家蚕精子介导转基因技术体系的优化  

The Improvement on the Sperm Mediated Gene Transfer System for the Silkworm,Bombyx mori

作　　者：张业顺[1] 夏定国[2] 张国政[1] 唐顺明[2] 沈兴家[2] 赵浩勤[2] 郭锡杰[1] 

机构地区：[1]江苏科技大学,镇江212003 [2]中国农业科学院蚕业研究所农业部家蚕生物技术重点开放实验室,镇江212018

出　　处：《蚕业科学》2007年第2期201-206,共6页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划"973"项目(编号2005CB-121000);江苏省自然科学基金项目(编号BK2004206)

摘　　要：为了构建高效的家蚕转基因技术,对采用piggyBac转座载体的家蚕精子介导转基因的主要技术参数进行优化,结果表明外源DNA的稀释剂和注射方式对导入效率影响较大,G0代未整合的外源DNA在5龄期前基本被降解破坏。推荐的家蚕精子介导基因转移技术方案为:处女蛾交尾囊注射6～8μL以0.1×TE稀释的2 g/Lpig-gyBac转座子载体DNA后正常交配产卵,根据G0代5龄期及蛾期PCR检测标记基因为阳性的蛾区自交,G1代阳性个体自交以获得纯合后代。To improve the ratio of sperm mediated gene transfer for the silkworm, Bombyx mori, using piggyBac transposon vector, the influence of four factors, i. e., the foreign DNA concentration, the diluent agents for the foreign DNA, the injection methods and the different plasmids were investigated and optimized. The results showed that both the injection methods and the diluent agents played important roles in the foreign DNA transfer efficiency for the silkworm. The introduced foreign DNAs,which didn＇t integrate into the silkworm gehome, were destroyed mostly before the fifth instar larval stage in Go generation. Thus the optimal conditions for the sperm mediated gene transfer system were suggested as follows; the concentration of piggyBac transposon vector was 2 g/L diluted with 0. 1× TE and 6 -8μL solution were injected into a virgin moth＇s bursa copulatrix. Then, the treated moths mated with normal males and laid eggs naturely. The GFP positive silkworms were identified by PCR method in the 5th instar and the moth stage in Go generation and reared for successive generation （G1）, The transgenic homozygous silkworms were obtained by sib mating of the positive G1 adults from the positive moth batch.

关 键 词：家蚕 精子介导转基因 交尾囊注射 转座子载体DNA PCR检测 

分 类 号：Q78[生物学—分子生物学]