CN103937822B - The sequence of locust I type chitinase gene and the application of dsRNA thereof - Google Patents
The sequence of locust I type chitinase gene and the application of dsRNA thereof Download PDFInfo
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- CN103937822B CN103937822B CN201410069843.7A CN201410069843A CN103937822B CN 103937822 B CN103937822 B CN 103937822B CN 201410069843 A CN201410069843 A CN 201410069843A CN 103937822 B CN103937822 B CN 103937822B
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Abstract
The present invention provides the sequence of a kind of locust I type chitinase gene (cht5) and the application of dsRNA thereof, and it can make locust dead by reticent I type chitinase gene.After the I type chitinase gene of Locustamigratoria (Linnaeus) is cloned, checking order, obtain the I type chitinase gene that sequence is SEQ ID NO:1；Then the genetic fragment selecting sequence to be SEQ ID NO:2, for the synthesis of double-stranded RNA (dsRNA).After this dsRNA injects the body cavity of locust, locust growth course shows as development delay, and cannot cast off a skin smoothly next age of entrance, cause death.The present invention is the target new based on the RNA Pest control disturbed offer.
Description
Technical field
The present invention relates to biological technical field.It is specifically related to the sequence of locust I type chitinase gene and the application of dsRNA thereof.
Background technology
Insect is the formidable enemy of China's agricultural production, chronic administration chemical insecticide, causes that pesticide residues, environmental pollution are serious, Drug resistance occurs in locust, endanger human body health etc. series of problems.Along with the development of society, in the urgent need to novel insect pest control method.
Disturb (RNAi) Pest control technology just at high speed development based on RNA in recent years.The method has following advantage: 1) select the gene that insect is single-minded is disturbed, to higher mammal and and the mankind be safe；2) pest-resistant have specificity, to non-target organism without lethal effect；3) environment is nontoxic.
For the phenomenon design Pest control scheme of casting off a skin that locust is unique, both can efficiently kill insect, turn avoid environmental pollution, safe and reliable.Insect exoskeleton is mainly made up of chitin, and chitinase genes of insects is responsible for chitinous degraded, so that insect molting.Locust I type chitinase gene (cht5) plays a key effect when casting off a skin, and utilizes its important biomolecule function to carry out Pest control, time saving and energy saving, safe and efficient.
Summary of the invention
It is an object of the invention to provide the sequence of a kind of locust I type chitinase gene and dsRNA application in lethal insect thereof.
A kind of locust I type chitinase gene provided by the invention, its nucleotide sequence is SEQIDNO:1.This sequence is by the search to Locustamigratoria (Linnaeus) transcript profile, obtains 1 Locustamigratoria (Linnaeus) I type chitinase gene fragment, and after end sequence rapid amplifying (RACE), clone obtains full length cDNA sequence further.The long 1587bp of this sequence, comprises 1515bp open reading frame, encodes 505 aminoacid.
A kind of locust I type chitinase gene fragment provided by the invention, its nucleotide sequence is SEQIDNO:2, being design forward primer SEQIDNO:3 and downstream primer SEQIDNO:4 according to SEQIDNO:1, obtain SEQIDNO:2 by pcr amplification, it contains T7 promoter.Further according to T7RiboMAXTMExpressRNAiSystem(Promega) test kit illustrates in vitro transcription synthesis dsRNA.
The application in lethal locust of dsRNA of SEQIDNO:2 synthesis: inject above-mentioned dsRNA to locust body cavity, result shows: this dsRNA can the mrna expression of reticent locust I type chitinase gene LmCht5-1 specifically, and cause Locustamigratoria (Linnaeus) to cast off a skin difficulty and dead.
Accompanying drawing explanation
Occur casting off a skin after the dsRNA of Locustamigratoria (Linnaeus) injection SEQIDNO:2 synthesis in Fig. 1: 5 ages the phenotype of difficulty and death.Left side is the comparison of injection dsGFP, and right side is the dsRNA of injection SEQIDNO:2 synthesis.
Fig. 2: behind dsRNA24 hour of Locustamigratoria (Linnaeus) injection SEQIDNO:2 synthesis, the mRNA silence situation of Locustamigratoria (Linnaeus) I type chitinase gene LmCht5-1, β-actin is as reference gene.* P < 0.01.
Detailed description of the invention
Embodiment 1: the sequence of Locustamigratoria (Linnaeus) I type chitinase gene and the acquisition of dsRNA thereof
1, the sequence of Locustamigratoria (Linnaeus) I type chitinase gene obtains
1) sequence of Locustamigratoria (Linnaeus) I type chitinase gene is in the search of Locustamigratoria (Linnaeus) transcript profile data base
Based on Locustamigratoria (Linnaeus) transcript profile data base, adopt bioinformatics method that the sequence of Locustamigratoria (Linnaeus) I type chitinase gene is scanned for, after sequence analysis and comparison, obtain the sequence fragment of 1 Locustamigratoria (Linnaeus) I type chitinase gene altogether, this cDNA sequence disappearance 3' end.
2) full length cDNA sequence of Locustamigratoria (Linnaeus) I type chitinase gene obtains
Based on obtaining Locustamigratoria (Linnaeus) I type chitinase gene fragment, adopt primerpremier5.0 software design 3 ' RACE primer.All primers are by the synthesis of Shanghai English Weihe River victory base biology company limited.Extract Locustamigratoria (Linnaeus) 5 nymph in age total serum IgE, synthesize RACE template with RACE test kit (clontech), transfer for full length cDNA sequence.After PCR, obtaining product purification, Cloning Transformation, in escherichia coli, sends to order-checking.After interpretation of result, splicing obtains Locustamigratoria (Linnaeus) I type chitinase gene LmCht5-1cDNA full length sequence, and its sequence is SEQIDNO:1.
2, Locustamigratoria (Linnaeus) I type chitinase gene dsRNA synthesis
1) design of Locustamigratoria (Linnaeus) I type chitinase gene dsRNA primer
Based on obtained Locustamigratoria (Linnaeus) I type chitinase gene sequence SEQIDNO:1, adopt primerpremier5.0 software design.Design dsRNA synthetic primer, its sequence respectively SEQIDNO:3 and SEQIDNO:4.All primers are by the synthesis of Shanghai English Weihe River victory base biology company limited.
2) synthesis of Locustamigratoria (Linnaeus) I type chitinase gene dsRNA
Above-mentioned primer SEQIDNO:3 and SEQIDNO:4 synthesizes PCR primer, warpSVGelandPCRClean-UpSystem(Promega) after kits, according to T7RiboMAXTMExpressRNAiSystem(Promega) test kit illustrates in vitro transcription synthesis dsRNA.DsRNA concentration adopts microplate reader SpectraMax190 to measure, and is concentrated into final concentration of 3 μ g/ μ l.
Embodiment 2: Locustamigratoria (Linnaeus) I type chitinase gene dsRNA lethal 5 age Locustamigratoria (Linnaeus)
1, Locustamigratoria (Linnaeus) I type chitinase gene dsRNA injection
The dsRNA of 1 μ l(3 μ g) SEQIDNO:2 is expelled between 5 age Locustamigratoria (Linnaeus) the 2nd age in days nymph two, three uromeres with 25 μ l specification microsyringes, altogether injection 40, male and female half and half.The dsGFP of injection same volume concentration is to matched group body.Locustamigratoria (Linnaeus) after injection is placed in 30 DEG C of constant temperature biochemical cultivation cases and raises.
2, inject after dsRNA 5 age Locustamigratoria (Linnaeus) phenotype observation
5 age nymph after injection dsRNA, injection dsGFP matched group starts after 6 days to cast off a skin and all successfully slough off to adult, and on average the time of casting off a skin is 15-20 minute, sloughs off to adult polypide form vigor normal, and starts normally to take food after half a day.The process group nymph totally 40 of injection dsLmCht5-1, compared with matched group nymph, wherein has 26 nymphs finally can not complete to cast off a skin to cause death, and mortality rate reaches 65.0%.There is phenotype (Fig. 1) three kinds different in 26 dead nymphs: when the first is casted off a skin for the nymph of a locust, only pronotary and head are a little along crestal line cracking, and ftracture partially visible new epidermis, and back is swelled, and wing bud opens, and is continued for death；The second phenotype is that the new and old epidermis of appendage cannot separate, and new wing also cannot be sloughed off from old epidermis wing bud；The third is then that pronotary crestal line cracking part increases, and the new and old epidermis of abdomen end can separate, it is apparent that ghost, and the distortion of whole polypide is until dead.The probability that three of the above phenotype occurs is essentially identical.
3, Locustamigratoria (Linnaeus) I type chitinase gene silence detection
After dsRNA injection in 5 ages, after selecting injection, 24h silence efficiency detects, and whole worm polypide extracts object as RNA, and often group arranges and repeats 3 biologys.Total serum IgE the reverse transcription of extracting each sample become the first chain cDNA, Real-timePCR testing goal gene (LmCht5-1) and the relative expression quantity of house-keeping gene (β-actin), to calculate the silence efficiency (Fig. 2) of genes of interest.
Claims (4)
1. a Locustamigratoria (Linnaeus) I type chitinase gene, its nucleotide sequence is SEQIDNO:1.
2. a Locustamigratoria (Linnaeus) I type chitinase gene fragment, its nucleotide sequence is SEQIDNO:2.
3. the dsRNA of Locustamigratoria (Linnaeus) I type chitinase gene fragment synthesis as claimed in claim 2.
4. dsRNA application in lethal Locustamigratoria (Linnaeus) as claimed in claim 3.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104293810B (en) * | 2014-09-01 | 2018-02-02 | 太原理工大学 | Gypsymoth chitinase gene and dsRNA based on gene silent technology |
| CN104630247B (en) * | 2015-02-13 | 2018-07-24 | 山西大学 | Insect chitin deacetylate enzyme gene 1 and its application in control of insect |
| CN104789571B (en) * | 2015-04-07 | 2017-12-05 | 山西大学 | The application of Retroactive genes and its dsRNA in control of insect |
| CN105734068A (en) * | 2016-03-22 | 2016-07-06 | 山西大学 | LmCas9 gene, LmCas9 gene vector and construction method and application of LmCas9 gene vector |
| CN109251929A (en) * | 2018-04-27 | 2019-01-22 | 杭州贝英福生物科技有限公司 | A kind of siRNA interfering striped rice borer chitinase gene CsCht10 |
| CN109504684B (en) * | 2018-12-20 | 2021-07-27 | 山西大学 | Application of Ran gene and dsRNA thereof in pest control |
| CN109810993B (en) * | 2019-03-25 | 2022-05-31 | 临沂大学 | Locust beta-D-glucoside hydrolase gene and application thereof |
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| CN101805746A (en) * | 2010-03-26 | 2010-08-18 | 山西大学 | Chitinase genes of insects and application of dsRNA thereof |
| CN101831445A (en) * | 2010-04-30 | 2010-09-15 | 山西大学 | Insect chitin synthase 2 gene and application |
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| CN101805746A (en) * | 2010-03-26 | 2010-08-18 | 山西大学 | Chitinase genes of insects and application of dsRNA thereof |
| CN101831445A (en) * | 2010-04-30 | 2010-09-15 | 山西大学 | Insect chitin synthase 2 gene and application |
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