CN115678903A - 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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- 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
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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 (double-stranded ribonucleic acid) and application thereof in the aspect of RNAi (ribonucleic acid interference) mediated pest control, wherein the sogatella furcifera Ago1 gene is prepared by adopting an RNA interference technology, and a higher lethal phenomenon occurs after dsRNA (double-stranded ribonucleic acid) of the sogatella furcifera Ago1 gene is injected into a 4-year-old nymph, so that the 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 (double-stranded ribonucleic acid) and application thereof in the aspect of RNAi (ribonucleic acid interference) mediated pest control.
Background
The Sogataria furcifera (Horv th) belongs to Delphacidae of Hemiptera planthopper, is one of pests seriously harmed in rice production, and mainly uses nymphs and adults to stab rice stem juice to cause nutrient loss; meanwhile, viruses such as Southern rice black-streaked dwarf virus (SRBSDV) are also transmitted. As a migratory insect pest, the sogatella furcifera is easy to outbreak under the suitable environmental condition, and brings serious economic loss to the rice production. The control of sogatella furcifera still mainly depends on chemical pesticides at present, however, the long-term unreasonable and excessive use of the chemical pesticides causes the sogatella furcifera to generate drug resistance, kill natural enemies and pollute the environment. Therefore, the research and development of the novel environment-friendly pesticide have great significance to the prevention and control work of the sogatella furcifera.
RNA interference (RNAi) is a phenomenon in which small dsRNA causes sequence-specific degradation of mRNA of a target gene, resulting in silencing or down-regulation of the amount of expression of the target gene. RNAi has become an important tool for studying insect gene function, and this technology has become a new means of pest control in the field of plant protection: the gene fragment in the insect body is utilized to inhibit the transcription level of the gene which is vital to the growth and development of the pests through RNAi so as to achieve the purpose of preventing and treating the pests. Therefore, the application of RNAi technology to develop novel pesticides has important significance for pest control.
Among the proteins in the RNAi pathway of insects are mainly Drosha proteins, dicer proteins, argonaute proteins, which serve as core elements of the RNA-induced silencing complex (RISC), and dsRNA-binding domain proteins. 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 is an important component of miRNA pathway in insect body, and can specifically recruit miRNA to participate in mRNA silencing, thereby regulating gene expression.
The functional research of miRNA in the aspects of insect growth and development, reproduction, resistance, immunity and the like is researched by interfering the Ago1 gene, and the gene is closely related to various insect growth and development processes. However, no relevant report is found in the current research aiming at the Ago1 gene of the Sogatella furcifera, and no report is found in the RNAi mediated pest control aiming at the Ago1 gene of the Sogatella furcifera.
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 (double-stranded ribonucleic acid) and application thereof in the aspect of RNAi (ribonucleic acid interference) mediated pest control, wherein the sogatella furcifera Ago1 gene is prepared by adopting an RNA interference technology, and a high lethal phenomenon occurs after the sogatella furcifera Ago1 gene dsRNA is injected into 4-year-old nymphs, so that the Ago1 gene can be used as a novel control target in pest control.
In order to realize the purpose, the invention adopts the technical scheme that:
an Ago1 gene of Sogatella furcifera, comprising SEQ ID NO:1, SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3, and (b) 3. The target gene sequence shown in the figure.
The nucleotide sequence of 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 nucleotide sequence of SEQ ID NO:2 is as follows:
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 nucleotide sequence of SEQ ID NO:3 is as follows:
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 gene fragment Ago1 of the Sogatella furcifera is disclosed, wherein the dsRNA is based on the nucleotide sequence SEQ ID NO:1, designing dsRNA upstream Primer and downstream Primer carrying T7 promoter by Primer Premier 6.0 software, wherein the sequences of the upstream Primer and the downstream Primer are respectively SEQ ID NO:4 and SEQ ID NO:5, then obtaining a PCR product through PCR amplification; purifying the obtained PCR product, and cloning and transforming the PCR product into escherichia coli; carrying out amplification culture on the bacterial liquid with correct sequencing, and extracting a plasmid containing a target gene fragment; taking the extracted plasmid as a template, carrying out PCR amplification on a primer with a T7 promoter, recovering and purifying a PCR product gel, and taking a High-concentration product recovered by the gel as the template, and synthesizing dsRNA by in vitro Transcription according to the instruction of a Transcript Aid T7 High Yield Transcription (Thermo) kit.
The nucleotide sequence of SEQ ID NO:4 is as follows: taatacgact cactataggg tggtcgtagt tgttcttcc.
The nucleotide sequence of SEQ ID NO:5 is as follows: taatacgact cactataggg aatgatctcc tggcgatg.
The sequencing is correct, and the nucleotide sequence in the DNA fragment is shown as SEQ ID NO:3.
the application of the Ago1 gene of the Sogatella furcifera in the aspect of RNAi-mediated pest control, wherein the in-vitro synthesized dsRNA comprises a substance which has a destructive effect on the molting process of the Sogatella furcifera. In the RNAi mediation, dsRNA synthesized in vitro is transferred into the nymph of the Sogatella furcifera at the 4 th age by a microinjection mode; after the target gene is specifically silenced, the molting process of the sogatella furcifera is damaged, and finally death is caused. The injection volume of the dsRNA is 0.1 mu l, the injection concentration is 100 ng/mu l, and the injection part is the junction of the chest and the middle chest of the nymphaea alba L.4.
Compared with the prior art, the invention has the following beneficial effects:
the sogatella furcifera contains SEQ ID NO: the 3 gene is a target gene and enters the sogatella furcifera body in a dsRNA microinjection mode, the phenotype is observed and the death number is counted after the injection, and the result experiment group has obvious phenotypes such as molting abnormality and wing malformation, the death rate is 87% after 10 days of interference, wherein the molting abnormality mortality rate accounts for 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 map of the Ago1 gene of Sogatella furcifera according to the present invention.
FIG. 2 is a schematic diagram showing the relative expression amounts of SfAgo1 genes (with SfRPL9 as a reference gene, dsSfAgo1 as an experimental group and dsGFP as a control group) after SfAgo1 gene dsRNA48 and 72h are injected into Laodelphax striatellus 4-year nymphs of the invention.
FIG. 3 is a graph plotting survival rate of Sogatella furcifera nymphs after the Sogatella furcifera 4-year old nymphs of the invention are injected with dsRNA 1-10d of SfAgo1 gene fragment (wherein dsGFP is a control group, and SfAgo1 is an experimental group).
FIG. 4 is a graph comparing the results of dsSfAgo1 and dsGFP injection into Sogatella furcifera 4-instar nymphs on the effect of Bai Beifei on the ecdysis development of lice.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The sogatella furcifera Ago1 gene realized by the invention comprises the following components in percentage by weight: 1, SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3, and (b) 3. The target gene sequence shown in the figure.
The nucleotide sequence of 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 nucleotide sequence of SEQ ID NO:2 is as follows:
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 nucleotide sequence of SEQ ID NO:3 is as follows:
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 gene fragment Ago1 of the Sogatella furcifera is disclosed, wherein the dsRNA is based on the nucleotide sequence SEQ ID NO:1, designing dsRNA upstream Primer and downstream Primer carrying T7 promoter by Primer Premier 6.0 software, wherein the sequences of the upstream Primer and the downstream Primer are respectively SEQ ID NO:4 and SEQ ID NO:5, then PCR products are obtained through PCR amplification; purifying the obtained PCR product, and cloning and transforming the PCR product into escherichia coli; carrying out amplification culture on the bacterial liquid with correct sequencing, and extracting a plasmid containing a target gene fragment; taking the extracted plasmid as a template, carrying out PCR amplification on a primer with a T7 promoter, recovering and purifying a PCR product gel, and taking a High-concentration product recovered by the gel as the template, and synthesizing dsRNA by in vitro Transcription according to the instruction of a Transcript Aid T7 High Yield Transcription (Thermo) kit.
The amino acid sequence of SEQ ID NO:4 is as follows: taatacgact cactataggg tggtcgtagt tgttcttcc.
The nucleotide sequence of SEQ ID NO:5 is as follows: taatacgact cactataggg aatgatctcc tggcgatg.
The practice of the present invention will now be described with reference to the following examples.
Example 1: acquiring a sogatella furcifera Ago1 gene, analyzing a structural domain and verifying a target gene fragment.
1. And (3) acquiring a gene sequence of the Ago1 of the sogatella furcifera.
On the basis of the sogatella furcifera genome and transcriptome database, the gene Ago1 of the sogatella furcifera is searched by Geneius R9 software, and the sequence of the gene Ago1 of the sogatella furcifera is obtained after the sequence comparison and analysis by NCBI database BLAST: SEQ ID NO:1.
2. and (3) carrying out bioinformatics analysis on the Ago1 gene of the Sogatella furcifera.
According to the cDNA sequence of the gene Ago1 of the sogatella furcifera, the nucleotide sequence SEQ ID NO:1, obtaining the amino acid sequence shown as SEQ ID NO:2, respectively. The amino acid sequence analysis of the Bai Beifei louse Ago1 gene by using 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 with a theoretical isoelectric point of 9.17. The protein instability stability factor was 47.90, which was assumed to be an unstable protein. Utilizing ScanProsite database (https)// position. Expay. Org /) a predicted conserved domain, which contains 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: dsRNA gene fragment verification and synthesis
(1) Based on the nucleotide sequence SEQ ID NO:1, designing an upstream Primer containing a T7 promoter by using Primer Premier 6.0 software, wherein the upstream Primer is SEQ ID NO:4 and the downstream primer SEQ ID NO:5. since the green fluorescent protein GFP is not present in insects, the GFP gene was selected as a control gene for dsRNA synthesis. The designed specific primer is sent to Beijing Optimalaceae biotechnology limited 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) Selecting and mixing nymphs of the Sogatella furcifera and adults, placing the mixture into a 1.5mL centrifuge tube without RNA enzyme, extracting Total RNA of the Sogatella furcifera by using an RNA extraction Kit HP Total RNA Kit (Omega company, USA) according to the instruction Kit specification, and then reversely transcribing the Total RNA into first-strand cDNA by using a reverse transcription Kit PrimeScript RT Reagent Kit and gDNA Eraser (TaKaRa company) according to the operation instruction.
(3) The synthesized dsRNA primer is subjected to PCR amplification by taking the synthesized cDNA as a template. The total volume of the PCR reaction system is 25 mu L, and the PCR reaction system comprises: 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, extension at 72 ℃ for 30s, and 34 cycles; finally, extension is carried out for 5min at 72 ℃.
(4) The above 1. Mu.L of product was run on a 1% agarose gel to give a desired band of consistent size. The PCR product was recovered and purified by E.Z.N.A.gel Extraction Kit (Omega, USA), and the specific procedures were described in the Kit. The gel recovered product was ligated to pMD18-T vector (system: 2. Mu. L, solation I2.5. Mu.L recovered product and 0.5. Mu.L pMD18-T vector, incubated overnight at 16 ℃), transformed into E.coli DH 5. Alpha. Competent cells, and cultured in a shaker for 3-4 h. Then, 50. Mu.L of the bacterial solution was aspirated and added to LB solid medium containing ampicillin (AMP +), and the mixture was smeared with a glass rod, and then inverted and placed in a 37 ℃ incubator overnight for culture. Taking 1 mu L of bacterial liquid to carry out bacterial liquid PCR detection, and sucking 500mL of bacterial liquid with correct detection and sending the bacterial liquid to Beijing Optimalaceae Biotech Limited for sequencing. 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 reserved bacterial liquid with correct sequencing is added into 5mL LB liquid culture medium containing ampicillin and placed on a shaking table at 37 ℃ and 180rpm for shaking culture overnight. Plasmid extraction was performed using the E.Z.N.A.plasmid Midi (Omega, USA) kit, with the procedures as described in the specification.
(6) PCR amplification was performed using the extracted plasmid as a template and upstream and downstream primers carrying a T7 promoter, and the reaction system and conditions were as described in (3) above. The PCR product was checked for size of amplified fragments by 1% agarose gel electrophoresis, and then recovered and purified using E.Z.N.A.gel & PCR Clean Up Kit (Omega, USA), and the concentration of the purified product was measured using a Nanodrop 2000spectrophotometer (Thermo Fisher) to ensure that the dsDNA concentration was not less than 300ng/L.
(7) The recovered dsDNA was used as a template, and dsRNA was synthesized in vitro using a TranscriptAID T7 High Yield Transcription Kit (Thermo). Before synthesis, 5 × Transcriptaid Reaction Buffer was thawed at room temperature, and other reagents were thawed on ice as well. After all reagents are thawed, the mixture is gently mixed evenly and then is centrifuged for a short time. The specific reaction system is as follows: 2 μ L ATP, 2 μ L GTP, 2 μ L UTP, 2 μ L CTP, 4 μ L5X TranscriptAID Reaction Buffer 4 μ L dsDNA template 1 μ g, T7 Enzyme Mix 2 μ L, nucleic-free Water to 20 μ L. The solutions were added sequentially to a 1.5mL centrifuge tube, gently mixed, centrifuged briefly, and then incubated for 8h at 37 ℃ on a PCR instrument. After the reaction is finished, 2 mu L of DNase I is added into the reaction system, mixed uniformly and centrifuged for a short time, and the mixture is placed on a PCR instrument and incubated for 10min at 37 ℃. Then 2 μ L EDTA was added to the reaction system, gently mixed, centrifuged briefly, and then placed on a PCR instrument to incubate at 65 ℃ for 10min, terminating the reaction.
(8) dsRNA was purified using a GeneJET RNA Purification Kit from Thermo, and the purified dsRNA was used as a template for synthesizing dsRNA, and dsRNA was synthesized in vitro using a Transcript Aid T7 High Yield Transcription (Thermo) Kit and then purified using the Kit.
(9) Taking the purified dsRNA product, carrying out 1% agarose gel electrophoresis to detect a target band, and detecting the concentration of the dsRNA by using a Nanodrop 2000 nucleic acid concentration analyzer. Storing in a refrigerator at-80 deg.C.
Example 3: and (3) carrying out a dsRNA injection interference experiment on the Ago1 gene fragment of the sogatella furcifera.
1. And (3) dsRNA injection of gene fragments of Ago1 genes of sogatella furcifera.
1d nymphs with consistent sizes and healthy 4 ages were used as experimental insects. First, 2% agarose plates were prepared for testing CO for Sogatella furcifera 2 Anaesthetize and mount ventrally up on agarose plates. The injection was carried out using an IM-31 microinjector, the injection needle being drawn from a capillary glass tube using a PUL1000 needle drawing instrument (World Precision Instruments, FL, USA). The anterior and medial chest were connected at the injection point, the dose of dsRNA was approximately 100 ng/head, and dsGFP was injected as a negative control group, with 53 test insects per group, and 3 biological replicates were set. After injection, the test insects were placed in two-way glass test tubes containing fresh rice seedlings and kept in a climatic chamber at temperature (25 ℃. + -. 1 ℃), relative humidity (70%. + -. 10%), light cycle (1698.
And 2, detecting the silencing efficiency of the SfAgo1 gene.
Based on the injection assay described above, 10 surviving test worms were collected for each treatment, 3 biological replicates, after injection of dsSfAgo1 or dsGFP 48 and 72 h. Extracting total RNA and carrying out reverse transcription to obtain cDNA, taking SfRPL9 as an internal reference gene, and detecting the transcription level of SfAgo1 after RNAi by qPCR. The results show that the relative expression of dsSfAgo1 in the experimental group was significantly reduced compared to the control group 48 and 72h after injection, and was down-regulated by 74% and 73%, respectively, as shown in fig. 2.
The 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 sogatella furcifera mortality after dsRNA injection.
After injection of either dsRNA or dsGFP, sogatella furcifera mortality was observed and recorded daily for both experimental and control groups. The results show that the mortality rate of sogatella furcifera in the experimental group injected with dsSfAgo1 is up to 87% after 10d injection, compared with the control group injected with dsGFP (the mortality rate is only 3%), the lethal effect is obvious, and the results are shown in FIG. 3.
4. Observation of sogatella furcifera phenotype after dsRNA injection.
After dsGFP is injected into 1d nymphs of the sogatella furcifera at the age of 4, 97 percent of the control groups can successfully molt and eclose into adults, and the adults after molting have good development state. After dsSfAgo1 is injected, the sogatella furcifera has high lethality, and the normal death of the larvae only accounts for 13 percent of the test larvae; and the death caused by the failure of the whole molting process due to the malformation of the polypide or the opening of the old cuticle reaches 74 percent. In addition, fin teratogenicity was statistically 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 present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The Sogatella furcifera Ago1 gene is characterized by comprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3, and (b) 3. The target gene sequence shown in the figure.
2. The Sogatella furcifera Ago1 gene according to claim 1, characterized in that said sequence of 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。
3. the Sogatella furcifera Ago1 gene according to claim 2, characterized in that said sequence of SEQ ID NO:2 is as follows:
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*。
4. the Sogatella furcifera Ago1 gene according to claim 3, characterized in that said sequence of SEQ ID NO:3 is as follows:
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。
5. a method for synthesizing dsRNA in vitro by adopting the Ago1 gene segment of the sogatella furcifera is characterized in that the method is based on the nucleotide sequence SEQ ID NO:1, designing dsRNA upstream Primer and downstream Primer carrying T7 promoter by Primer Premier 6.0 software, wherein the sequences of the upstream Primer and the downstream Primer are respectively SEQ ID NO:4 and SEQ ID NO:5, then PCR products are obtained through PCR amplification; purifying the obtained PCR product, and cloning and transforming the PCR product into escherichia coli; carrying out amplification culture on the bacterial liquid with correct sequencing, and extracting a plasmid containing a target gene fragment; taking the extracted plasmid as a template, carrying out PCR amplification on a primer with a T7 promoter, and after recovering and purifying a PCR product gel, taking a high-concentration product recovered by the gel as a template to synthesize dsRNA through in vitro transcription.
6. The method for in vitro synthesis of dsRNA using the Ago1 gene fragment of Sogatella furcifera according to claim 5, wherein the nucleotide sequence of SEQ ID NO:4 is as follows:
taatacgact cactataggg tggtcgtagt tgttcttcc；
the nucleotide sequence of SEQ ID NO:5 is as follows:
taatacgact cactataggg aatgatctcc tggcgatg。
7. the application of the sogatella furcifera Ago1 gene fragment in the in vitro synthesis of dsRNA of claim 5, wherein the sequencing is correct and is the nucleotide sequence in the DNA fragment of SEQ ID NO:3.
8. the use of the above-mentioned fragment of the Ago1 gene of Sogatella furcifera for the in vitro synthesis of dsRNA according to claim 5, characterized in that the in vitro synthesis of dsRNA comprises substances having a disruptive effect on the molting process of Sogatella furcifera.
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