家蚕精子介导转基因技术体系的优化

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。

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

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 genome,were destroyed mostly before the fifth instar larval stage in G0 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 6~8 μL 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 G0 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.