转植酸酶基因家蚕的制作及表达检测

家蚕Bombyx mori丝腺具有高效合成蛋白质的特性，开发在丝腺特异表达外源蛋白质的生物反应器具有重要的意义。本研究利用piggyBac来源的两种载体pPIGA3GFP和pBac{3×P3-EGFPaf}，建立了稳定的家蚕转基因技术体系； 然后，利用一株黑曲霉来源的植酸酶基因，构建了在家蚕后部丝腺特异表达的融合表达载体pBac 3×P3-EGFP+ FibLphyADsRed]，注射蚕卵后，在53个G1蛾区中检测到3个有荧光蚕的蛾区。经Southern blot和反向PCR验证，转基因表达盒整合到家蚕染色体上。RT-PCR结果显示，植酸酶基因特异性地在后部丝腺表达，其表达模式与家蚕轻链丝素基因一致。结果表明我们成功获得了在后部丝腺特异表达植酸酶融合蛋白的转基因蚕，这为进一步开发家蚕生物反应器，利用转基因蚕生产各种重组蛋白具有积极的促进作用。

Transgenic breeding of silkworms for the production of recombinant phytases

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.