcription Kit PrimeScript RT Reagent Kit and gDNA Eraser (TaKaRa Co.) according to the instructions.
(3) By the combination ofThe synthesized dsRNA primer is subjected to PCR amplification by taking the cDNA as a template. The total volume of the PCR reaction system was 25. Mu.L, including: 1. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), 12.5. Mu.L of PCR Mix, 3. Mu.L of cDNA template, ddH 2 O7.5. Mu.L. PCR reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 65℃for 30s, elongation at 72℃for 30s,34 cycles; finally, the extension is carried out for 5min at 72 ℃.
(4) The 1. Mu.L of the above product was run on a 1% agarose gel to obtain a desired band of uniform size. The PCR products were recovered and purified by E.Z.N.A.gel Extraction Kit (Omega Co., U.S.A.), see Kit instructions for specific procedures. The gel recovery product was ligated to pMD18-T vector (the system was recovery product 2. Mu. L, solation I2.5. Mu.L and pMD18-T vector 0.5. Mu.L incubated overnight at 16 ℃) and transformed into E.coli DH 5. Alpha. Competent cells, which were cultured for 3-4 h in a shaker. Then, 50. Mu.L of the bacterial liquid was aspirated, and the resulting mixture was added to LB solid medium containing ampicillin (AMP+) and smeared with a glass rod, followed by pouring into a 37℃incubator for overnight culture. And (3) taking 1 mu L of bacterial liquid for bacterial liquid PCR detection, sucking 500mL of bacterial liquid for detecting correctness, and sending to Beijing qingke biotechnology Co. And comparing the sequencing result by SeqMan software to obtain a DNA fragment with the length of 368bp, wherein the nucleotide sequence of the fragment is shown as SEQ ID NO 3.
(5) The correctly sequenced pre-strain solution is added into 5mL LB liquid medium containing ampicillin, and placed on a shaking table at 37 ℃ and 180rpm for shake culture overnight. Plasmid extraction was performed using the E.Z.N.A.plasmid Midi (Omega Co., U.S.A.) kit, with reference to the instructions for the procedure.
(6) PCR amplification was performed using the extracted plasmid as a template and the upstream and downstream primers carrying the T7 promoter, and the reaction system and conditions were as described in (3) above. The size of the amplified fragment of the PCR product was checked by 1% agarose gel electrophoresis, and then the PCR product was recovered and purified using E.Z.N.A.gel & PCR Clean Up Kit (Omega Co., USA), and the concentration of the purified product was checked by Nanodrop 2000spectrophotometer (Thermo Fisher), ensuring that the dsDNA concentration was not less than 300ng/L.
(7) dsDNA recovered was used as template for in vitro synthesis of dsRNA using Transcriptaid T7 High Yield Transcription Kit (Thermo) kit. Before synthesis, 5X TranscriptAid Reaction Buffer was thawed at room temperature and other reagents were thawed on ice. After all reagents were thawed, gently mixed and centrifuged briefly. The specific reaction system is as follows: ATP 2. Mu.L, GTP 2. Mu.L, UTP 2. Mu.L, CTP 2. Mu.L, 5X TranscriptAid Reaction Buffer 4. Mu.L dsDNA template 1. Mu.g, T7 Enzyme Mix 2. Mu.L, nucleic-free Water make up to 20. Mu.L. The above solutions were added sequentially into a 1.5mL centrifuge tube, gently mixed, centrifuged briefly, and then placed on a PCR apparatus for incubation at 37℃for 8h. After the reaction is finished, 2 mu L of DNase I is added into the reaction system, and after the DNase I is uniformly mixed, the mixture is subjected to short centrifugation, and the mixture is placed on a PCR instrument for incubation at 37 ℃ for 10min. Then, 2. Mu.L of EDTA was added to the reaction system, gently mixed, centrifuged briefly, and then incubated on a PCR instrument at 65℃for 10min to terminate the reaction.
(8) dsRNA was purified using a Thermo company GeneJET RNA Purification Kit kit, and the purified dsRNA was used as a template for dsRNA synthesis, and dsRNA was synthesized in vitro using a trans Aid T7 High Yield Transcription (Thermo) kit, followed by purification using the kit.
(9) The purified dsRNA product was subjected to 1% agarose gel electrophoresis to detect the target band, and the concentration of dsRNA was detected using a Nanodrop 2000 nucleic acid concentration analyzer. Placing in a refrigerator at-80deg.C for use.
Example 3: dsRNA injection of Bai Beifei lice Ago1 gene fragment interfered with experiments.
1. dsRNA injection of Bai Beifei lice Ago1 gene fragment.
A 4-year-old 1d nymph of uniform and healthy size was used as the test insect. Firstly, preparing 2% agarose plate, and using CO for testing sogatella furcifera 2 The abdomen was fixed up on agarose plates for anesthesia. The injection was performed using an IM-31 mini-syringe, the injection needle being drawn from a capillary glass tube using a PUL1000 needle puller (World Precision Instruments, FL, USA). The injection site was the site where the anterior and middle chest were joined, the dose of dsRNA injection was about 100 ng/head, and 53 subjects were injected in each group with dsGFP as a negative control group, and 3 biological replicates were set. After injection, the test insects are put into the deviceThe seedlings were placed in a two-pass glass tube with fresh rice seedlings in a climatic chamber at a temperature of 25.+ -. 1 ℃ and a relative humidity of 70%.+ -. 10% and a lighting period of 16L: 8D.
Detection of silencing efficiency of the sfago1 gene.
Based on the injection assay described above, 10 heads of surviving test insects were collected for each treatment and 3 organism replicates were obtained after injection of dsSfAgo1 or dsGFP for 48 and 72 hours. Total RNA was extracted and reverse transcribed into cDNA, and SfAgo1 transcript levels after RNAi were detected by qPCR using SfRPL9 as an internal reference gene. The results showed that the relative expression levels of dsSfAgo1 were significantly reduced in the experimental group compared to the control group after 48 and 72h injection, respectively, by 74% and 73%, and the results are shown in fig. 2.
qPCR primers were as follows:
Y-SfAgo1-F：TCGCATGGAGCTTGAGGTGACA SEQ ID NO 8
Y-SfAgo1-R：CGTCGGGTGAGGAGAAGAAGGA SEQ ID NO 9
Y-SfRPL9-F：GGGCGAGAAGTACATCCGTAGG SEQ ID NO 10
Y-SfRPL9-R：GCGGCTGATCGTGAGACATCTT SEQ ID NO 11
3. statistics of death of sogatella furcifera after injection of dsRNA.
Following dsRNA or dsGFP injection, the death of the sogatella furcifera in the experimental and control groups was observed and recorded daily. The results showed that 10d after injection, the mortality rate of the experimental group injected with dsSfAgo1 was as high as 87%, which was evident in the lethal effect compared with the control group injected with dsGFP (mortality rate was only 3%), and the results were compared as shown in FIG. 3.
4. Observations of the sogatella furcifera phenotype after dsRNA injection.
After the Bai Beifei lice 4-age 1d nymphs are injected with dsGFP, 97% of control groups can successfully molt and eclosion into adults, and the development state of the adults after molting is good. After dsSfAgo1 is injected, the high-mortality phenomenon occurs in the sogatella furcifera, and the normal death of the insect body only accounts for 13% of the tested insects; and the malformation of the insect body or the opening of old epidermis can not complete the whole ecdysis process, so that death is caused, and the death rate reaches 74%. In addition, the rate of fin deformity was found to be 25% in surviving individuals (n=20). The phenotype is shown in FIG. 4. Therefore, the SfAgo1 gene is verified to obviously inhibit the normal molting development of the sogatella furcifera, and the SfAgo1 gene is an ideal target gene of the sogatella furcifera in RNAi-mediated pest control.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (1)
1. The application of the sogatella furcifera Ago1 gene in RNAi-mediated pest control is characterized in that the pest is controlled by synthesizing dsRNA in vitro, wherein the pest is sogatella furcifera, and the sequence of the dsRNA is shown as SEQ ID NO: as shown in figure 3, the number of the holes in the steel plate is,
the SEQ ID NO:3 is:
tggtcgtagt tgttcttcca ggaaaaactc cagtatatgc tgaagtgaag cgagtgggcg
atacagtgct gggaatggcg acccagtgtg tgcaggccaa aaatgtcaac aagacttccc
cacagaccct ttccaatctt tgtctaaaga tcaatgtcaa gcttggtgga atcaacagca
tacttgttcc tagcattaga cccaaggtgt tcaatgagcc ggtgatattc ctgggtgccg
acgtgacgca cccgccggcc ggcgacaaca agaagccgtc gattgcggca gtggtcgggt
cgatggacgc gcatcctagc cggtatgcag ccactgtgcg cgtgcaacag catcgccagg
agatcatt。
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| 飞蝗Argonaute1的分子特性及生物学功能;王艳丽等;中国农业科学;20160927;3569-3577 * |
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