Gene Cloning And Expression Of Silkworm BmGSTz2 And BmGSTd3

Glutathione S-transferases (GSTs) is one of the three enzymatic super families which function as detoxification. They can catalyze the combination between Thiol of reduced glutathione and various electrophiles, increasing the solubility of endogenetic and ectogenic toxicants, which helps to expel the toxicants from organisms. Besides the detoxification, GSTs also act on intercellular material transport, hormonal synthesis and elimination of Reactive Oxygen Specices (ROS) induced injury. Moreover, GST plays an important role in chemical therapy of cancer and drug-fastness. In addition, their polymorphisms also correlate with susceptibility of disease. In insects, the GST functions as a crucial role in pesticide detoxifcation and resistance forming.Organic phosphorus (OPs) has been widely used to prevent lepidopteran pests. However, the OPs resistance of insects increases along with heavy use of organic phosphorus. 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 two GSTs, namely BmGSTz2 and 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 BmGSTz2 was taken using prokaryotic expression system.The results are as follows:1 cloning and expression profiles analysis of BmGSTz2 and BmGSTd3 The CDS length of BmGSTz2 and BmGSTd3 (NCBI accession numbers: EF565386&DQ355374) were 651bp and 663bp respectively. For the tissue expression pattern, expression level of BmGSTz2 was the highest in fat body, and relative low in hemolymph, brain, silkgland, epidermis and ovary, whereas no expression was detected in midgut and tetise. BmGSTd3 expressed in eight tissues such as hemolymph, expression level in midgut and epidermis were relative high, whereas that in hemolymph was a little low. In the course of phoxim stress, the transcription level of BmGSTz2 was induced as a whole, however, that of BmGSTd3 held the line comparatively.2 Gene structure and regulatory elements of BmGSTz2 and BmGSTd3BmGSTz2 located on nscaf2888 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 829bp, and that of introns 18652bp, the longest intron is 13942bp. BmGSTd3 located on nscaf2279 is also 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 938bp, and that of introns 5442bp, the longest intron is 3243bp. Both of the BmGST genes have E.coli rare codon. The transcription regulatory elements are analyzied as follows: 5' UTR length of BmGSTz2 is 945bp, and 15 possible transcription regulatory elements have been found altogether, which are XRE(1), ARE(3), TRE(8) and NF-kB(3); 5' UTR length of BmGSTd3 is 831bp, and there are two introns inserted in 5' UTR. 12 possible transcription regulatory elements have been found altogether, including XRE(2), ARE(4), TRE(5) and NF-kB(1).3 Analysis of deduced amino acid sequence of BmGSTz2 and BmGSTd3Comparability analysis of sequences results as follows: BmGSTZ2 have 38.7%～43.9%, 15.1%～21.2%, 13.3%～20.5% and 13.3%～20.5% identities with Zeta family members, Epsilon family members, Dlata family members and Theta family members, respectively. While BmGSTD3 have 35.0%～51.3%, 30.7%～37.3%, 18.4%～23.9% and 15.1%～19.2% identities with Dlata family members, Epsilon family members, Theta family members, and Zeta family members, respectively. Analysis of phylogenic tree showed that BmGSTZ2 belongs to the Zeta family, while BmGSTD3 belongs to the Delta family. The physical chemistry of protein analyzed by bioinformatics tools are as follows: molecular weight of BmGSTZ2 is 24.7 kDa, pI is 8.56, there are 9 potential phosphated sites, namely, S28,S175,T41,T81,T141,T208,Y49,Y133,Y200; molecular weight of BmGSTd3 is 24.7 kDa, pI is 5.15, and there are 8 potential phosphated sites, namely, S82,S84,S161,S167,S175,T56,Y149,Y196. Both BmGSTZ2 and BmGSTD3 are non-secretory proteins. Forecast results towards secondary structures of protein are as follows: BmGSTZ2 and BmGSTD3 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 withoutβsheet. Both BmGSTZ2 and BmGSTD3 have a conservative Ser catalyse site.4 Prokaryotic expression of BmGSTz2We subcloned the CDS of BmGSTz2 to pET-28a(+) vector to construct recombined expressive plasmid, Then the recombined plamid was transformed in BL21(DE3) bacterial strain. Single clone is cultivated in medium to OD₆₀₀=0.6～0.8, induced with IPTG (final concentration=1mM) at 20℃and 37℃, respectively. 250rpm for 5h. Through SDS-PAGE electrophoresis analysis , the target gene produce protein band around 27 kDa, while the control did not. The result showed that the protein encoded by BmGSTz2 was expressed by Ecoli.