家蚕转基因技术中若干因素对转基因效率的影响

Effect of several factors on the transformation efficiency in the transgenic technology of silkworm,Bombyx mori

建立高效、稳定的家蚕Bombyx mori转基因技术对于推进家蚕功能基因组研究,解决蚕丝产业重大问题以及向非绢丝产业拓展等具有重要意义。本文在已建立的基于piggyBac的家蚕转基因技术基础上,探索了多个影响转基因效率的因素。结果显示:以家蚕品种大造(P50)为供试材料、pBac[GOI]为供体质粒、pHA4PIG为辅助质粒,以眼睛和神经组织特异启动子3×p3启动的红色荧光蛋白基因DsRed为报告基因,在蚕卵产下后2～3h进行注射,综合效果最佳,孵化率和转化率分别达到62.7%和34.8%;荧光筛选的最佳时期在胚胎发育第5到第8天;在2000～8000bp之间时,外源片段的长度对转化率并无太大影响。本研究建立的技术体系,有望为家蚕功能基因研究、品种分子改良和家蚕生物反应器的开发奠定基础,并为其他鳞翅目昆虫转基因技术的建立提供参考。

Efficient transgenic technology of silkworm is the key to promote the tunctional research of silkworm genome, and to solve the major problems in the sericuhure industry. On the basis of the established transgenic technology in silkworm, we investigated the effect of several factors on transformation efficiency. The results showed that when taking P50 as a recipient strain, pBac[ GOI] as a donor plasmid and pHA4PIG as a helper plasmid, and red fluorescent protein gene, DsRed, driven by an eye-specific promoter, 3 × p3, as a reporter gene, microinjection into the eggs during 2 - 3 h after oviposition increased the transformation efficiency. The hatching rate of injected eggs and the transformation rate were 62.7% and 34.8% , respectively. The best time for fluorescent screening was the 5th to 8th day of embryo. The results also showed that when varied from 2 000 to 8 000 bp the length of inserts had no significant effect on transformation rate. The results will set a model for transgensis of other lepidopteran insects and will contribute to the functional genomics study of the silkworm and the development of molecular bioreactors involved.