CN101818159B - Insect chitin synthetase 1 gene segment, dsRNA and application thereof - Google Patents
Insect chitin synthetase 1 gene segment, dsRNA and application thereof Download PDFInfo
- Publication number
- CN101818159B CN101818159B CN2010101636251A CN201010163625A CN101818159B CN 101818159 B CN101818159 B CN 101818159B CN 2010101636251 A CN2010101636251 A CN 2010101636251A CN 201010163625 A CN201010163625 A CN 201010163625A CN 101818159 B CN101818159 B CN 101818159B
- Authority
- CN
- China
- Prior art keywords
- dsrna
- insect
- chitin synthetase
- gene
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides an insect chitin synthetase 1 gene segment, dsRNA and application thereof. The chitin synthetase 1 gene segment of which the sequence is SEQ ID NO:1 is synthesized according to a common part of two chitin synthetase gene sequences with access numbers of GU067730 and GU067731 respectively in an NCBI database; and the dsRNA is synthesized by using the segment. After the synthesized dsRNA is injected into the body cavity of an insect, the insect dies from molting difficulties. Therefore, a new way is provided for the development of a safe and nuisanceless pest preventing and controlling 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 and dsRNA thereof and the dsRNA application in the insect that causes death.
A kind of insect chitin synthetase 1 gene fragment provided by the invention, its nucleotide sequence are SEQ ID NO:1.
The method that a kind of insect chitin synthetase 1 gene fragment provided by the invention obtains, be respectively the common ground of two chitin synthetase sequences of GU067730 and GU067731 according to accession number in the ncbi database, design upstream primer sequence is SEQ ID NO:2, the downstream primer sequence is SEQ ID NO:3, obtains SEQ ID NO:1 by pcr amplification.
With the insect chitin synthetase 1 gene fragment is template, by the synthetic dsRNA of test kit.
The application of dsRNA in the insect that causes death: injection dsRNA is to the insect body cavity, and the result shows: dsRNA can reticent specifically insect body in the mRNA of chitin synthetase 1 gene express, the insect molting difficulty is dead.
Description of drawings
Fig. 1: the influence of behind Asiatic migrotory locust nymph injection in the 2 ages dsRNA insect growth being grown.(1 control group, 2 and 3 are the experimental group of injection dsRNA) for injection dsGFP.The experimental group insect of injection dsRNA is dead because of the difficulty of casting off a skin.
Fig. 2: behind Asiatic migrotory locust nymph injection in the 2 ages dsRNA to the influence of chitin synthetase 1 (LmCHS1) genetic transcription.β-actin is internal control gene (1 control group for injection dsGFP, 2 experimental group for injection dsRNA).
Embodiment
Embodiment 1: the acquisition of Asiatic migrotory locust chitin synthetase 1 gene fragment and dsRNA thereof
1) acquisition of Asiatic migrotory locust chitin synthetase 1 gene fragment
Be respectively the common ground of two chitin synthetase gene sequences of GU067730 and GU067731 according to accession number in the ncbi database, adopt primer premier5.0 software design Auele Specific Primer, the upstream primer sequence is SEQ ID NO:2, the downstream primer sequence is SEQ ID NO:3, 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 Asiatic migrotory locust 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.The M-MLV ThermoScript II becomes the first chain cDNA with carrying RNA reverse transcription, and as template, pcr amplification obtains chitin synthetase 1 gene fragment,
SV Gel and PCRClean-Up System (Promega) test kit carries out purifying with obtaining chitin synthetase 1 gene fragment.
2) acquisition of Asiatic migrotory locust chitin synthetase 1 gene fragment synthetic dsRNA
With 1) chitin synthetase 1 gene fragment that obtains of step is template, according to T7RiboMAX
TMThe explanation of Express RNAiSystem (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 2: the deadly Asiatic migrotory locust experiment of chitin synthetase 1 gene fragment synthetic dsRNA
1, Asiatic migrotory locust chitin synthetase 1 gene fragment synthetic dsRNA injection
The nymph of choosing the 4th day big or small homogeneous of 2 Asiatic migrotory locusts in age, 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 the dsRNA of injection chitin synthetase 1 gene fragment is 3 μ g, and the control group of dsGFP (3 μ g) is set, every group 15 cephalont, and 3 biology repeat, and amount to 45.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, the observation of Asiatic migrotory locust phenotype behind the injection dsRNA
Continue to raise after the nymph injection finishes, and observe in time.Injection dsRNA Asiatic migrotory locust experimental group and control group there is no significant difference in food ingestion, build variation and body weight gain.The control group migratory locusts all can cast off a skin smoothly, and ftractureing to slough off fully to health from crestal line only needs short several minutes, in the injection dsRNA experimental group nymph, has the part nymph to grow and deformity occurs, because of the difficulty death of casting off a skin.Phenotype is seen Fig. 1.
3, Asiatic migrotory locust chitin synthetase 1 gene silencing effect detection
Get above-mentioned deformity but dead 3 age nymph, receive 6 of worms for every group, male and female half and half, experimental group and control group are all got 3 biology and are repeated.Adopt the Trizol method to extract total RNA, adopt the M-MLV ThermoScript II to obtain the first chain cDNA, carry out RT-PCR as template, whether the expression amount that detects chitin synthetase 1 mRNA reduces.The result shows that the expression amount of experimental group chitin synthetase 1 mRNA significantly reduces.See Fig. 2.
4, the observation of Asiatic migrotory locust death condition behind the injection dsRNA
The Asiatic migrotory locust experimental group mortality ratio of injection dsRNA is 98%.This control group (mortality ratio is 0%) with injection dsGFP is compared, and lethal effect is obvious.
SEQUENCE?LISTING
＜110〉University Of Shanxi
＜120〉a kind of insect chitin synthetase 1 gene fragment and dsRNA thereof and application
<160>3
<170>PatentIn?version?3.5
<210>1
<211>405
<212>DNA
<213>Locusta?migratoria?manilensis(Meyen)
<220>
<221>gene
<222>(1)..(405)
<400>1
taatacgact?cactataggg?ctttattgct?gcctgccttc?accctcaaga?attctggtgt 60
attgttcctg?gaataattta?cttgctctct?attccatcta?tgtatcttct?cctcattttg 120
tactcactca?tcaacctgaa?tgttgtttca?tggggaacaa?gagaggttgc?agtgaagaaa 180
acaaagaagg?aaatagagca?agaaaagaaa?gaagctgaag?aagcaaagaa?gaaggccaga 240
caaaaatctc?tcttgggctt?tctccaagga?ggtggtggtg?gtgatgacaa?tgatgaagga 300
tccattgaaa?tctcatttgc?tggcctcttc?aaatgcatgt?ttttcacaca?tccaaaacca 360
attgatgaga?ggcagcagtt?gcttacccta?tagtgagtcg?tatta 405
<210>2
<211>40
<212>DNA
<213>Locusta?migratoria?manilensis(Meyen)
<220>
<221>gene
<222>(1)..(40)
<400>2
taatacgact?cactataggg?ctttattgct?gcctgccttc 40
<210>3
<211>40
<212>DNA
<213>Locusta?migratoria?manilensis(Meyen)
<220>
<221>gene
<222>(1)..(40)
<400>3
taatacgact?cactataggg?taagcaactg?ctgcctctca 40
Claims (3)
1. insect chitin synthetase 1 gene fragment, its nucleotide sequence is SEQ ID NO:1.
2. a kind of insect chitin synthetase 1 gene fragment synthetic dsRNA as claimed in claim 1.
3. the application of a kind of insect chitin synthetase 1 gene fragment synthetic dsRNA as claimed in claim 2 in the Asiatic migrotory locust that causes death by RNAi.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101636251A CN101818159B (en) | 2010-04-30 | 2010-04-30 | Insect chitin synthetase 1 gene segment, dsRNA and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101636251A CN101818159B (en) | 2010-04-30 | 2010-04-30 | Insect chitin synthetase 1 gene segment, dsRNA and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101818159A CN101818159A (en) | 2010-09-01 |
| CN101818159B true CN101818159B (en) | 2011-09-07 |
Family
ID=42653435
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101636251A Expired - Fee Related CN101818159B (en) | 2010-04-30 | 2010-04-30 | Insect chitin synthetase 1 gene segment, dsRNA and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101818159B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876691B (en) * | 2012-09-21 | 2013-10-30 | 山西大学 | Beta-N-acetylglucosaminidase genes of insects and application thereof |
| 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 |
| CN104404042B (en) * | 2014-11-14 | 2018-05-01 | 重庆大学 | The application of a kind of Asiatic migrotory locust atp synthase beta subunit gene and its dsRNA in control of insect |
| 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 |
Family Cites Families (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 |
| CN101343637B (en) * | 2007-07-10 | 2011-09-28 | 中山大学 | Method for feeding dsRNA restraint insect gene expression |
-
2010
- 2010-04-30 CN CN2010101636251A patent/CN101818159B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101818159A (en) | 2010-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101805746B (en) | Chitinase genes of insects and application of dsRNA thereof | |
| CN109504685B (en) | E93 gene and application of dsRNA thereof in pest control | |
| CN101831447B (en) | Insect chitin synthase 1A gene segment, dsRNA and application thereof | |
| CN103088040A (en) | UDP-(Uridine Diphosphate)-N-acetylglucosamine pyrophosphorylase (UAP) gene of insect and applications of dsRNA (double-stranded Ribose Nucleic Acid) of gene | |
| CN103937822B (en) | The sequence of locust I type chitinase gene and the application of dsRNA thereof | |
| CN101818159B (en) | Insect chitin synthetase 1 gene segment, dsRNA and application thereof | |
| CN105695476A (en) | Migratory locust wing epidermis protein gene and application thereof in pest control | |
| CN104630247A (en) | Insect chitin deacetylate enzyme genes 1 and application of insect chitin deacetylate enzyme genes 1 in pest control | |
| CN104673812A (en) | Insect cytochrome P450 gene and application thereof | |
| CN101831444B (en) | Insect chitin synthetase 1 gene fragment and dsRNA and application thereof | |
| CN103088031A (en) | Insect fin plate dysplasia gene segment as well as dsRNA (Ribose Nucleic Acid) and application thereof | |
| CN104774843B (en) | Applications of the Knickkopf genes dsRNA in control of insect | |
| CN104046641B (en) | Application of type II chitinase gene specificity dsRNA (double strand ribonucleic acid) of insect | |
| CN104789571B (en) | The application of Retroactive genes and its dsRNA in control of insect | |
| CN104450755B (en) | The application of a kind of Asiatic migrotory locust atp synthase α subunit genes and its dsRNA in control of insect | |
| CN101831446B (en) | Insect chitin synthase 1B gene segment, dsRNA and application thereof | |
| CN109628459B (en) | HR4 gene and application of dsRNA thereof in pest control | |
| CN104404042B (en) | The application of a kind of Asiatic migrotory locust atp synthase beta subunit gene and its dsRNA in control of insect | |
| CN110358777B (en) | Migratory locust HMGR gene and application of dsRNA thereof in migratory locust control | |
| CN102876691B (en) | Beta-N-acetylglucosaminidase genes of insects and application thereof | |
| CN101831445B (en) | Insect chitin synthase 2 gene and application | |
| CN107699569A (en) | Migratory locusts coria epidermal protein gene 1 and its application in locust control | |
| CN110106177B (en) | dsRNA of locusta migratoria fatty acid elongase gene LmElo as well as preparation method and application thereof | |
| CN110172463A (en) | Application of the migratory locusts Knickkopf 3-5 ' gene dsRNA in control of insect | |
| CN110923240B (en) | Function of insect adult disc growth factor and application of dsRNA (double-stranded ribonucleic acid) thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110907 Termination date: 20170430 |