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Compound type piggyBac recombinant vector as well as preparation method and application of compound type piggyBac recombinant vector

A recombinant vector and composite technology, which can be used in recombinant DNA technology, introduction of foreign genetic material using vectors, animal husbandry, etc., and can solve problems such as lepidopteran species that have not yet been applied

Inactive Publication Date: 2015-06-03
SOUTHWEST UNIVERSITY
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Problems solved by technology

However, this strategy to achieve stable transgene integration by deleting piggyBac flanking sequences has not been applied to silkworms and other Lepidoptera species so far.

Method used

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  • Compound type piggyBac recombinant vector as well as preparation method and application of compound type piggyBac recombinant vector
  • Compound type piggyBac recombinant vector as well as preparation method and application of compound type piggyBac recombinant vector
  • Compound type piggyBac recombinant vector as well as preparation method and application of compound type piggyBac recombinant vector

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Embodiment Construction

[0054] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. For the experimental methods not specified in the examples, the conventional conditions are generally followed, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.), or the conditions suggested by the manufacturer.

[0055] The practical diapause silkworm strain 871 (white cocoon) of the Chinese lineage is preserved in the laboratory of the inventor. Vector pSLfa1180fa(Carsten Horn.et al2000), pBac{3×P3-DsRedaf}(AiChun Zhao.et al2010), pSL{3×P3-EGFP-SV40}(Dingpei Long.et al2012), pHA3PIG(Toshiki Tamura.et al2000), pMLS104 (Mark L. Siegal. et al1996) and pBac{3×P3-DsRedaf}-R3 (AiChun Zhao. et al2010) were all preserved by the inventor's laboratory.

[0056] 1. Construction of composite piggyBac recombinant vector

[0057] Composite piggyBac recombinant vector pBac{R1-3×P3-DsRed-SV40-L...

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Abstract

The invention discloses a compound type piggyBac recombinant vector as well as a preparation method and an application of the compound type piggyBac recombinant vector. According to the recombinant vector, a target gene expression frame, selection marker gene expression frames I and II, piggyBac transposase expression frame regulated by a heat shock promoter and truncated type piggyBac transposon arms L2 and R2 are inserted between wild type piggyBac transposon arms R1 and L1, integration of target gene, selection marker gene, piggyBac transposase expression frame sequence in silkworm genome can be conveniently mediated, complete deletion of all of transposon sequences and selection marker gene sequences in progeny transgenosis silkworm genome can be realized through establishing and screening a transgenosis silkworm line with high target gene expression and then inducing piggyBac to express in the transgenosis silkworm by heat shock treatment, replacement of other exogenous gene and the target gene can be conveniently realized through box type exchange reaction mediated by phiC31 integrase by surplus target gene expression frame anchored by attP locus, and then fixed point integration of other exogenous genes in the genome locus is further realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a piggyBac recombination vector and a preparation method thereof, and also relates to the application of the recombination vector in preparing a stable, replaceable and high-efficiency transgenic silkworm expression system. Background technique [0002] Since the first report of insect genetic transformation mediated by P factor in Drosophila melanogaster in 1982, the transposon-based genetic transformation system has been widely used in insects and other invertebrates over the past 30 years. and applied research. Among them, the most widely used and mature transposon system is the piggyBac transposon derived from Trichoplusia ni. piggyBac is a typical type II transposon system, and its internal region sequence encodes a transposase with 594 amino acids. The transposase can act on the terminal inverted repeat (Terminal inverted repeat, TIR) at both ends of the piggyBac transposon and its...

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/85A01K67/04
Inventor 赵爱春龙定沛陆威健鲁成向仲怀
Owner SOUTHWEST UNIVERSITY
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