Cloning of Bombyx mori Cytoplasimic Actin Gene Promoter and Construction of piggyBac Transposon Expression Vector
Li Wei1 Wang Yu1 Zhang Shiying1 Zhu Lingqiao1 Huang Min1 Liu Huifen2
(1.Department of Chemistry and Life Sciences, Sichuan Normal University, Chengdu 610066, China; 2.Sichuan Tianyou Biology Engineering Stock Company, Chengdu 610078,China)
Abstract：Abstract: In order to construct silkworm (Bombyx mori ) transposon expression vector as a tool in research on the stable transgenesis of B. mori , the cytoplasmic actin A3 gene promoter ( named A3 ) from silkworm genome DNA was amplified by PCR and sequenced, the results showed that two typical constructive expression regulated elements: SRE (serum response element) and ActE1(active element 1) were included. Its sequence similarity compared with cytoplasmic actin A3 gene from European B. mori strain homology is 94%. Promoter A3 was fused with recombined transposase gene. And a nonautonomous helper plasmid was obtained. Promoter A3 fragment contained signal sequence and partial coding region was fused with green fluorescent protein (gfp ) gene, The fused fragment with A3 promoter and gfp was then inserted into a cloning site between two synthetic transposon piggyBac terminal inverted repeats in a plasmid pXLBac. A silkworm transposon piggyBac vector was constructed successfully. The transposon expression vector consists of the piggyBac inverted terminal repeats, between which the recombined gfp gene derived by A3 promoter was flanked. Both transposon vector and helper plasmid were co-injected into the embryos of Bombyx mori , the constructive expression transposase in the helper palsmid might function and make the DNA fragment between the piggyBac inverted terminal repeats of transposon vector inserted into the host genome chromosome. This bipartite vector-helper system had advantages as followed: the transposon vector was only 6 400 bp and might facilitate further DNA manipulation; between the terminal inverted repeats, the transposon vector was designed with a multiply clone site, which was more convenient to be inserted exogenous gene of interest and construct expression plasmid. Nowadays, an interesting gene was inserted into the transposon vector and was tested for gene transfer vector function as a part of bipartite vector-helper system in Bombyx mori.
