Effect On The Development Of Bombyx Mori By Transfering Egt Gene

The Bombyx mori is one member of the holometabolous class of insects, which possesses four distinguished developmental stages, i.e., egg, larva, pupa, and adult. In order to achieve fresh cocoon filature and raise the quality of silk, people want to prolong the pupal stage by artificial methods.In this experiment, we transformed ecdysteroid UDP-glueosyhransferase (EGT) gene of Bombyx mori nucleopolyhedrovirus(BmNPV) into the genome of silkworm and studied the effect on development of silkworms.A recombinant vector containing a red fluorescent protein gene (Discosoma red fluorescent protein, DsRed) under the control of hsp23.7 promoter was constructed, and then, was transfected into BmN cells. The result showed that hsp23.7 promoter can drive the DsRed gene transient expression in the silkworm and BmN cells; A new type transgenic vector based on piggybac transposon was constructed with BmNPV egt gene driven by hsp23.7, and a neomycin resistance gene (neo) driven by the BmNPV immediately early promoter ie-1, the vector was transferred into Bombyx mori eggs by using sperm-mediated gene transfer. The transgenic silkworms were obtained succeefully by screening for gfp and neo genes, and verified them by PCR, DNA dot-blot hybridization and western blotting. The result of developmental survey indicated that the hatching rate of eggs for transgenic silkworm were about 60%, was lower than that of non-transgenic silkworm. The larval stage of transgenic silkworm extended three days compairing to that of the non-transgenic silkworm, The pupal stage of ransgenic silkworm had four days longer than that of non-transgenic silkworm in generation G2; The measurement of ecdysone showed that the level of ecdysone in the transgenic silkworm was 90% lower than that of non-transgenic silkworm, the results demonstrated that the egt gene expression in transgenic silkworm could declined the level of ecdysone in silkworm, and delay the development of the silkworm. It was suggested the development of pupa into moth will be blocked if egt gene was expressed specifically in the pupal stage.