Studies On The Fusion Expression Of Gfp And Fib-l In Transgenic Silkworm Based On Gene Targeting

The silkworm (Bombyx mori) belonging to Lepidoptera Saturniidae, can be raised on a large-scale, and its genetic background is more clearly. Their silk glands have the powerful ability to synthesis and secrete silk protein. In order to improve the silkworm in molecular level, the study using two fragments from fibroin light chain gene (fib-L) as the homogenous arms, the green fluorescent protein(gfp) gene with no promoter was cloned into the gap between the two homologous arms, then the gene-targeted vector pSK-FibL-L-GFP-FibL-R in which gfp gene fused with the fib-L gene was obtained. The vector was transfected into the BmN cells. The positive result in BmN cells indicated the vector was successfully constructed. The targeting vector was then introduced into the eggs of silkworm, and the transgenic silkworms were verified by PCR and RT-PCR after being screened for the gfp gene. Western blotting analysis using an antibody against GFP demonstrated a specific band, about 70 kD, in the silk glands of transgenic silkworms. These results showed that the heterologous gene could be introduced at the desired locus of the silkworm genome by gene targeting and expressed successfully.In addition, we used two fragments (one include the sequence between exon5 and exon7, about 1.2kb; another include the sequence of exon7 and its following partial, about 0.5kb)from fib-L amplified from genome of silkworm and cloned into PSK as homologous arms; then the gfp gene and artificialaynthesis silkworm antibiotic peptide gene were inserted into the gap between the two homologous arms, making gfp-cec genes were fused with Fib-L; the DsRed gene with ie-1 promoter was cloned followed by the right homologous arm as the negative chosing factor. Then the targeted vector pSK-Fib.L-L-GFP-cec-Fib.L-R-IE-DsRed-PolyA, not similar with the vector mentioned above, was obstained. The vector was transfected into the BmN cells and the positive result indicated the vector was successfully constructed. The vector was also introduced into the eggs of silkworm. After identifying of the flucrescent silkworm, the transgenic silkworm, which can spit flucrescent and antibiosis silk, was obtained. This research provides a new approch to improve silkworm in molecular level.During our study, the suppressor of cytokine signaling gene (SOCS -2A) in JAK/STAT pathway was obstained by RT-PCR. The sequencing results indicted the 792 bp fragment consisted of 4 exons, encoded 263 amino acids and the molecular was 28.8 kD. Structural analysis of BmSOCS-2A showed it conteined 5αhelix structures, 1 typical SH2 domain and 1 SOCS domain. The gene chip analysis indicted the expression level of BmSOCS-2 was higher in silkworm ganad than in other organs. The evolution analysis illustrated SOCS were clustered by different types which may produce SOCS gene families before speciation of insect.