Cloning And Eukaryotic Expression Of The GSTe4 Gene Of Silkworm, Bombyx Mori

Glutathiones S-tansferases(GSTs,EC 2.5.1.18) is a super enzyme with multifunctions encoded by several genes.It's widely distributed in animal,plant,yeast,bacteria and other organism.Insect GSTs played important roles in organophosphate,organochlorine,and pyrethroid insecticides. Pesticide plays a very important role in pest control.However,with the substantial use of pesticides, the resistance of insect to insecticide increased,people had to develop new products or increase pesticide usage dose.This is not only restricted economic development,but also gives the environment a serious burden and affects the economic and human durative development.In the world,more than50%pests of agriculture and forestry belong to Lepidoptera.Bombyx mori is a model organism of Lepidoptera.It is the only insect whose genome was sequenced in Lepidoptera.Study on GST genes of Bombyx mori is not only provided theory foundation for resistant breeding of silkworms,but also established important foundation for biological control of Lepidopteran pests.We forecasted one GSTs gene,namely BmGSTd4,according to genome and EST database of Bombyx mori through bioinformatics methods.We designed primers according to EST assembling results,and cloned the sequences,and then we analyzed these sequences in detail, detected tissue expression by RT-PCR.Eukaryotic expression of BmGSTe4 was taken using of baculovirus expression vector eukaryotic expression system..At last,we verified activity of the expression product and Carried out western blot hybridization.The main results are followed.1.Gene cloning and sequence analysis of BmGSTe4 geneThe CDS length of BmGSTe(?) was 654 bp.The results from sequencing prove the correctness of cloning gene.Gene structure analysis:BmGSTe4 is composed of 5 exons and 4 introns.The whole length of introns is 3 631 bp,the longest intron is 1 949 bp,the shortest is 214 bp.Transcription regulatory elements were predicted by neural network promoter.One TATA box.and 14 possible transcription regulatory elements.which are XRE(1),ARE(6),TRE(5) and NF-kB(2),have been found after searching in the upstream of 2 500 bp..Phylogenetic analysis:of BmGSTe4 showed that there have four family members,Dlata family members,Epsilon family members,Theta family members,and Zeta family members,BmGSTe4 belongs to the Zeta family.2.Expression profiles analysis of BmGSTe4The BmGSTe4 express only in epidermis and brain,and expression level was the higher in epidermal than brain.lt indicate that the BmGSTe4 gene has a high specificity expression in tissues.3.Eukaryotic Expression of BmGSTe4GeneTransfect Bac-BmGSTe4 into sf9 cell by cellfectin,Bac-BmGSTe4 transfect into sf9 and SDS-PAGE analysis showd that the molecular weight of BmGSTe4 was 27 kD,it is similar with the theoretic molecular weight.4.Western blotDetected cell eukaryotic Expression of BmGSTe4 by SDS-PAGE electrophoresis,then cut the plastic between 62 kD and 14 kD to switch to the membrane and did western blotting hybridization. The hybridization signal in the sample showed that target gene had been expressioned in cells.5.Determination of BmGSTe4 activityWe had preliminarily determinaed the activityof BmGSTe4 by CDNB,the result showed that the recombinant GST protein has good biological activity.The recombinant BmGSTe4 protein was successfully expressed in Bac-to-Bac baculovirus expression system,and the biological function was good.It is a good bases for BmGSTe4 protein function research.