Effect Of Transferring BmKIT3~R Gene Into Genome On Development And Survival Rate Of Bombyx Mori

In order to verify the effect of BmKIT3~R, scorpion venom (Chinese Scorpion Buthus martensii Karsch) on the development and survival of insects and find a new way to control pests, we carried on researches about effects of transferring BmKIT3~R gene into genome on development and survival Rate of silkworms, using ,Lepidoptera model organism Bombyx mori as a model. A transient expression vector with BmKIT3~R gene under the control of hsp20.4 promoter(heat shock promoter 20.4)was injected into pupae and recombinant BmKIT3~R expressed in E. coli was injected into larvae at fifth instar, respectively. The former could delay the development of pupae, while the latter could lead to silkworms paralysis. It indicated that the development of silkworms could be repressed by the toxicity of BmKIT3~R. On this basis, a novel transformation vector derived from transposon piggyBac with the BmKIT3~R gene controlled by hsp20.4 promoter and a neomycin resistance gene controlled by baculovirus early promoter was constructed successfully. The vector was transferred into Bombyx mori eggs by sperm-mediated gene transferring methods. Screened with GFP and antibiotics G418, combined identification of PCR and Dot hybridization, transformed silkworms were obtained. The results showed that both the number of laid eggs for transformed silkworms and the hatching rate decreased. During the G3 generation, the number of laid eggs was 274 per moth. The hatching rate and final survival rate were about 63%, and 47.5%, respectively. 18.9% of the survived pupae died before developing into moths, but the dead pupae withered, not decayed. Furthermore, about 99% of transformed silkworms of G4 generation died before the third instar. Western blotting was performed with anti-BmKIT3~R antiserum. A specific band could be detected in tissues from poisoning transformed silkworms. The results showed that hsp20.4 promoter also possessed activity of constitutive expression promoter, and poisoning of the transformed silkworms was caused by the expression of BmKIT3~R gene. It could be presumed that the development of pupa into moth will be ceased if BmKIT3~R gene was expressed specifically in pupal stage.Analogous poisoning was also found in the hybrid progeny between the G4 generation of transformation silkworms and the wild strain (Bombyx mandarina Moore), and the significant reduction of survival rate suggested that population quantity of pests could be effectively controlled by transferring BmKIT3~R gene into the genome of pests. What's more, it can be speculated that the development of pupa into moth would be ceased and reeling with fresh cocoon would come true, if BmKIT3~R was specially expressed in pupal stage.In addition, the promoter of bmhsp19.9 (heat shock promoter 19.9) was cloned (GenBank no.:FJ447563) and a transient expression vector with the reporter gene DsRed (Discosoma red fluorescent protein, DsRed/drFP583) under the control of bmhsp19.9 promoter was constructed. The result of transient expression showed that the promoter could induce transient expression of DsRed gene in both BmN cells and silkworm tissues.