The Optimization Of The Transgenic Elements And Their Application For The Gene Functional Research In Silkworm

The silkworm, Bombyx mori, is not only the biological foundation of the sericulture, but also an important model for Lepidoptera. Moreover, it has been already explored as a new bioreactor and used as the alternative material for new type of insectical industry. Until recently, the research about the silkworm has achieved a higher scholarship value and important realistic meaning, especially the genetical linkage maps, the whole-genome sequence. As a result, the research on functional genome is underway. There are many excellent methods for the study of functional genome, and one of them is the transgenic technology. In our study, we carried out research based on this technology and the results obtained are as follows:1. The relationship between the internal sequence of piggyBac and its transgenic efficiencySince the transgenic silkworm based on the piggyBac transposon was reported, more and more researchers highlight its use in bioreactor of silkworm and functional analysis on genes of economical value. But this technology is time consuming and laborious, and the transgenic efficiency is relatively low, inhibiting its extensive application. To improve its transgenic efficiency, we have optimized the internal sequences. A series of piggyBac ID synthetic deletion plasmids containing A3EGFP are compared for germline transformation in BmN cells and silkworm. Our analyses have identified a better combination of ID sequence, by which we got a higher transgenic efficiency,2-7 times the original vector. This optimized system can be a potential tool for our future transgenic study.2. Clonning and functional analysis of the Bombyx mori hsp70 promoterWe have got two PCR fragments named hsp70-538 (538bp) and hsp70-305 (305bp) directly upstream of Bombyx mori hsp 70 gene. By comparing analysis, these two fragments possess the consensus of HSE (Heat Shock Element) CTnGAAnnTTCnAG in the upstream sequence. Applying the transient expression assay in BmN cells using the dual-luciferase vector, these two fragments exhibited the heat shock ability. We also validated it in silkworm by transgenic analysis. So these two fragments can be recognized as the heat-induced promoter. This information can be used for biological analysis of gene function.3. The application of IRES on expression system in silkwormIRES can induce a cap-independent translation and has been greatly used in genetic engineering. To investigate its possible use in genetic research of silkworm, we have got four fragments of IRES by chemical synthesis or PCR amplification and reconstructed them into pFastBac donor plasmid containing dual luciferase genes. Through Bac-to-Bac we got these four kinds of recombinant baculoviruses. Quantization by fluorescence spectrophotometry of the luciferase proteins produced in Sf9 cells and silkworm indicated that the IRES593 has the higher activity than others'and highest in the middle silk gland, higher in the fat body and high in the post silk gland. These results suggested that the IRES can be used in the development of expression vectors for production of multiprotein complexes in silkworm.4. The promoter trap studies of gene function in the silkwormPromoter trap system is a highly throughput method. It can creat mutants and also be a tag for the disrupted gene. Combined the above results, we constructed two kinds of transgenic silkworm. One is supposed to provide the transposase steadily. Owing to the low remobilization rates observed with hsp70 inducible transposase, we reengineered the transposase with a constitutive promoter A3. With this construct, we observed somatic excision. The other kind of transgenic silkworm is designed to harbor the non-autonomous transposon with a promoterless EGFP gene. There may exist a piggyBac transposase promoter in the left hand, so we choose another vector, whose direction is opposite to the transposase gene as the potential promoter trap.5. Insertion site analysis of piggyBac transposon in silkwormFor the better use of piggyBac transposon, we investigated the insertion site in the transgenic silkworm by amplifying the flanking sequences through inverse PCR method. The results showed that 6% of the insertion sites happened in the intron,12% in the exon,8% in the 5'end, 3% in the 3'end, 18% in the repetitive sequence, and 53% in the intergenic region.All of the results described above including an optimized transposon, a heat-induced promoter, the application of IRES in expression system and the relevant technology about the promoter trap system have been useful for promoting the transgenic research, and elucidating functional genome based on promoter trap analysis.