Cloning And Eukaryotic Expression Of BmGSTe3 Gene Of Silkworm (Bombyx Mori)

Silkworm is not only one of the most important economic insects in agriculture but also the crucial experimental insects. Glutathiones S-tansferases(GSTs,EC 2.5.1.18) is a super enzyme encoded by several genes and possessing multifunctions. It's widely distributed in animal, plant,yeast, bacteria and other organism, it's play a important role in detoxification of endogenous and exogenous toxic substance.with the rapid development of economic society ,More and more organic pesticide been used in agricultural production .the insect resistance to insecticides also had a remarkable increased . For Silkworm is the mode organism of lepidopteran insects, the research on expressive mode of GST of Bombyx mori (BmGST) and regulative mechanism can settle theoretical foundation for clarifying the relationship between GST and pesticide resistance, and preventing pests in farming and foresting. In order to make clear the expression and function of GST in silkworm and clarify deeper mechanism of the pesticide resistance, we forecasted one GSTs gene, namely BmGSTd3, according to genome and EST database of Bombyx mori through bioinformatics methods. We designed primers according to EST assembling results, and cloned the sequences, then we analyzed these sequences in detail, detected tissue expression and OPs stress profiles by RT-PCR. At the end, Heterologous expression of BmGSTe3 was taken using baculovirus expression vector eukaryotic expression system. Our research preliminarily established a new way to express the GST protein through insect cell. The results are as follows:1. Gene cloning and sequence analysis of BmGSTe3 geneThe CDS length of BmGSTe3 was 654bp. The results from sequencing prove the correctness of cloning gene. Gene structure analysis: BmGSTe3 is composed of 5 exons and 4 introns, and all the boundaries between exons and introns accord with typical GT-AG rule. The whole length of exons is 512bp, each length is less than 250bp,and the whole length of introns is 4792bp,the longest intron is 1771bp,the shortest is 788bp.transcription regulatory element analysis: after predicted by neural network promoter,it has one TATA box.and 15 possible transcription regulatory elements have been found altogether after search in the upstream of 2500bp.which are XRE (1),ARE(4),TRE(5) and NF-kB(5).phylogenetic analysis: Analysis of phylogenic tree showed that there have four family members, Dlata family members, Epsilon family members, Theta family members, and Zeta family members , BmGSTe3 belongs to the Zeta family. Protein secondary structure prediction: BmGSTe3 contain both N-terminal and C-terminal domains. N-terminal is composed of 7 motifs, namelyβ-α-β-α-β-β-α, while C-terminal is composed of 5αhelixs, BmGSTD3 have a conservative Ser catalyse site.2. Expression profiles analysis of BmGSTe3For the tissue expression pattern,BmGSTe3 express only in hemolymph and brain,expression level of BmGSTz2 was the higher in brain than hemolymph, it indicate that the BmGSTe3 gene has a high level of tissue specificity.3. Construction of The recombinant transfer plasmid and BEVS expressing plasmidAfter enzyme digestion of recombinant cloning plasmid PMD18-T/BmGSTe3 and transfer plamid pFastBacHTa by BamHI and Nco I,target fragment recycling and connection in 16℃for night, then transformed in DH5α, plasmid extraction from bacterial liquid of positive clones , It was identified by Enzyme digestion that the GSTe3 gene was successfully inserted into the vector pfastbacHT A.He recombined transfer plamid was transformed in DH10Bac, It was verified by PCR that the GSTe3 genewas successfully inserted into the site of the shuttle vector.numed Bac-BmGSTe3.4. Eukaryotic Expression of BmGSTe3 GeneTransfect Bac-BmGSTe3 into sf9 cell by cellfectin, Bac-BmGSTe3 transfect into sf9 and SDS-PAGE analysis showd that the molecular weight of BmGSTe3 was 26kD,it is similar with the theoretic molecular weight.5. Determination of BmGSTe3 ActivityWe had preliminarily Determination of BmGSTe3 Activity by CDNB, the result show that the recombinant GST protein have good biological activity.The recombinant BmGSTe3 protein was successfully expressed in Bac-to-Bac baculovirus expression system, and the biological function was good. It is a good bases for after protein function research..