CN108823222A - White backed planthopper chitin synthetase 1（SfCHS1）The application of genetic fragment and its dsRNA in control of insect - Google Patents
White backed planthopper chitin synthetase 1（SfCHS1）The application of genetic fragment and its dsRNA in control of insect Download PDFInfo
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- CN108823222A CN108823222A CN201810653955.5A CN201810653955A CN108823222A CN 108823222 A CN108823222 A CN 108823222A CN 201810653955 A CN201810653955 A CN 201810653955A CN 108823222 A CN108823222 A CN 108823222A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01016—Chitin synthase (2.4.1.16)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Abstract
The invention discloses a kind of application of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment and its dsRNA in control of insect, the partial sequence of chitin synthetase 1 (SfCHS1) gene is obtained by analysis white backed planthopper transcript profile database, it is cloned and is sequenced on this basis, the nucleotide sequence of acquisition such as sequence table SEQ ID NO：Shown in 3, it is based on sequence SEQ ID NO：3 synthesize the dsRNA of the gene, provide candidate gene for the strategy of insect pest control that future white backed planthopper mediated rnai is led, while providing theoretical foundation for research and development novel pesticide.Belong to insect genes field of engineering technology.
Description
Technical field
The present invention relates to insect genes field of engineering technology, especially a kind of white backed planthopper chitin synthetase 1
(SfCHS1) application of genetic fragment and its dsRNA in control of insect.
Background technique
White backed planthopper Sogatallafurcifera (Horv á th) is under the jurisdiction of Semiptera Hemiptera Delphacidae
Delphacidae is a kind of important pests in the Rice Production of the Asian-Pacific area.It mainly passes through the direct piercing and sucking rice of adult, nymph
Strain juice, causes paddy growth slow, and tiller delay, empty grain increases, and can cause rice strain withered when aggrieved serious；In addition, white back
Plant hopper or southern rice black-streaked dwarf disease virus (southern rice black-streaked dwarfvirus,
SRBSDV primary vehicle), caused by endanger it is even more destructive.Instantly the prevention and control of white backed planthopper still depend on
Chemical insecticide, and a large amount of unreasonable uses of chemical insecticide constitute serious prestige to the health of ecological environment and the mankind
The side of body, while but also white backed planthopper produces different degrees of resistance to a variety of insecticides, it would therefore be highly desirable to develop a kind of novel
The strategy of environmentally friendly prevention and treatment white backed planthopper.
RNA interference (RNAinterference, RNAi) is a kind of important gene silencing means of discovered in recent years, is logical
The external double-stranded RNA (double-strandRNA, dsRNA) for synthesizing one section with target gene homology is crossed, is imported in insect bodies, is made interior
Degradation of homologous mRNA in source property target gene, to achieve the purpose that blocking gene is translated.RNAi is insect genes functional study
Effective means, its successful application is development environment friendly, the novel strategy of insect pest control of good economical benefit provides new think of
Road and new method.
Chitin is the chief component of insect cuticle and middle intestines funnel.The epidermis of insect is also due to chitin and tan
Change the presence of albumen and be in rigid structure, this undoubtedly limits the growth of insect.In growth and development process, insect is subjected to
It periodically sloughs off old epidermis and forms new epidermis, essence of this process dependent on chitin biosynthesis and degradation in insect bodies
Really control.Chitin synthetase (chitin synthase, CHS) is that final step is biochemical anti-in insect chitin synthesis process
The key enzyme answered adjusts behavior, development by metamorphosis and breeding of insect etc..By the shape for upsetting or blocking insect chitin synthetase
At so that occurring phenomena such as Dysecdysis, survival rate reduction, reproductive capacity decline in insect bodies.Due to the mankind with it is other high
Chitin is not present in animal body, so the key enzyme during chitin synthesis is comparatively safe as the target of control of insect,
But relevant report is had no for the research of white backed planthopper chitin synthetase 1 at present, it also has no for white backed planthopper chitin
The report of the dsRNA of synthetase 1 gene.
Summary of the invention
The object of the present invention is to provide a kind of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment and its dsRNA
Application in control of insect, to realize environmentally friendly prevention and treatment white backed planthopper.
To achieve the above object, the present invention provides a kind of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment,
Its nucleotide sequence such as SEQ ID NO：Shown in 3.
Its acquisition methods is as follows：Based on the segment that white backed planthopper transcript profile database obtains, the upstream for designing specificity is drawn
Object 1SEQ ID NO：1 and downstream primer 1SEQ ID NO：2, it is obtained by PCR amplification.
The present invention also provides a kind of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment synthesis dsRNA with
And the method for synthesizing the dsRNA：Based on the nucleotide sequence SEQ ID NO：3, utilize 6.0 software of Primer Premier
Design has the upstream primer 2SEQ ID NO of T7 promoter：4 and downstream primer 2SEQ ID NO：5, it is obtained by PCR amplification
One segment length is the DNA fragmentation of 491bp, which is named as intermediate DNA fragmentation, the nucleotides sequence of intermediate DNA fragmentation
Column such as SEQ ID NO：6.By nucleotide sequence SEQ ID NO：6 genetic fragment PCR product glue recycling with after purification, with this
Glue recycling product be template according toSynthesis is transcribed in vitro in RNAi Kit (Thermo) kit specification
dsRNA。
Compared with prior art, advantages of the present invention is as follows：
White backed planthopper can effectively be prevented using the dsRNA that genetic fragment of the present invention synthesizes, by synthesizing one in vitro
The double-stranded RNA (double-strandRNA, dsRNA) of section and target gene homology imports in insect bodies, makes in endogenous target gene
Degradation of homologous mRNA is the novel pest of development environment friendly, good economical benefit to achieve the purpose that blocking gene is translated
Control strategy provides new approaches and new method；It is in use, be transmitted to white backed planthopper body for dsRNA by microinjection
Interior, detection dsRNA expresses the mRNA of SfCHS1 gene, and observes the influence of dsRNA Whitebacked Planthopper growth and development.As a result table
It is bright：Inject dsRNA can specifically silencing white backed planthopper chitin synthetase 1 (SfCHS1) gene mRNA expression, simultaneously
The lopsided phenotype that polypide has apparent husking difficult, and death is eventually led to, the death rate reaches 100% after interference 5 days.The present invention
Candidate gene is provided for the strategy of insect pest control that the following white backed planthopper mediated rnai is led, while the present invention is to research and develop novel kill
Worm agent provides theoretical foundation.
Detailed description of the invention
Fig. 1 is the SfCHS1 gene relative expression quantity after the dsRNA of 5 age white backed planthopper nymphs injection SfCHS1 gene.18S
RNA is reference gene, and dsGFP is control group, and dsCHS1 is experimental group.
Fig. 2 is the cumulative survival rate after the dsRNA of 5 age white backed planthopper nymphs injection SfCHS1 gene.Wherein dsGFP is pair
According to group, dsCHS1 is experimental group.
Specific embodiment
Below by specific embodiment, the invention will be further described, specific as follows：
Embodiment：White backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment and its dsRNA
The acquisition of white backed planthopper chitin synthetase 1 1. (SfCHS1) genetic fragment
1) preliminary analysis is carried out using bioinformatics technique Whitebacked Planthopper transcript profile database, passes through ncbi database
The programs such as Blast determine the partial nucleotide sequence for obtaining white backed planthopper SfCHS1 gene.Utilize Primer Premier 6.0
The upstream primer 1SEQ ID NO of software design specificity：1 and downstream primer 1SEQ ID NO：2, all primers are raw by raw work
The synthesis of object engineering (Shanghai) limited liability company.
2) 5 age of white backed planthopper nymph 15 are chosen, is placed in 1.5mL without in the centrifuge tube of RNA enzyme, after liquid nitrogen frozen quickly
It is ground into fine powder, the extraction of total serum IgE is carried out with TRIzol method, concrete operation step is carried out referring to TRIzol kit specification.
Then the first chain is synthesized according to the specification of Sangon company AMV First Strand cDNA Synthesis Kit kit
cDNA。
3) using the cDNA of above-mentioned synthesis as template, upstream primer 1SEQ ID NO is utilized：1 and downstream primer 1SEQ ID
NO：2 carry out PCR amplification.PCR reaction system total volume is 25 μ L, including：Upstream and downstream primer 1 (10 μM) each 1 μ L, 10 × LAPCR
Buffer(Mg2+Plus) 2.5 μ L, dNTP Mixture (2.5mM), 4 μ L, cDNA2 μ L, Taq enzyme 0.25 μ L and 14.25 μ of redistilled water
L.PCR reaction condition is：94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 30 are followed
Ring；Last 72 DEG C of extensions 10min.
4) PCR product electrophoresis in 1% Ago-Gel judges in gel imager testing goal band length and tentatively
It whether is expected purpose band.Glue then is cut to purpose band, is passed throughQuick Gel Extraction Kit
Kit carries out PCR product recycling and purifying, and concrete operation step is referring to kit specification.Glue recovery product is connected to
(system is 2 μ L of recovery product, 2.5 μ L and pMD18-T carrier of Solation I, 0.5 μ L, and 16 DEG C were incubated on pMD18-T carrier
Night), subsequent linked system is converted to DH5 α competent cell, after the screening of blue hickie and PCR are examined, will examine correctly clone
After LB liquid medium (containing ampicillin) overnight incubation, Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced.
5) sequencing result is subjected to check and correction and sequence assembly by SeqMan software, and utilizes Blast journey in the website NCBI
Sequence carries out sequence homology comparison, obtains white backed planthopper SfCHS1 genetic fragment, nucleotide sequence such as SEQ ID NO：3 institutes
Show.
The synthesis of the dsRNA of white backed planthopper chitin synthetase 1 2. (SfCHS1) genetic fragment
1) it is based on SfCHS1 genetic fragment SEQ ID NO obtained：3, it is set using 6.0 software of Primer Premier
Meter has the upstream primer 2SEQ ID NO of T7 promoter：4 and downstream primer 2SEQ ID NO：5, all primers are raw by raw work
The synthesis of object engineering (Shanghai) limited liability company.
2) using the cDNA of above-mentioned synthesis as template, PCR amplification is carried out using the dsRNA primer of above-mentioned synthesis.PCR reaction
System total volume is 25 μ L, including：Each 1 μ L, 10 × LAPCR Buffer (Mg of upstream and downstream primer (10 μM)2+Plus) 2.5 μ L,
DNTP Mixture (2.5mM) 4 μ L, cDNA 2 μ L, 14.25 μ L of 0.25 μ L of Taq enzyme and redistilled water.PCR reaction condition is：94℃
Initial denaturation 3min；94 DEG C of denaturation 30s, 62 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30 recycle；Last 72 DEG C of extensions 10min.
3) 1 μ LPCR product electrophoresis in 1% Ago-Gel is taken, detects gained band length simultaneously just in gel imager
Step judges whether it is expected purpose band, then carries out glue recycling to PCR product and verifies and survey with purifying, clone, bacterium solution PCR
Sequence.
4) after sequence verification is errorless, 200 μ L sequencing bacterium solution is taken to be added to 20mL LB liquid medium (green containing ammonia benzyl
Mycin) in, it is placed in 37 DEG C of shaking table, 220rpm shake culture is stayed overnight.It utilizesPlasmid MiniPrep Kit reagent
Box (the full formula gold in Beijing) carries out plasmid extraction, and specific steps are completed referring to specification.
5) it using the plasmid of extracting as template, usesRNAi Kit (Thermo) kit is to specifications
It is transcribed in vitro and synthesizes and purify to obtain the dsRNA of white backed planthopper SfCHS1 genetic fragment.
6) it takes the dsRNA product of above-mentioned purifying to carry out 1% agarose gel electrophoresis and detects its unicity, and use Nanodrop
2000spectrophotometer (Thermo Fisher) detects its concentration.It saves spare to -80 DEG C of refrigerators.
Experimental example：The lethal experiment of dsRNA of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment
1. the dsRNA of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment is injected
Choose the dsRNA that first day 5 age size is uniform, the consistent nymph of health status is used to inject above-mentioned synthesis.First
The agarose plate for preparing 1%, by white backed planthopper in CO2Processing lower abdomen be fixed on agarose plate upwards, using aobvious
Microinjection instrument Nanoliter 2010Injector (World Precision Instruments) is injected, and when injection is wanted
The position that mild manipulation, shirtfront and mesothorax connect is as injection point.The amount for injecting dsRNA is 100ng/ head, and to inject dsGFP
As a control group, every group injection 50 are arranged 3 biology and repeat.After injection, test worm is put into equipped with fresh rice seedling
In test tube, it is placed in temperature (25 ± 1) DEG C, relative humidity 70% ± 5%, photoperiod 16L:In the growth cabinet of 8D.
2. the silence efficiency of white backed planthopper chitin synthetase 1 (SfCHS1) gene detects
Based on above-mentioned test injection, after injecting dsRNA or dsGFP 72h, test worm 10 of every group of collection survival, setting
3 biologies repeat.Total serum IgE is extracted, and reverse transcription is cDNA, the destination gene expression feelings after RNAi is detected using qPCR technology
Condition.The result shows that compared with the control group of injection dsGFP, white backed planthopper injects the experimental group of the dsRNA of SfUAP after 72h
Relative expression quantity significantly reduce, and reduce up to 80%.As a result as shown in Figure 1.
3. injecting the observation of white backed planthopper death condition after dsRNA
After 5 first day age nymphs inject dsRNA or dsGFP, the white backed planthopper death condition of observation experiment group and control group.
The result shows that the experimental group white backed planthopper death rate for injecting dsRNA reaches 100% after injection 5 days, pair of this and injection dsGFP
It is compared according to group (death rate 8%), lethal effect is obvious.As a result as shown in Figure 2.
4. injecting the observation of white backed planthopper phenotype after dsRNA
Experimental group nymph totally 150 for injecting dsRNA, 100% individual cannot complete the transformation of nymph to adult, i.e., in plumage
It is dead before changing.
Sequence table
<110>Guizhou University
<120>White backed planthopper chitin synthetase 1（SfCHS1）The application of genetic fragment and its dsRNA in control of insect
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>White backed planthopper (Sogatalla furcifera)
<400> 1
gcacgagacc aacattagg 19
<210> 2
<211> 18
<212> DNA
<213>White backed planthopper (Sogatalla furcifera)
<400> 2
agagaatgag cagcaggt 18
<210> 3
<211> 2214
<212> DNA
<213>White backed planthopper (Sogatalla furcifera)
<400> 3
gaattggctg aacgtgaaaa agatcccgat gaatattacg agacaatctc agttcacacg 60
gatgcaagca gtacaactcc gaaaacagtg aaaaagtctg atagtatcac aaggatctat 120
gcatgcgcta ccatgtggca cgaaactaaa gaggagatga tggagatgtt gaagtctatt 180
ctgagaatgg atgaagatca gtgcgccaga cgagttgctc agaaatactt gagagtggtt 240
gatccagact actacgaatt cgaaacacac attttcatgg acgacgcatt cgaaatcagt 300
gacgtgaacg acgactggtg tcaggtgaat cgtttcgtga agctgctggt gtcgatcatc 360
gatgaggccg cgtcacatgt gcacgagacc aacattagga tcaggccgcc caccaaatac 420
ccaactgtct acggtggtcg tctggtctgg accctgcctg gaaagactaa gatgatctgt 480
catctcaagg acaaagcaaa gatcaggcac aggaaacgtt ggagccaggt gatgtacatg 540
tactaccttt tgggtcaccg attgatggaa ctgcccatat cagtcgagag aaaggaagtg 600
atggctgaga gcactttcct gttgacactt gatggtgata tcgacttcca gcctcatgct 660
gttagactgc tcattgattt gatgaaaaag aacaagaact tgggagctgc ttgtggaaga 720
attcatccag ttggatcagg acccatggta tggtatcaga tgttcgagta tgcgattggt 780
cattggcttc agaaggcaac agagcatatg attggttgtg tcctttgtag tccgggatgt 840
ttctcacttt tccgtggaaa ggctttgatg gatgacaacg ttatgagaag atacacaacc 900
agatctgacg aagcaagaca ttacgtgcaa tacgatcagg gagaggacag atggctgtgc 960
accctgctgc tgcaaagagg ctaccgagtg gagtactcag cggccagtga tgcctacacg 1020
cactgccccg aaagtttcaa cgagttcttc aaccagcgtc ggcgatgggt gccatccacc 1080
atggccaaca tcatggacct gctagccgac tacaagcaca cagtcaagat caacgacaac 1140
atttcgctgc cctacatctg gtatcagatt atgttgatgg gaggtacgat tctcggccct 1200
ggaacgatat tccttatgtt ggtgggagct ttcgtggctg ctttcaggat tgacaactgg 1260
actagcttct attacaacat tatacctata ttcttcttca tgttggtgtg tttcacttgc 1320
aaatcagaaa tacagctact tgtggcgcaa atattctcaa cggcgtacgc actgataatg 1380
atggctgtga tagtgggtac ggcgctgcag ctgggtgagg acggtcccgg ctctccgtcg 1440
gccattttcc tctcggctat gttaggctcg ttcttcatag cggcggtgtt acatccgcag 1500
gagttcaagt gtatcacgcc gttcgttatc tacatgctgt ccattccgtc catgtacctg 1560
ctgctcattc tctactcgat catcaatctc aacgtggtct cctggggtac tcgagaagtg 1620
gcggtcaaga aaactaaaaa ggaactggaa gaagaaaaaa aagccgagga agctgcaaag 1680
aaaaaagcga aatcgagatc actgctcggt tttcttcaga acgagaacat tggccttgga 1740
aataatgacg aggaaggatc tctggagctt tcatttgctg gcttgttcag atgtatgttc 1800
tgcacctatc catctccagt cgatgaaaag cagcagctca tgagaatagc cgagtcattg 1860
gaggcaatgg aaaagaggct ggacaccatg gaaaaggtga tggatccaaa ctgtcaggtg 1920
attggacgtc gtcgaaccac ttcggcgagt agtcgcggcg atcaccacgg aggggcgctg 1980
catgcgctca ctgaggaccc cgccgaggag gaccagatgc atgacgataa cagtgatacc 2040
atgtccacca tggctacaga acatagaatt gaaaggaatg acgatgtcaa tccgtactgg 2100
attgaagacc gtgatttgaa gaaaggaccc gtcaatttct tgtctgcttc tgaaaaccaa 2160
ttctggaaag agctccttga taaatatcta ttccctatcg atgaagacaa ggct 2214
<210> 4
<211> 40
<212> DNA
<213>White backed planthopper (Sogatalla furcifera)
<400> 4
taatacgact cactataggg ctgacgaagc aagacattac 40
<210> 5
<211> 42
<212> DNA
<213>White backed planthopper (Sogatalla furcifera)
<400> 5
taatacgact cactataggg cactatcaca gccatcatta tc 42
<210> 6
<211> 491
<212> DNA
<213>White backed planthopper (Sogatalla furcifera)
<400> 6
ctgacgaagc aagacattac gtgcaatacg atcagggaga ggacagatgg ctgtgcaccc 60
tgctgctgca aagaggctac cgagtggagt actcagcggc cagtgatgcc tacacgcact 120
gccccgaaag tttcaacgag ttcttcaacc agcgtcggcg atgggtgcca tccaccatgg 180
ccaacatcat ggacctgcta gccgactaca agcacacagt caagatcaac gacaacattt 240
cgctgcccta catctggtat cagattatgt tgatgggagg tacgattctc ggccctggaa 300
cgatattcct tatgttggtg ggagctttcg tggctgcttt caggattgac aactggacta 360
gcttctatta caacattata cctatattct tcttcatgtt ggtgtgtttc acttgcaaat 420
cagaaataca gctacttgtg gcgcaaatat tctcaacggc gtacgcactg ataatgatgg 480
ctgtgatagt g 491
Claims (4)
1. a kind of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment, it is characterised in that：Its nucleotide sequence such as SEQ
ID NO：Shown in 3.
2. a kind of white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment as described in claim 1, acquisition methods are such as
Under：Based on the segment that white backed planthopper transcript profile database obtains, the 1 SEQ ID NO of upstream primer of specificity is designed：1 and downstream
1 SEQ ID NO of primer：2, it is obtained by PCR amplification.
3. a kind of dsRNA, it is characterised in that：The dsRNA is closed by white backed planthopper chitin synthetase 1 (SfCHS1) genetic fragment
At.
4. a kind of dsRNA as claimed in claim 3, which is characterized in that synthetic method is as follows：Based on nucleotide sequence SEQ ID
NO：3, the upstream primer 2SEQ ID NO of T7 promoter is had using software design：4 and downstream primer 2SEQ ID NO：5, lead to
It crosses PCR amplification and obtains the DNA fragmentation that a segment length is 491bp, the nucleotide sequence of the segment such as SEQ ID NO：6, by nucleosides
Acid sequence SEQ ID NO：6 genetic fragment PCR product glue recycling with after purification, using the glue recycling product as template according toSynthesis dsRNA is transcribed in vitro in RNAi Kit (Thermo) kit specification.
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| CN114561411A (en) * | 2022-03-14 | 2022-05-31 | 贵州大学 | Dicer1 gene and application of dsRNA thereof in pest control |
| CN114854774A (en) * | 2021-02-04 | 2022-08-05 | 南京吉星生物技术开发有限公司 | Chordoporus punctatus chitin synthase 1(CHS1) gene and application thereof in aspect of RNAi mediated pest control |
| CN115678903A (en) * | 2022-11-03 | 2023-02-03 | 贵州大学 | Sogatella furcifera Ago1 gene, method for synthesizing dsRNA and application thereof |
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| CN114854774A (en) * | 2021-02-04 | 2022-08-05 | 南京吉星生物技术开发有限公司 | Chordoporus punctatus chitin synthase 1(CHS1) gene and application thereof in aspect of RNAi mediated pest control |
| CN114561411A (en) * | 2022-03-14 | 2022-05-31 | 贵州大学 | Dicer1 gene and application of dsRNA thereof in pest control |
| CN115678903A (en) * | 2022-11-03 | 2023-02-03 | 贵州大学 | Sogatella furcifera Ago1 gene, method for synthesizing dsRNA and application thereof |
| CN115678903B (en) * | 2022-11-03 | 2024-04-02 | 贵州大学 | Sogatella furcifera Ago1 gene, method for synthesizing dsRNA and application thereof |
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