The Transgenic Research On Regulation Of Silkworm Development

The mulberry silkworm Bombyx mori (Lepidoptera: Bombycidae) is the tamed member of the millions of insects and reared economically in large scale. The larva of silkworm has a pair of highly developed silk glands with the ability to synthesize fibroin. Recent, expression of foreign protein use transgenic silkworm are more common, however, there are a lots reports of by transgenic method research development of silkworm.In this study,a new transgenic vector base on piggyBac transposon for silkworm was constructed by silkworm heat shock protein 20.4 gene promoter (heat shock promoter 20.4) fragment and baculovirus egt gene (Ecdysone UDP-glucosyl transferase, EGT). The Bombyx mori hsp20.4 promoter and baculovirus egt gene (Ecdysone UDP-glucosyl transferase, EGT) were cloned and their motif were analyzed through bioinformatics. Recombined hsp20.4 promoter with red fluorescent protein gene (Discosoma red fluorescent protein, DsRed/drFP583) and hsp20.4 promoter with BmNPV egt gene respectively to form two different recombinant vectors. The former transferred into silkworm BmN cells and tissues while the latter which was injected into silkworm pupa. Transgenic vectors which contain egt gene under the control of hsp20.4 promoters were constructed and used in the transgenic research of BmN cells and silkworms.As a result, hsp20.4(EU350579) promoter and egt gene(EU350576)were cloned for generate transgenic vectors .expression of transient indicating the hsp20.4 promoter sequence possess heat shock protein gene promoter activity in BmN cell and silkworm, and show that production of egt gene could delay the pupal development of silkworm by heat inducing. The transgenic vector were constructed by combining hsp20.4 promoters with egt gene, neor and the GFP reporter gene already included in the original vector. BmN cells were transfected by transgenic vector transposon with neor and GFP double selection elements. Through screen by G418 at the final concentration of 800μg/mL for 1 month, a stable transformation cell strain was obtained. Approximate 70% of the BmN cells were observed emitting green fluorescence. SDS-PAGE analysis found specific bands of EGT (about 55KD). The constructed transgenic vector was used to conduct exploration of transgenic experiment on silkworm. The weak green fluorescence was observed in 2 transgenic larvae at 1 day 5nd instar silkworm.Analyse the egt gene show that baculovirus egt gene originated in the antennal-enriched UGT genes and the egt gene of NPV originated in egt gene of GV.pigA3GFP-En-IE-NEO-hsp20.4-IT3-PA.was Constructed.The transgenic vector constructed in this research may lead deep research on development of silkworm.