Abstract: In this study, we produced germline transgenic silkworm that spin cocoons containing recombinant phyase in the fibroin layer. Using the piggyback-derived vector pPIGA3GFp and pBac{3×P3-EGFPaf}, we successfully developed stable germline transformation in the silkworm Bombyx mori L. We further constructed a piggyback-based transformation vector that carried a fusion cDNA of phyase with partial fibroin light chain (FibL) and red fluorescent protein (DsRed). The fusion cDNA was driven by FibL gene promoter. Silkworm eggs were injected with the vectors, producing worms displaying red fluorescence in their silkglands and cocoons. Southern blot and inverse-PCR results indicated that the insertion fragment was recombined with the chromosome of silkworm. RT-PCR analysis showed that the phytase gene was expressed especially in the posterior silkgland, and this expression pattern was the same as that of fibroin light chain gene. These results showed that we successfully got phy-transgene silkworm. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in the silkglands.

Key words: Bombyx mori, transgenic silkworm, piggyBac, transgene, green fluorescent protein (GFP), phytases