家蚕转基因载体pBacA3EG的构建及其表达

Construction and expression of the transgenic vector pBacA3EG in the silkworm Bombyx mori

以家蚕Bombyxmori肌动蛋白A3(actin3)启动子、增强性绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)基因及SV40的多聚腺苷酸识别序列为元件,经多次克隆,将其插入到piggyBac转座载体中。经PCR、酶切鉴定及测序表明各元件已按正确的方式插入到piggyBac载体中。将构建好的piggyBac表达载体显微注射到胚盘形成前期的蚕卵中,在胚胎早期发育的第3天,通过体视荧光显微镜检测到蚕卵内发出较强的绿色荧光。结果表明该载体构建正确且能在蚕卵中进行表达。家蚕转基因载体的体外瞬时表达不但是成功进行家蚕转基因所必需的第一步,而且其自身也可以应用于基因的功能研究,为家蚕后基因组研究奠定了基础。

Using three basic elements, i. e., actin 3 promoter of Bombyx mori, enhanced green fluorescent protein （EGFP） gene and the identified sequence of polyadenylation acid in SV40, the piggyBac transposon vector.pBacA3EG was constructed. This vector was further confirmed by PCR, enzyme digestion and sequencing analysis, and the results showed that all elements had been inserted into the piggyBac vector correctly. Stronger green fluorescent was observed under the stereoscopic fluorescence microscope three days after the reconstructed vector was injected into the preblastodermic eggs. The results showed that the vector was constructed correctly and expressed in silkworm eggs. The transient expression of silkworm transgenic vector is not only the first step necessary for silkworm transgene, but also can be applied in gene function analysis, which establishes the foundation for functional studies of silkworm genome.