CN103966242A - Novel Chrysopa septempunctata P450 gene and application thereof - Google Patents
Novel Chrysopa septempunctata P450 gene and application thereof Download PDFInfo
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- CN103966242A CN103966242A CN201310027131.4A CN201310027131A CN103966242A CN 103966242 A CN103966242 A CN 103966242A CN 201310027131 A CN201310027131 A CN 201310027131A CN 103966242 A CN103966242 A CN 103966242A
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Abstract
The invention discloses a DNA sequence of a novel P450 gene of an important natural enemy insect--Chrysopa septempunctata and a protein sequence coded by the gene. The DNA sequence and the protein sequence fill a gap in lack of molecular information of the P450 gene of neuropteron, provide a novel gene for research on action mechanism and the like of the P450 gene in drug resistance of natural enemy insects and have good application prospects in the field of integrated control of insects.
Description
Technical field
The invention belongs to genetically engineered and protein engineering field, relate to clone and the application of chrysopa septempunctata (Chrysopaseptempunctata) P450 family gene.
Background technology
Chrysopa septempunctata is the Important Natural Enemy of the multiple agriculture and forestry injurious insects such as aphid, tetranychid, lepidopteran ovum and low instar larvae, is a kind of natural enemy insect that has using value in biological control of insect pests.Cytochrome P 450 monooxygenases system, has another name called mixed-functional oxidase, is the supergene family albumen that a class has protoheme binding site.At present, the insect P450 having found mainly contains the function of two aspects: the one, and biosynthesizing and the degraded of catalyzing endogenous property material (as moulting hormone, neotonin, sterol, lipid acid and telergone etc.), the normal function of the body that sustains life; The 2nd, for exogenous material (as sterilant, plant secondary substance and mutagenic compound precursor etc.), play removing toxic substances and the effect activating.Wherein Cytochrome P450 is the main mechanism that most insects develops immunity to drugs to sterilant to Metabolism of pesticides effect enhancing.Insect has obtained the ability that adapts to various complex environments in long-term evolutionary process; in this process; the induction of detoxication enzyme system (especially P450 enzyme system); can help insect removing toxic substances poisonous and be unfavorable for various external sources and the interior source compound of existence to self, thereby strengthen its self-protection and the adaptive faculty of environment to external world.
A large amount of uses of chemical pesticide, the pest species developing immunity to drugs and quantity are in continuous increase, along with molecular biological develop rapidly, insect P450 gene constantly separated, clone and realize vivoexpression, its structure, function, expression regulation and deepen continuously with the relation of insect resistance research, but relevant research in formidable enemy insect, also be there is no.Therefore the present invention be take chrysopa septempunctata as material, four kinds of chrysopa septempunctata P450 genes have been cloned, and the aminoacid sequence of its coding, homology and evolutionary degree are analyzed, for studying function and the Study on Evolution of P450 gene family, provide new P450 gene fragment, for the mechanism of action of research P450 gene in natural enemy insect resistance provides new genetic material and method.
Summary of the invention
The present invention includes following content:
1. a kind of primer and cloning process thereof of cloning chrysopa septempunctata P450 gene is provided.
2. the present invention clones and obtains four new P450 gene fragments from chrysopa septempunctata larva.
3. the invention provides the application method of chrysopa septempunctata P450 gene.
Beneficial effect
The present invention be take chrysopa septempunctata larva as material, 4 of chrysopa septempunctata P450 gene cDNA sequences have been cloned, utilize reverse transcriptase polymerase chain reaction (RT-PCR) to be studied chrysopa septempunctata P450 gene, and protein sequence feature, homology, the evolutionary degree of its coding are analyzed, for further expression, the regulation and control of research chrysopa septempunctata P450 gene are laid a good foundation, for producing, P450 gene involved in insect resistance provides reference, simultaneously also for neuraopteron P450 gene functional research and Study on Evolution provide novel material.This serial genes also can be applicable to the fundamental researchs such as natural enemy insect resistance mechanism of action simultaneously, for cultivating resistance natural enemy insect Dominant variety and utilization thereof, provides fundamental basis.
The present invention is further illustrated will to quote non-limiting example below.
Embodiment
Example 1
The clone of chrysopa septempunctata P450 gene intermediate segment
1, extract total RNA: adopt Trizol method one step to extract total RNA of chrysopa septempunctata larva.
2, cDNA the first chain is synthetic: with reference to the cDNA of Beijing Quanshijin Biotechnology Co., Ltd the first chain synthetic agent box specification sheets, carry out, be specially: get total RNA3 μ l, oligo (dT)
18primer 1 μ l, 2 * TS ReactionMix10 μ l, Transcript RT/RI Enzyme mix1 μ l, RNase-free H
2o adds to 20 μ l, mixes, and 42 ℃ of 30min, 85 ℃ of 5min, 4 ℃ are cooling, and-20 ℃ save backup.
3, the intermediate segment of pcr amplification chrysopa septempunctata P450 gene: according to the conserved sequence design degenerated primer (primer is as follows) of insect P450 gene, take cDNA as template, the intermediate segment of pcr amplification chrysopa septempunctata P450 gene;
S1：5’-ACNYTYATGTTYGARGGHCAYGAYAC-3’
A1：5’-CDATRCARTTTCKKGRHCCDGC-3’
Chain polymerization enzyme reaction (pcr amplification) reaction reagent used form and reaction conditions as follows: first by following reagent mix together,
Template cDNA1 μ l
Deoxynucleotide substrate dNTP4 μ l
Ex-Taq archaeal dna polymerase 0.5 μ l
10 * Taq DNA polymerase buffer liquid, 5 μ l
Forward primer (10 μ M) 2 μ l
Reverse primer (10 μ M) 2 μ l
ddH
2O35.5μl
Cumulative volume 50 μ l
Reaction conditions is: first 94 ℃ of denaturations are 3 minutes, then circulate as follows: 94 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 1 minute, carry out altogether 35 circulations, last 72 ℃ are extended 10 minutes, 4 ℃ save backup.
4, PCR product purification: utilize sepharose to reclaim test kit (purchased from Beijing Tian Gen biotechnology company) to the recovery of PCR product, purifying, concrete operation step is undertaken by test kit specification sheets.
5, PCR product connects, transforms and identifies: get the above-mentioned PCR purified product of 5 μ l, be connected in pMD19-T simple carrier (Dalian precious biotechnology company limited product), reaction system is as shown in pMD19-T simple support agent box, then be transformed into competent escherichia coli cell Top10, be coated with and contain on the LB flat board of Amp in 37 ℃ of standing cultivations, picking list bacterium colony is cultivated, and on use cloning vector pMD19-T simple, universal primer M13-47 and RV-M carry out bacterium liquid PCR evaluation containing positive colony of P450 intermediate segment.
6, positive colony of step 5 gained is served to Hai Sheng work Bioisystech Co., Ltd and carry out sequencing, institute's calling sequence and GenBank database sequence are compared.
Example 2
The induction of Provado to chrysopa septempunctata P450
With Provado, chrysopa septempunctata third-instar larvae is induced, and the chrysopa septempunctata larva RNA of the rear 4h.8h.16h of extraction induction, after dnase digestion, reverse transcription becomes cDNA, with real time fluorescent quantitative, the changing conditions of different time P450mRNA is detected, and usings actin gene as internal reference.With 2
-Δ Δ CTmethod is calculated the relative expression quantity of P450 gene, with the expression amount of control group, is made as 1, after induction different time be expressed as the variation multiple (table 1) with respect to control group.
After table 1. induction different time be expressed as the variation multiple with respect to control group
In sum, the invention discloses chrysopa septempunctata P450 gene (P450-1-4) and coded protein thereof.This gene is the reported first in Neuroptera natural enemy insect chrysopa septempunctata, this gene of examples prove of the present invention provides new P450 gene fragment for studying function and the Study on Evolution of P450 gene family, for the mechanism of action of research P450 gene in natural enemy insect resistance provides new genetic material and method, in integrated pest control field, have a good application prospect.
Claims (4)
1. a cDNA sequence for 4 P450 genes of chrysopa septempunctata, is characterized in that, its nucleotide sequence is as described in SEQ IDNO:1-SEQ ID NO:4.
2. 4 P450 genes of chrysopa septempunctata described in claim 1, its aminoacid sequence is as described in SEQ ID NO:5-SEQID NO:8.
3. the cloning process of chrysopa septempunctata P450cDNA described in claim 1, comprises the following steps:
(1) from chrysopa septempunctata larva, extract total RNA, then the synthetic of cDNA the first chain carried out in reverse transcription;
(2) according to the conserved sequence design degenerated primer of insect P450 gene family gene:
S1：5’-ACNYTYATGTTYGARGGHCAYGAYAC-3’
A1：5’-CDATRCARTTTCKKGRHCCDGC-3’
(3) take cDNA as template, the intermediate segment of pcr amplification chrysopa septempunctata P450 gene;
(4) purification step (3) gained PCR product, sepharose reclaims object fragment, getting purified product is connected on pMD19-T simple carrier, then transform competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, picking list bacterium colony is cultivated, and on use cloning vector pMD19-T simple, universal primer M13-47 and RV-M carry out bacterium liquid PCR evaluation containing positive colony of P450 intermediate segment.
(5) positive colony of step 4 gained is served to Hai Sheng work Bioisystech Co., Ltd and carry out sequencing, institute's calling sequence and GenBank database sequence are compared.
4. the application of chrysopa septempunctata P450 gene cDNA in the anti-medicine research of chrysopa septempunctata described in claim 1, with Provado, chrysopa septempunctata third-instar larvae is induced, extract the rear 4h of induction, 8h, the total RNA of chrysopa septempunctata of 16h, and remove genomic dna with Takara DNA enzyme, and establish juice fluorescent quantitation primer according to these four P450 genes, use the method for fluorescent quantitation to analyze the situation of transcribing of rear these 4 the P450 genes of Provado induction, using actin gene as internal reference.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274238A (en) * | 2015-11-19 | 2016-01-27 | 浙江省农业科学院 | Screening method of chilo suppressalis reference genes under stress of chlorantraniliprole and application of chilo suppressalis reference genes |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20000032060A (en) * | 1998-11-12 | 2000-06-05 | 김강권 | Feed component for chrysopa pallens ramber imago and method to breed chrysopa pallens ramber by using the same |
| CN102017929A (en) * | 2009-09-10 | 2011-04-20 | 中国科学院动物研究所 | Method for artificially feeding ladybirds or lacewings by using lepidoptera insect larvae |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20000032060A (en) * | 1998-11-12 | 2000-06-05 | 김강권 | Feed component for chrysopa pallens ramber imago and method to breed chrysopa pallens ramber by using the same |
| CN102017929A (en) * | 2009-09-10 | 2011-04-20 | 中国科学院动物研究所 | Method for artificially feeding ladybirds or lacewings by using lepidoptera insect larvae |
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| NCBI: "GENBANK AB354061.1", 《GENBANK》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274238A (en) * | 2015-11-19 | 2016-01-27 | 浙江省农业科学院 | Screening method of chilo suppressalis reference genes under stress of chlorantraniliprole and application of chilo suppressalis reference genes |
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