利用同源重组改变家蚕丝心蛋白重链基因  

Altering Fibroin Heavy Chain Gene of Silkworm Bombyx mori by Homologous Recombination

作　　者：赵昀[1] 陈秀[2] 彭卫平 董良[1] 黄君霆[2] 陆长德[1] 

机构地区：[1]中国科学院上海生命科学院生物化学与细胞生物学研究所,上海200031 [2]中国农业科学院蚕业研究所,镇江212018

出　　处：《生物化学与生物物理学报》2001年第1期112-116,共5页

基　　金：国家高技术"86 3"计划资助项目!No .86 3 10 3 13 0 2 0 6&&

摘　　要：在家蚕丝心蛋白重链基因 5′和 3′端序列之间插入以绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因(gfp)与人工合成丝心蛋白样基因的融合基因 ,利用电穿孔方法导入蚕卵中。卵孵化、发育和结茧后 ,用紫外灯检查 ,在约 5 40 0个茧中有 73个“亮茧” ,茧蛋白在ELISA反应中可以与GFP的多克隆抗体反应。“亮茧”对应的蚕蛾进行交配、制种。对其后代进行了基因鉴定 ,Southern杂交的结果表明 ,gfp基因和人工合成丝心蛋白样基因都存在于家蚕基因组DNA中且发生了预期的同源重组事件。上述结果说明“亮茧”这一表型能用于筛选转基因蚕 。A gene unit, which encoded fibroin like peptide, was synthesized and constructed. The unit was multimerized to about 2 400 bp using Bam HI and Bgl II at each end of the unit, then was fused with gfp reporter gene. The fusion gene, flanked by the 5′ and 3′ sequence of the fibroin heavy chain gene of silkworm Bombyx mori , was transferred into the eggs of silkworm by electroporation. After the silkworms developed and spinned silk, 73 out of about 5 400 cocoons were brighter than normal ones under UV light. The protein extracted from the brighter cocoon could react with the GFP polyclonal antibodies. Genomic DNA from these silkworms and their progenies were analyzed. The integration of gfp gene into genomic DNA of silkworm and the occurrence of expected homologous recombination event had been proved by Southern hybridization. It was shown that gfp fibroin like fusion gene had integrated into the genomic DNA of silkworm by homologous recombination and the phenotype of “brighter cocoon” could be used to select transgenic silkworms.

关 键 词：家蚕 绿色荧光蛋白 人工合成丝心蛋白样基因 转基因 同源重组 

分 类 号：S881[农业科学—特种经济动物饲养]