CN103966242B - Chrysopa septempunctata P450 new gene and application thereof - Google Patents
Chrysopa septempunctata P450 new gene and application thereof Download PDFInfo
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- CN103966242B CN103966242B CN201310027131.4A CN201310027131A CN103966242B CN 103966242 B CN103966242 B CN 103966242B CN 201310027131 A CN201310027131 A CN 201310027131A CN 103966242 B CN103966242 B CN 103966242B
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Abstract
The invention discloses the protein sequence of a kind of Important Natural Enemy insecticide chrysopa septempunctata (Chrysopa septempunctata) P450 new gene DNA sequence and coding thereof, fill up the shortage of P450 gene molecule information in neuraopteron, provide new gene for researchs such as P450 gene mechanism of action in natural enemy insect Drug resistance.Have a good application prospect in integrated pest control field.
Description
Technical field
The invention belongs to genetic engineering 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, Lepidoptera ovum and low instar larvae, is a kind of natural enemy insect of great using value in biological control of insect pests.Cytochrome P 450 monooxygenases system, has another name called mixed-functional oxidase, is the class supergene family albumen with heme binding sites.At present, it has been found that insecticide P450 mainly have the function of two aspects: one is biosynthesis and degraded, the normal function of the body that sustains life of catalyzing endogenous property material (such as ecdyson, juvenile hormone, sterol, fatty acid and telergone etc.)；Two is the effect playing removing toxic substances and activation for exogenous material (such as insecticide, plant secondary substance and mutagenic agent precursor etc.).Wherein metabolism of pesticides effect enhancing is the main mechanism that insecticide is developed immunity to drugs by most insects by Cytochrome P450.Insecticide obtains the ability adapting to various complex environments in long-term evolutionary process; in the process; the induction of Chlorogenic Acid (especially P450 enzyme system); can help insecticide removing toxic substances that self is poisonous and be unfavorable for various external sources and the endogenous compounds of existence, thus strengthening the adaptive capacity of its self-protection and environment to external world.
A large amount of uses of chemical pesticide, the pest species developed immunity to drugs and quantity are being continuously increased, develop rapidly along with molecular biological, insecticide P450 gene constantly separated, clone and realize vivoexpression, its structure, function, expression regulation and the relation with insect resistance research deepen continuously, but but without relevant research in formidable enemy insecticide.Therefore the present invention is with chrysopa septempunctata for material, four kinds of chrysopa septempunctata P450 genes are cloned, and the aminoacid sequence of its coding, homology and evolutionary degree are analyzed, function and Study on Evolution for studying P450 gene family provides new P450 genetic fragment, provides new genetic material and method for research P450 gene mechanism of action in natural enemy insect Drug resistance.
Summary of the invention
The present invention includes herein below:
1. provide a kind of primer cloning chrysopa septempunctata P450 gene and cloning process thereof.
2. the present invention clones from chrysopa septempunctata larva and obtains four new P450 genetic fragments.
3. the invention provides the application process of chrysopa septempunctata P450 gene.
Beneficial effect
The present invention is with chrysopa septempunctata larva for material, clone chrysopa septempunctata P450 gene cDNA sequence 4, utilize reverse transcriptase polymerase chain reaction (RT-PCR) that chrysopa septempunctata P450 gene has been studied, and the protein sequence feature of its coding, homology, evolutionary degree have been analyzed, for studying the expression of chrysopa septempunctata P450 gene further, regulation and control are laid a good foundation, produce to provide reference for P450 gene involved in insect Drug resistance, also provide new material for neuraopteron P450 gene functional research and Study on Evolution simultaneously.This serial genes also apply be applicable to the basic research such as natural enemy insect Drug resistance mechanism of action simultaneously, provides fundamental basis for cultivating Drug resistance natural enemy insect Dominant variety and utilization thereof.
The present invention is further illustrated will to quote non-limiting example below.
Detailed description of the invention
Example 1
The clone of chrysopa septempunctata P450 gene intermediate segment
1, total serum IgE is extracted: adopt the total serum IgE of Trizol method onestep extraction chrysopa septempunctata larva.
2, cDNA the first chain synthesis: carry out with reference to Beijing Quanshijin Biotechnology Co., Ltd cDNA the first chain synthetic agent box description, particularly as follows: take total serum IgE 3 μ l, oligo (dT)18Primer 1 μ l, 2 × TSReactionMix10 μ l, TranscriptRT/RIEnzymemix1 μ l, RNase-freeH2O adds to 20 μ l, and mixing, 42 DEG C of 30min, 85 DEG C of 5min, 4 DEG C of coolings ,-20 DEG C save backup.
3, the intermediate segment of pcr amplification chrysopa septempunctata P450 gene: design degenerate primer (primer is as follows) according to the conserved sequence of insecticide P450 gene, with cDNA for template, the intermediate segment of pcr amplification chrysopa septempunctata P450 gene；
S1:5 '-ACNYTYATGTTYGARGGHCAYGAYAC-3 '
A1:5 '-CDATRCARTTTCKKGRHCCDGC-3 '
Reaction reagent composition and reaction condition used by chain polymerization enzyme reaction (pcr amplification) are as follows: first mixed by following reagent,
Template cDNA1 μ l
Deoxydization nucleotide substrate dNTP4 μ l
Ex-TaqDNA polymerase 0.5 μ l
10 × Taq DNA polymerase buffer 5 μ l
Forward primer (10 μMs) 2 μ l
Reverse primer (10 μMs) 2 μ l
ddH2O35.5μl
Cumulative volume 50 μ l
Reaction condition is: first 94 DEG C of denaturations 3 minutes, then carries out circulation as follows: 94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 1 minute, carry out 35 circulations altogether, last 72 DEG C of extensions 10 minutes, 4 DEG C save backup.
4, PCR primer purification: utilizing agarose gel to reclaim test kit (purchased from Beijing Tian Gen biotechnology company) to PCR primer recovery, purification, concrete operation step is undertaken by test kit description.
5, PCR primer connects, converts and identifies: take the 5 above-mentioned PCR purified products of μ l, it is connected to pMD19-Tsimple carrier (Dalian treasured biological engineering company limited product), reaction system is such as shown in pMD19-Tsimple support agent box, then competent escherichia coli cell Top10 it is transformed into, in 37 DEG C of quiescent culture in coating and the LB flat board containing Amp, picking list bacterium colony is cultivated, and uses universal primer M13-47 and RV-M on cloning vehicle pMD19-Tsimple to carry out the bacterium solution PCR qualification positive colony containing P450 intermediate segment.
6, positive colony of step 5 gained is served Hai Sheng work Bioisystech Co., Ltd and carries out sequencing, gained sequence and GenBank database sequence are compared.
Example 2
The imidacloprid induction to chrysopa septempunctata P450
With imidacloprid, chrysopa septempunctata third-instar larvae is induced, and extract the chrysopa septempunctata larva RNA of 4h.8h.16h after induction, after dnase digestion, reverse transcription becomes cDNA, with real time fluorescent quantitative, the situation of change of different time P450mRNA is detected, using actin gene as internal reference.With 2-ΔΔCTMethod calculate P450 gene relative expression quantity, be set to 1 with the expression of matched group, after induction different time the change multiple (table 1) being expressed as relative to matched group.
Table 1. induction after different time be expressed as the change multiple relative to matched 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, present example proves that this gene is for studying the function of P450 gene family and the P450 genetic fragment that Study on Evolution offer is new, provide new genetic material and method for research P450 gene mechanism of action in natural enemy insect Drug resistance, have a good application prospect in integrated pest control field.
Claims (4)
1. the cDNA sequence of a chrysopa septempunctata P450 gene, it is characterised in that described in its nucleotide sequence such as SEQIDNO:1.
2. the aminoacid sequence that the cDNA sequence of chrysopa septempunctata P450 gene described in claim 1 is corresponding, it is characterised in that as described in SEQIDNO:5.
3. the cloning process of the cDNA of chrysopa septempunctata P450 gene described in claim 1, it is characterised in that comprise the following steps:
(1) extracting total serum IgE from chrysopa septempunctata larva, then reverse transcription carries out the synthesis of cDNA the first chain；
(2) degenerate primer is designed according to the conserved sequence of insecticide P450 gene family gene:
S1:5 '-ACNYTYATGTTYGARGGHCAYGAYAC-3 '
A1:5 '-CDATRCARTTTCKKGRHCCDGC-3 '
(3) with cDNA for template, the intermediate segment of pcr amplification chrysopa septempunctata P450 gene；
(4) purification step (3) gained PCR primer, agarose gel reclaims purpose fragment, take purified product and be connected on pMD19-Tsimple carrier, then competent escherichia coli cell Top10 is converted, 37 DEG C of slat chain conveyor, picking list bacterium colony is cultivated, and uses universal primer M13-47 and RV-M on cloning vehicle pMD19-Tsimple to carry out the bacterium solution PCR qualification positive colony containing P450 intermediate segment；
(5) positive colony of step 4 gained is served Hai Sheng work Bioisystech Co., Ltd and carries out sequencing, gained sequence and GenBank database sequence are compared.
4. the application in chrysopa septempunctata drug resistance is studied of the cDNA of chrysopa septempunctata P450 gene described in claim 1, it is characterized in that, with imidacloprid, chrysopa septempunctata third-instar larvae is induced, extract the chrysopa septempunctata total serum IgE of 4h, 8h, 16h after inducing, and remove genomic DNA with TakaraDNA enzyme, and set juice fluorescent quantitation primer according to this P450 gene, after using the method for fluorescent quantitation that imidacloprid is induced, the situation of transcribing of this P450 gene has been analyzed, using actin gene as internal reference.
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| 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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| 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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| 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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| GENBANK AB354061.1;NCBI;《GENBANK》;20080207 * |
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