The Impact On BmNPV Propagation In Vitro By Transgnic Interfere With Ie-2 And Lef-3 Genes

BmNPV (Bombyx mori nucleopolyhedrovirus) is one of the most common pathogeny in the silkworm which does great harm to sericulture. So the research on BmNPV is of great scientific and practical value. The ie2 and lef3 are selected as suppressing target genes which are essential for baculovirus propagation. Using the transposition vector piggyBac-A3EGFP to construct transgenic RNAi vector that can stabilize transcription dsRNA of target genes(pigA3EGFP-39KPie2, pigA3EGFP-39Klef3, pigA3EGFP-IE1Plef3) transfect to BmE cell strain, we obtain the transgenic cell that can stabilize to form the dsRNA by pressure selection with G418. The inhibition effects of the tested dsRNAs on the replication and propagation of Bm-BacPAK6 and BmNPV were investigated. Significant viral inhibition effect was achieved when BmE cells transfected with transgenic RNAi vector. These results revealed that the combination of RNAi and transgenic technology would be a new method for breeding antiviral silkworm strain in the future.The results of experiment as follow:1. Cloning of the target genesCloning the fragment of target genes ie2 and lef3, which are essential for baculovirus propagation.Two fragments is the+623～+1186bp(564bp) region of ie-2 and the+233～+926 (694bp) region.of lef-3.2. Construction of the transgenic RNAi vectorsUsing the piggyBac-based transgenic RNAi vectors with two different promoter: 39KP and IE1P. three transgenic RNAi vectors were constructed:pigA3-EGFP-39KPie2,pigA3-EGFP-39KPlef3 and pigA3-EGFP-IE1Plef3. These vectors have EGFP as the report gene and neo as the resistance selection tagging.3. Selection and obtain the cell line of the transgenic interferenceWe obtained the steady transgenic cell line that cans constitutively expression the dsRNA of target gene by transfection the transgenic interference vectors to BmE cells.4. The impact of transgenic interference to BmNPV propagation in vitroThe transgenic cells were infected by the Bm-BacPAK6 and BmNPV for studing the ability of cell lines resistance to BmNPV. The effect of the transgenic RNAi was: pigA3-EGFP-39KPie2> pigA3-EGFP-IElPlef3> pigA3-EGFP-39KPlef3...