CN101831444A - Insect chitin synthetase 1 gene fragment and dsRNA and application thereof - Google Patents
Insect chitin synthetase 1 gene fragment and dsRNA and application thereof Download PDFInfo
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- CN101831444A CN101831444A CN 201010163607 CN201010163607A CN101831444A CN 101831444 A CN101831444 A CN 101831444A CN 201010163607 CN201010163607 CN 201010163607 CN 201010163607 A CN201010163607 A CN 201010163607A CN 101831444 A CN101831444 A CN 101831444A
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Abstract
The invention provides an insect chitin synthetase 1 gene fragment and an application of dsRNA thereof. By cloning and measuring the sequence of Qxya chinensis chitin synthetase 1, a chitin synthetase gene 1 of which the nucleotide sequence is SEQ ID NO:1 is obtained, and a chitin synthetase gene fragment of which the sequence is SEQ ID NO:2 is selected for synthesizing the dsRNA. After the dsRNA is injected into the body cavity of an insect, the insect dies because of molting difficulty. The invention provides a new approach for developing a safe and nuisance-free insect control method.
Description
Technical field
The present invention relates to biological technical field.Be specifically related to the application in the deadly insect of insect chitin synthetase 1 gene fragment and dsRNA thereof and dsRNA.
Background technology
It is the important restraining factors of China's farm output that Agricultural pests are caused harm, and the long-term application chemical insecticide causes a series of problems: 1) resistance appears in insect, and dosage strengthens, and the control cost improves; 2) pesticide residue cause environmental pollution serious; 3) non-target organism there is considerable influence.Existing biotic pesticide are used wider in pest control, but the desinsection time is long and effect is slow.
It is gene silencing phenomenon behind a kind of specific transcriptional that is caused by double stranded rna molecule that RNA disturbs (RNAi), obtains the Nobel prize in 2006.The discovery of RNAi not only provides the breakthrough on the method for the functional study of gene, also is that new approach has been opened up in the control of human disease treatment and crop pest simultaneously.2007, two pieces of papers of " Nature Biotechnology " magazine priority have been reported to use and have been expressed the important lethal gene V-ATPase of target insect A, the dsRNA transgenic plant of CYP6AE14 and GST1 are as a kind of novel method (Baum et al, 2007 of controlling disease and pest; Mao et al, 2007).2008, Price and Gatehouse proposed the pest control strategy based on RNAi.Pest control based on the RNA perturbation technique has following advantage: 1) selecting the single-minded gene of insect is disturbed, is safe to the higher animal and the mankind; 2) pest-resistant have a specificity, and non-target organism is not had lethal effect; 3) nontoxic to environment.
Studies show that the RNA perturbation technique can effectively be controlled special plant insect, has important development prospect in field of pest control.And realize that carrying out the effective key of controlling of insect based on RNAi is that screening is to the efficient lethal dsRNA of insect.
Chitin synthesizes and metabolism is the distinctive biological phenomena of insect constant pitch main drive thing, because the mankind and other higher animal do not have chitin, so the insect chitin synthesis system has been acknowledged as the target of novel pesticide effect.Chitin synthetase plays keying action in the final step of insect chitin synthetic, adopts the RNA perturbation technique, and it is significant to carry out the application of chitin synthetase dsRNA in pest control.
Summary of the invention
The purpose of this invention is to provide a kind of insect chitin synthetase 1 gene fragment, the application in the insect that causes death by gene fragment synthetic dsRNA and dsRNA.
A kind of insect chitin synthetase 1 gene fragment 1 provided by the invention, its nucleotide sequence are SEQ ID NO:1.After being the degenerated primer by the design insect chitin synthetase, pcr amplification obtains, called after OcCHS1, and its length is 312bp, 104 amino acid of encoding.
A kind of insect chitin synthetase 1 gene fragment 2 provided by the invention, its nucleotide sequence are SEQ ID NO:2, are according to SEQ ID NO:1 design upstream primer SEQ ID NO:3 and downstream primer SEQ ID NO:4, obtain by pcr amplification.Further utilize related kit to synthesize dsRNA.
The application of dsRNA in the insect that causes death: injection dsRNA is to the insect body cavity, and the result shows: the mRNA that dsRNA can reticent specifically chitin synthetase 1 gene expresses, and insect is because of the difficulty death of casting off a skin.
Description of drawings
Fig. 1: the influence of behind Chinese rice grasshopper nymph injection in the 3 ages dsRNA insect growth being grown.The experimental group of the injection dsRNA dead phenotype of difficulty (1 control group, the experimental group of 2 injection dsRNA) that occurs casting off a skin for injection dsGFP.
Embodiment
Embodiment 1: the acquisition of Chinese rice grasshopper chitin synthetase 1 gene fragment 1
1) PCR primer design
Amino acid conserved sequence according to known insect chitin synthetase has designed following degenerated primers: upstream primer: CHS1F 5 '-GAYGGNGAYATHGAYTT-3, downstream primer: CHS1R 5 '-TCYTCNCCYTGRTCRTA-3 '; All primers are synthetic by the prompt basic biological company limited in the English Weihe River, Shanghai.
2) acquisition of the total RNA of Chinese rice grasshopper
Choose the body surface of big or small consistent male and female half and half Chinese rice grasshopper nymph in 5 age, one group of four-head is frozen in the liquid nitrogen, RNA to be extracted, and the concrete operations step of extracting RNA is with reference to TaKaRa Trizol test kit.
3) the Chinese rice grasshopper first chain cDNA is synthetic
The first chain cDNA synthesis step is with reference to SMART
TMRACE cDNA Amplification test kit.
4) pcr amplification
With the above-mentioned first chain cDNA is template, carries out pcr amplification according to Taq polymerase (TIANGEN) specification sheets, and the fragment that is increased is further cloned, checked order.
5) sequential analysis
Utilize relevant online software and software analysis institute calling sequences such as GENEDOC, gene seeker in the Expasy website, in the NCBI website, use the BLAST function to carry out the sequence homology comparison, determine that the gene fragment that is obtained is a Chinese rice grasshopper chitin synthetase 1 gene fragment 1.
Embodiment 2: the acquisition of Chinese rice grasshopper chitin synthetase 1 gene fragment 2
The nucleotide sequence of the Chinese rice grasshopper chitin synthetase 1 gene fragment 1 that obtains according to embodiment 1, adopt the specific primer of primerpremier5.0 software design, the upstream primer sequence is SEQ ID NO:3, the downstream primer sequence is SEQ IDNO:4, and all primers are synthetic by the prompt basic biological company limited in the English Weihe River, Shanghai.Choose big or small consistent male and female half and half Chinese rice grasshopper nymph in 5 age, one group of four-head is frozen in the liquid nitrogen, RNA to be extracted, and the concrete operations step is with reference to TaKaRa Trizol test kit.The M-MLV ThermoScript II becomes the first chain cDNA with carrying RNA reverse transcription, and as template, pcr amplification obtains the chitin synthetase gene fragment,
SV Gel and PCR Clean-Up System (Promega) test kit carries out purifying.
Embodiment 3: the acquisition of the dsRNA of Chinese rice grasshopper chitin synthetase 1 gene fragment 2
With the chitin synthetase 1 gene fragment 2 of embodiment 2 acquisitions, according to T7RiboMAX
TMThe explanation of Express RNAi System (Promega) test kit, in-vitro transcription is synthesized dsRNA.The dsRNA that obtains detects its unicity with 1.5% agarose gel electrophoresis, with microplate reader (Molecular Devices SpectraMax 190, Menlo Park, CA, USA) with its quantitatively to final concentration be 1 μ g/ μ L.Be saved to-70 ℃ standby.
Embodiment 4: the deadly Chinese rice grasshopper experiment of the dsRNA of chitin synthetase 1 gene fragment 2
1) dsRNA of Chinese rice grasshopper chitin synthetase 1 gene fragment 2 injection
The nymph of choosing big or small homogeneous in the 4th day 3 ages, healthy state unanimity is used to inject above-mentioned synthetic dsRNA.25 μ l specification microsyringes are used for injection, can not be firmly excessive during injection, and along the direction of blood flow, valve will be avoided as injection point in the junction of flank portion the 2nd to 3 uromere.The amount of injection dsRNA is 3 μ g, and the control group of dsGFP (3 μ g) is set, each 25 of experimental group and control groups.After injection finishes, with insect with the beaker of 1L as for raising (illumination: interlunation=14h: 10h, 30 ± 2 ℃ of temperature, humidity 60%) in the growth cabinet, give fresh wheat seedling and suitable illumination, water spray maintenance humidity.
2) observation of Chinese rice grasshopper phenotype behind the injection dsRNA
Continue to raise after the nymph injection finishes, and observe in time.Injection dsRNA Chinese rice grasshopper experimental group has the part nymph dead because of the difficulty of casting off a skin.
3) observation of Chinese rice grasshopper death condition behind the injection dsRNA
The Chinese rice grasshopper experimental group mortality ratio of injection dsRNA is 80%.This control group (mortality ratio is 0%) with injection dsGFP is compared, and lethal effect is obvious.
SEQUENCE?LISTING
＜110〉University Of Shanxi
＜120〉insect chitin synthetase 1 gene fragment and dsRNA thereof and application
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atcggttgtg?tcctctgtag?ccctggatgt?ttctctctct?tcagagggaa?gtcacttatg 240
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tacgaccagg?gc 312
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gggaagtcac?ttatggatga?ttgtgtgatg?aacaaatata?caacacgatc?agaggaagct 300
cgacactatg?tgcaatacga?ccaggcccta?tagtgagtcg?tatta 345
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Claims (4)
1. insect chitin synthetase 1 gene fragment 1, its nucleotide sequence is SEQ ID NO:1.
2. insect chitin synthetase 1 gene fragment 2, its nucleotide sequence is SEQ ID NO:2.
3. a kind of insect chitin synthetase 1 gene fragment 2 synthetic dsRNA as claimed in claim 2.
4. the application of a kind of insect chitin synthetase 1 gene fragment 2 synthetic dsRNA as claimed in claim 3 in the insect that causes death.
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| CN2010101636073A CN101831444B (en) | 2010-04-30 | 2010-04-30 | Insect chitin synthetase 1 gene fragment and dsRNA and application thereof |
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| CN101831444B CN101831444B (en) | 2012-01-11 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630247A (en) * | 2015-02-13 | 2015-05-20 | 山西大学 | Insect chitin deacetylate enzyme genes 1 and application of insect chitin deacetylate enzyme genes 1 in pest control |
| CN105219790A (en) * | 2014-05-30 | 2016-01-06 | 华中农业大学 | A kind of sickle-like bacteria chitin synthase gene C hs3b and application |
| CN105685092A (en) * | 2015-12-30 | 2016-06-22 | 中山大学 | Chitin-synthesized inhibitory pesticide and preparing method and application thereof |
| CN106011138A (en) * | 2016-08-10 | 2016-10-12 | 中国农业大学 | DNA molecule for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene and application thereof |
| CN106282183A (en) * | 2016-08-09 | 2017-01-04 | 中山大学 | A kind of miRNA analogies and chitin synthesis suppress class biological pesticide and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5124149A (en) * | 1990-11-07 | 1992-06-23 | The United States Of America As Represented By The Secretary Of Agriculture | Compositions and methods for biocontrol using fluorescent brighteners |
| CN101343637A (en) * | 2007-07-10 | 2009-01-14 | 中山大学 | Method for feeding dsRNA restraint insect gene expression |
| CN101370940A (en) * | 2006-01-12 | 2009-02-18 | 德福根有限公司 | DsRNA as insect control agent |
-
2010
- 2010-04-30 CN CN2010101636073A patent/CN101831444B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5124149A (en) * | 1990-11-07 | 1992-06-23 | The United States Of America As Represented By The Secretary Of Agriculture | Compositions and methods for biocontrol using fluorescent brighteners |
| CN101370940A (en) * | 2006-01-12 | 2009-02-18 | 德福根有限公司 | DsRNA as insect control agent |
| CN101343637A (en) * | 2007-07-10 | 2009-01-14 | 中山大学 | Method for feeding dsRNA restraint insect gene expression |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219790A (en) * | 2014-05-30 | 2016-01-06 | 华中农业大学 | A kind of sickle-like bacteria chitin synthase gene C hs3b and application |
| CN104630247A (en) * | 2015-02-13 | 2015-05-20 | 山西大学 | Insect chitin deacetylate enzyme genes 1 and application of insect chitin deacetylate enzyme genes 1 in pest control |
| 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 |
| CN105685092A (en) * | 2015-12-30 | 2016-06-22 | 中山大学 | Chitin-synthesized inhibitory pesticide and preparing method and application thereof |
| CN105685092B (en) * | 2015-12-30 | 2019-01-11 | 中山大学 | A kind of chitin synthesis inhibits insecticides and preparation method and application |
| CN106282183A (en) * | 2016-08-09 | 2017-01-04 | 中山大学 | A kind of miRNA analogies and chitin synthesis suppress class biological pesticide and application thereof |
| CN106282183B (en) * | 2016-08-09 | 2019-03-05 | 中山大学 | A kind of miRNA analogies and chitin synthesis inhibit class biological pesticide and its application |
| CN106011138A (en) * | 2016-08-10 | 2016-10-12 | 中国农业大学 | DNA molecule for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene and application thereof |
| CN106011138B (en) * | 2016-08-10 | 2019-04-09 | 中国农业大学 | The DNA molecular of the hairpin RNA of expression inhibiting grain aphid chitin synthetase 1 gene and its application |
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| CN101831444B (en) | 2012-01-11 |
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