Silkworm ecdysone oxidase EO gene

A technology of ecdysone and oxidase, which is applied in the fields of oxidoreductase, genetic engineering, plant gene improvement, etc., and can solve the problems of few researches on enzyme genes and researches that have not been reported yet.

Inactive Publication Date: 2012-12-12
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene encoding the enzyme has been poorly studied
Bombyx mori is an important economic insect, and it is also a model representative of Lepidoptera insects, but the research on the ecdysone oxidase gene identified as silkworm has not been reported yet

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Bioinformatics identification of ecdysone oxidase gene in silkworm:

[0072] Firstly, the Lepidoptera insect Spodoptera littoralis ( Spodoptera littoralis ) in ecdysone oxidase gene (SlEO accession number is AY035784) was used as query sequence, and Blast program was used to search for homologous genes in silkworm expression database (EST) and genome database. Through bioinformatics analysis, a gene with the highest matching degree was finally obtained. The protein sequence encoded by this gene is 56% similar to S1EO of Spodoptera littoralis, and the protein has a conserved ecdysone binding site (Leu 96 , Met 346 , Ala 446 , Thr 533 and Trp 536 ). Therefore, this gene is a candidate gene of silkworm ecdysone oxidase.

Embodiment 2

[0074] Cloning of the silkworm ecdysone oxidase gene:

[0075]Design specific primers based on the nucleotide sequence of the candidate gene, the upstream is: 5' GAATTC ATGGTTTGCGGGTTG 3', Downstream: 5' GCGGCCGC TCATGCGACATTGAC 3'; the underlined part is the restriction site, wherein the restriction site of the upstream primer is SnaB I, and the restriction site of the downstream primer is Not I. Extract the whole silkworm RNA of the 5th instar and 3 days of the silkworm, reverse it into cDNA with MLV reverse transcriptase (promega company), then use it as a template, and use the designed specific primers to amplify to obtain the target band, After the PCR product was recovered, it was ligated with the PMD19-T vector and transformed into Escherichia coli DH5a competent cells. After the positive clones were obtained, they were sent to Shanghai Sangon Bioengineering Company for sequencing. The sequencing results showed that the amplified sequence was consistent with the expe...

Embodiment 3

[0077] Eukaryotic expression and enzyme activity determination of silkworm ecdysone oxidase gene:

[0078] The T clone containing the target fragment and pPIC9K empty plasmid were double-digested with SnaB I and Not I respectively, and the target fragments were recovered respectively, connected with solution I ligase and transformed into Escherichia coli DH5a competent cells, and obtained by screening pPIC9K- BmEO was cloned, and then the cultured bacterial liquid was sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. The sequencing result was consistent with the sequence measured for the first time, and the correct clone was obtained; the plasmid pPIC9K-EO was extracted to be linearized and removed. Phosphorylation treatment, and then transform into Pichia pastoris competent by electric shock transformation method, then spread on MD plate and culture for two days, pick the first single colony, and then spot it on the YPD plate containing G418 for positive The cl...

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PUM

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Abstract

The invention discloses an ecdysone oxidase (EO) gene which is from silkworm and has an activity of oxidizing in-vivo ecdysone of an insect, and the gene is obtained by cloning in the silkworm by using the bioinformatics and bioengineering method. An expression product of the EO gene can effectively exert catalytic effect on the ecdysone to reduce the activity of the ecdysone, thereby effectivelyreducing the titer of the in vivo ecdysone of the insect. The EO gene is introduced to in vivo of the silkworm by using the transgenic technology to be expressed in high level in the pupal stage; therefore, the titer of the ecdysone is adjusted; furthermore, the development of the silkworm can be artificially controlled.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an ecdysone oxidase derived from silkworm and having the activity of oxidizing ecdysone in insects EO Gene. Background technique [0002] The metamorphosis of insects is mainly controlled by the synergistic control of molting hormone (MH) synthesized and secreted by the prothymus and juvenile hormone (JH) synthesized and secreted by the lateral pharynx. The main function of ecdysteroids is to induce the regular molt of insects at different developmental stages, thereby determining the changes in the size and shape of insect larvae. For complete metamorphosis insects, in the last instar of their larvae, the larvae stop secreting Juvenile hormone, under the action of high concentration of ecdysone, the larva completes the transition to pupa and begins the metamorphosis of insects. It can be seen from this that the titer of ecdysone changes regularly at different developmental stages....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/10C12N15/81C12N9/02
Inventor 张泽孙伟沈以红向仲怀
Owner SOUTHWEST UNIV
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