Molecular Design Of Fibrion Gene And Improve Silk Yielding Using UAS/Gal4System In Transgenic Silkwoorm

The silkworm Bombyx mori belonging to Lepidoptera Saturniidae, can be raised ona large-scale, and its genetic background is clear. Its silk glands can synthesize andsecrete silk protein. To improve the performance of silk fibre by molecular designingand genetic modification of the fibrion gene of silkworm in molecular level, a1.2kb（between exon5and exon7）and a0.5kb （between exon7and its following partial） of thesequence located at fibroin light chain （fib-L） gene as homologous arms, a greenfluorescent protein gene （gfp） and artificialaynthesis silkworm antibiotic peptide genewas flanked by fib-LL and fib-LR, the DsRed gene with ie-1promoter was clonedfollowed by the right homologous arm as the negative chosing factor, to generate agene-targeted vector: pSK-Fib.L-L-GFP-cec-Fib.L-R-IE-DsRed-PolyA, in which the gfpgene and fib-L gene were in same open reading frame. BmN cells with greenfluorescence but without red fluorescence could be found after being transfected withthe vector, indicating the vector was successfully constructed. The targeted vector wasintroduced into the eggs of silkworm using sperm-mediated gene transfer, somesilkworms with green fluorescent cocoons were screened out, PCR results confirmedthat the silkworm was transformant. In the posterior silk glands of the G6generationtransformation silkworms, a specific band representing the fusion protein of GFP andFib-L could be detected by Western blotting with an antibody against GFP; antibacterialexperiment was done with the silk fibre spanned by transformation silkworms showedless clonies than the control;the silk fibre spanned by transformation silkworms emit fluorescent under theexcitation of ultraviolet light. These results indicated that the gfp gene could beintegrated into the fib-L gene locus of silkworm genome by gene targeting and fusion protein of Fib-L and GFP was expressed successfully.In addition, the transgenic silkworm had a high silk yield and converting food intosilk based on a binary Gal4/UAS system from the Chinese Academy of Sciences inShanghai. But it is difficult to apply this method in sericulture because the Ras1^CAwasexpressed in non-diapause silkworm. So, we designed the Gal4transgenic silkwormhybridized with stock JingSong and screened out some silkworms withred fluorescent eyes, then the next generation transgenic silkworm was constantlybackcross with JingSong to generate G6-Gal4diapause JingSong silkworm.we also designed the UAS transgenic silkworm hybridized with stock HaoYue andscreened out some silkworms with green fluorescent eyes, then the next generationtransgenic silkworm was constantly backcross with HaoYue to generate G6-UASdiapause HaoYue silkworm.Then the transgenic Gal4JingSong silkworm ishybridized with the transgenic UAS HaoYue silkworm to get the Ras1^CAspecifically expressed. The results showed Ras1^CAoverexpression improved posteriorsilk gland weight and silk yield11%、1.3%, confirming substantial increment of silkproduction capacity in transgenic silkworm; But food consumption only increased0.57%, which has potential in sericulture. As measured by quantitative real-timePCR,Ras1^CAmRNA level in the posterior silk gland of Fil-Gal4/UAS-Ras1^CAsilkwormwas4.08fold higher than in controls. These results ensured Ras1^CAis successfullyoverexpressed in the posterior silk gland.