CN103966225A - Novel Chrysopa septempunctata vitellogenin gene and application thereof - Google Patents
Novel Chrysopa septempunctata vitellogenin gene and application thereof Download PDFInfo
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- CN103966225A CN103966225A CN201310027144.1A CN201310027144A CN103966225A CN 103966225 A CN103966225 A CN 103966225A CN 201310027144 A CN201310027144 A CN 201310027144A CN 103966225 A CN103966225 A CN 103966225A
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Abstract
The invention discloses a DNA sequence of a novel vitellogenin 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 vitellogenin gene of neuropteron, provide a novel gene for research on action mechanism of the vitellogenin gene in reproductive development regulation of Chrysopa septempunctata and functions of the vitellogenin gene. The DNA sequence and the protein sequence provide theoretical bases for mass rearing and effective utilization of Chrysopa septempunctata and have good application prospects in the field of integrated biological control of insects.
Description
Technical field
The invention discloses the protein sequence of the new gene DNA sequence of a kind of vitellogenin and coding thereof, belong to genetically engineered and protein engineering field.The present invention relates to clone and the application of a kind of chrysopa septempunctata (Chrysopa septempunctata) new gene of vitellogenin.
Background technology
Vitellogenin (Vitellogenin, Vg) is a kind of albumen being present in specifically in the sexually matured oviparity jenny of non-mammals blood, is the precursor of nearly all oviparous animal vitellin(Vt).Within 1969, in cherishing guppy skylight silkworm mori (Hyalophora cecropia), identify first this albumen, and by its called after Vg.Insect Vg derives from the mRNA coding of 6-7kb and the precursor protein of the 200KD that forms, after modified, become the natural vitellogenin being formed by several subunits, nutrition and the functional substance such as amino acid, fat, carbohydrate, VITAMIN, phosphorus, sulphur and trace element are provided for the embryo who is growing.
The research of Insect Vitellogenin is the focus of insect physiology and biochemical research in recent years.Up to the present, the Vg gene of existing various insects is cloned and identifies, mainly comprises the insects such as Dictyoptera, Hemiptera, lepidopteran and Hymenoptera, but in neuraopteron, does not also have Vg gene to be cloned.Chrysopa septempunctata is a kind of important Neuroptera natural enemy insect, can effectively control the agriculture and forestry injurious insects such as aphid, coccid, aleyrodid, is deeply subject to domestic and international biological and ecological methods to prevent plant disease, pests, and erosion worker's attention because its scope that takes food is wide, heavy, it is wide to distribute, quantity is many.Chrysopa septempunctata vitellogenin is closely related with this natural enemy insect nutritional-physiological and reproductive characteristic, therefore, and the significant and using value of research chrysopa septempunctata vitellogenin.First the present invention finds, has cloned vitellogenin gene in Neuroptera natural enemy insect chrysopa septempunctata, and the aminoacid sequence of its coding, homology and evolutionary degree are analyzed, for Vg gene function and macromole organic evolution research provides new gene, for this Important Natural Enemy insect breeds in a large number and effectively utilizes theoretical basis is provided.
Summary of the invention
The present invention includes following content:
1. the invention provides a kind of polynucleotide molecule SEQ ID N0.1 of the chrysopa septempunctata vitellogenin of encoding, described polynucleotide molecule comprises the nucleotide sequence being consistent with SEQ ID N0.1 and partly conforms to and merge the segment with identity function with the nucleotide sequence shown in SEQ ID N0.1.
2. the invention provides a kind of protein, described protein comprise with SEQ ID No.2 aminoacid sequence substantially and the aminoacid sequence that is consistent of part.
3. a method for vitellogenin gene application, comprises by monitoring vitellogenin gene expression amount, studies vitellogenin content, thus research vitellogenin on insect nutrition and reproduction as the method for egg laying amount impact.
Place use " substantially conforming to " this term that relates to aminoacid sequence in literary composition is to want to comprise the variation on those aminoacid sequences that can not cause the reduction of vitellogenin matter biological activity. these variations may comprise conservative amino acid replacement etc.
Beneficial effect
1. the invention discloses a kind of coding chrysopa septempunctata vitellogenin gene (csvg) and coded protein thereof.This gene is the reported first in chrysopa septempunctata (Chrysopa septempunctata), for further expression, the regulation and control of research chrysopa septempunctata vitellogenin gene are laid a good foundation; For research vitellogenin gene participates in chrysopa septempunctata reproductive development, breeds in a large number and effectively utilize theoretical direction is provided for this Important Natural Enemy insect; This gene function and coded product result of study thereof also can be applicable to the research of other natural enemy insect nutrition and reproduction aspect simultaneously, for the utilization of natural enemy insect in the integrated control of biological control and insect provided fundamental basis.
2. the invention provides chrysopa septempunctata vitellogenin gene and coded protein thereof, this vitellogenin gene is the first vitellogenin gene being cloned in neuraopteron, filled up the blank of neuraopteron vitellogenin gene clone, also for research macromole, evolved useful material is provided.
The present invention is further illustrated will to quote non-limiting example below.
Embodiment
Example 1
The clone of chrysopa septempunctata vitellogenin Vg gene cDNA, is undertaken by following steps: from chrysopa septempunctata emergence female insect, extract total RNA; Then by the reverse transcription of clontech3 '-RACE test kit operation steps, carry out the synthetic of cDNA the first chain; With degenerated primer S1 and test kit primer amplification chrysopa septempunctata vitellogenin gene 3 '-terminal fragment; The above step gained of purifying PCR product, sepharose reclaims object fragment, gets purified product and is connected on pMD19-T simple carrier, then transforms competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, carry out PCR with M13 universal primer to positive colony and detect evaluation; According to the result of cloning and sequencing, design 5 '-RACE Auele Specific Primer, carries out the amplification of 5 '-end sequence according to clontech5 '-RACE test kit operation steps; Purification step gained PCR product, sepharose reclaims object fragment, gets purified product and is connected on pMD19-T simple carrier, then transforms competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, carry out PCR with M13 universal primer to positive colony and detect evaluation; According to the positive colony sequencing result of above step gained, by measured gene order splicing, obtain chrysopa septempunctata vitellogenin full length gene sequence.Chrysopa septempunctata vitellogenin gene of the present invention (csvg) DNA sequence dna SEQ ID NO.1, for the first vitellogenin gene being cloned in neuraopteron, the sequence of complete of this gene, filled up the blank of neuraopteron vitellogenin gene clone, also for research macromole, evolved useful material is provided.The result of BLAST proves: this vitellogenin gene is a new gene.Chrysopa septempunctata vitellogenin gene provided by the invention and coded product thereof can be applicable to the research of insect nutrition and reproduction aspect, and this invention simultaneously also can be applied to natural enemy insect and expand in a large number numerous and batch production production.
Example 2
The DNA sequence dna SEQID NO:1 of the chrysopa septempunctata of above-mentioned acquisition (Chrysopa septempunctata) vitellogenin gene csvg, the protein of its coding contains 1810 amino acid, and its sequence is shown in SEQ ID NO:2.In database, compare, in the protein of vitellogenin gene csvg coding and database, the similarity of Lethocerus deyrollei, Nilaparvata lugens, Anopheles gambiae is respectively 49%, 42%, 40%, with Pimpla nipponica, Tribolium castaneum all under 40%.
Example 3
The application of the present invention as described in claim 1 and 2, utilize RNAi the Study of Interference technical result to show, disturb after chrysopa septempunctata (Chrysopa septempunctata) vitellogenin gene, the expression amount (table 1) of vitellogenin, the total amount of laying eggs and egg hatching rate (table 2) are all subject to larger impact, so this invention is significant to studying the physiological function of vitellogenin gene in chrysopa septempunctata.
Table 1, the impact on the situation of transcribing after RNAi interference Vg
Choose 4 sections of Vg as the object fragment of disturbing, by the method for microinjection, the adult sprouting wings latter 4 days is injected, to inject dsGFP in contrast, by the method for quantitative fluorescent PCR, the 5th day Vg mRNA level after disturbing detected.
Impact on egg hatching rate after table 2RNAi disturbs
| Interference region segment number | Egg hatching rate (%) after disturbing |
| V1 | 42.50％ |
| V2 | 43.33％ |
| V3 | 58.33％ |
| V4 | 36.67％ |
| GFP | 85.83％ |
RNAi measures egg hatching rate after disturbing, and after injection Vg dsRNA, the hatching rate of ovum is starkly lower than injection GFPdsRNA.
In sum, the invention discloses a kind of chrysopa septempunctata vitellogenin Vg gene (csvg) 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 can be applied to study natural enemy insect nutritional-physiological and reproductive characteristic, for this Important Natural Enemy insect breeds in a large number and effectively utilizes theoretical direction is provided, filled up the blank of neuraopteron vitellogenin gene clone.Also for research macromole, evolve useful material is provided.The recombinant protein of this gene and expression thereof is aspect natural enemy insect nutrition and reproductive study, have a good application prospect in the integrated control field of biological control and insect simultaneously.
Claims (4)
1. a cDNA of chrysopa septempunctata vitellogenin gene Vg, is characterized in that, its nucleotide sequence is as described in SEQ IDNO.1.
2. the protein of polynucleotide encoding as claimed in claim 1, its aminoacid sequence is as shown in SEQ ID No.2.
3. a chrysopa septempunctata vitellogenin gene cDNA clone method as claimed in claim 1, comprises the following steps:
(1) from chrysopa septempunctata, extract total RNA, then the synthetic cDNA of reverse transcription;
(2) according to holometabolan Vg gene conserved sequence design degenerated primer;
(3) take the cDNA that step (1) obtains is template, with primer step (2) Suo Shu, carries out pcr amplification, obtains amplified fragments;
(4) according to the result of 3 ' end sequence, design 5 '-RACE Auele Specific Primer;
(5) by the reverse transcription of clontech5 '-RACE test kit operation steps, carry out the synthetic of cDNA;
(6) take cDNA as template, 5 '-end sequence of pcr amplification chrysopa septempunctata Vg gene;
(7) by 3 ' and 5 ' end fragment splicing of described gene, obtain cDNA claimed in claim 1.
4. the application as described in claim 1 and 2, it is characterized in that: in the material of application, comprise polynucleotide sequence claimed in claim 1 or protein claimed in claim 2, for example, utilize the physiological function of RNAI the Study of Interference technical study vitellogenin gene.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762380A (en) * | 2015-03-27 | 2015-07-08 | 中国农业科学院植物保护研究所 | Application of vitellogenin gene to detection of habitat fitness of insects |
| CN106011144A (en) * | 2016-05-24 | 2016-10-12 | 江苏省农业科学院 | Spodoptera exigua vitellogenin (SEVg) as well as specific peptide chain, carrier, strain and application thereof |
| CN108251430A (en) * | 2017-05-05 | 2018-07-06 | 东北林业大学 | A kind of harmonia axyridia vitellogenin novel gene cloning and its method of expression |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN102851297A (en) * | 2012-07-23 | 2013-01-02 | 中国农业科学院植物保护研究所 | Myzuspersicae hunchback gene cDNA and application thereof |
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2013
- 2013-01-25 CN CN201310027144.1A patent/CN103966225A/en active Pending
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 |
| CN102851297A (en) * | 2012-07-23 | 2013-01-02 | 中国农业科学院植物保护研究所 | Myzuspersicae hunchback gene cDNA and application thereof |
Non-Patent Citations (1)
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| 陶淑霞: "大草蛉卵黄蛋白及卵黄发生的研究", 《中国博士学位论文全文数据库.农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762380A (en) * | 2015-03-27 | 2015-07-08 | 中国农业科学院植物保护研究所 | Application of vitellogenin gene to detection of habitat fitness of insects |
| CN106011144A (en) * | 2016-05-24 | 2016-10-12 | 江苏省农业科学院 | Spodoptera exigua vitellogenin (SEVg) as well as specific peptide chain, carrier, strain and application thereof |
| CN108251430A (en) * | 2017-05-05 | 2018-07-06 | 东北林业大学 | A kind of harmonia axyridia vitellogenin novel gene cloning and its method of expression |
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Application publication date: 20140806 |