| 家蚕胞质肌动蛋白基因启动子的克隆及piggyBac转座子表达载体的构建 |
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| 引用本文： | 李维,王宇,张世英,朱玲巧,黄敏,刘辉芬.家蚕胞质肌动蛋白基因启动子的克隆及piggyBac转座子表达载体的构建[J].农业生物技术学报,2003,11(2):173-178. |
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| 作者姓名： | 李维 王宇 张世英 朱玲巧 黄敏 刘辉芬 |
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| 作者单位： | 1. 四川师范大学化学与生命科学学院,成都,610066
2. 四川天友生物科技股份有限公司,成都,610078 |
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| 基金项目： | 四川天友发展总公司资助项目.A3细胞质肌动蛋白基因启动子序列 GenBank accession No.AF422795. |
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| 摘 要： | 摘要: 利用PCR方法从家蚕（Bombyx mori）总DNA中克隆到细胞质肌动蛋白基因（cytoplasmic actin）启动子片段，序列分析表明，该片段中含有典型的组成型表达的细胞质肌动蛋白基因A3（BmA3）调控序列：SRE元件（serum response element）和ActE1元件(active element 1)。该序列的核苷酸序列与来自欧洲的一个家蚕品种的序列同源性为94%。通过多次重组，将A3启动子片段(不含信号肽序列)与转座子 piggyBac的转座酶编码区融合构建成辅助质粒；将其(含信号肽序列和部分编码区)与报告基因多管水母绿色荧光蛋白基因gfp融合，插入到人工合成的转座子piggyBac两反向重复末端序列之间，进而构建成基于转座子piggyBac的转基因载体。研究中构建的转座子转基因载体仅6.4 kb，并在两反向重复末端序列之间设计了1个多克隆位点区，便于目的基因的插入。
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| 关 键 词： | 关键词:家蚕 胞质肌动蛋白基因启动子 转座子piggyBac 表达载体 |
| 修稿时间： | 2002年3月1日 |
Cloning of Bombyx mori Cytoplasimic Actin Gene Promoter and Construction of piggyBac Transposon Expression Vector |
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| 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. |
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