Cloning And Prokaryotic Expression Of Glutathione S-transferase And Metallothionein Genes In Crassostrea Hongkongensis

Crassostrea hongkongensis is one of the important shellfishes in South China Sea andhave become a monitoring animal for early warning of marine environment pollution in shore.All kinds of diseases and deaths have been took place in the breeding process, the existingresearch shows that Glutathione s-transferases(GSTs) play an important role in detoxicatingand oxidative stress to protect cells. GSTs also are the molecular instructions of pollution dueto the sensitive reaction response to environmental pollutants. Metallothionein(MT) playdouble roles in keeping the metal content of organic body physiological balance anddetoxificating heavy metals.Because the oyster GSTs and MT are involved in controlling deathof the oyster and monitoring polluted status of marine environment in which the oysterinhabited, it is necessary to get the GSTs and MT protein to separate and purificate GSTs andMT protein for further research of the GSTs and MT function. Then enough GSTs proteinshould be prepared. The present study aims to synthesize GSTs and MT with prokaryoticexpression method. The results showed that：Extract RNA of Crassostrea hongkongensis. Produce cDNA as the first chain by reversetranscripyion. Expand the target fragment by PCR with the primers of upstream anddownstream and connect PMD-18T vector. Change the product into DH5α. The GST-μ、GST-σand MT cDNA gene were cloned successfully. The coding region of GST-μ、GST-σ and MTwere654bp、629bp and245bp respectively. There were4、6and3variation sites of basicgroup by GST-μ、GST-σ and MT sequence comparing with the known sequence respectively.The similarity between the GST-μ、GST-σ、MT and the known sequence was99.39%、98.73%and98.68%respectively. There were3variation sites by MT sequence comparing with theknown sequence after translating into amino acids. The similarity between the amino acidswas96%.The recombinant expression plasmids pQE30/GST-μ and pQE30/GST-σ were constructedby means of linking the oyster GST-μ and GST-σ cDNA with prokaryotic expression vectorpQE30respectively. The recombinant expression plasmids pGEX-4T-1/MT was constructedby means of linking the oyster MT cDNA with prokaryotic expression vector pGEX-4T-1.They were identified by endonuclease digestion and DNA sequencing of the recombinant plasmids. Then, the recombinant plasmids pQE30/GST-μ、pQE30/GST-σ and pGEX-4T-1/MTwere transformed into E. coli M15and induced to express the fusion proteins with0.1mmol/LIPTG. The fusion proteins were identified by SDS-PAGE and Western blotting. The GST-μprotein quantity induced at30℃is the highest among those induced at25℃,30℃and37℃.Thus,30℃is the fittest inducing temperature. Meanwhile, the expression level induced for4hat30℃is up to the highest, so the fittest induction time was4h. The GST-σ protein quantityinduced at37℃is the highest among those induced at25℃,30℃and37℃. Thus,37℃is thefittest inducing temperature. Meanwhile, the expression level induced for6h at37℃is up tothe highest, so the fittest induction time was6h. The MT protein quantity induced at37℃isthe highest among those induced at25℃,30℃and37℃. Thus,37℃is the fittest inducingtemperature. Meanwhile, the expression level induced for8h at37℃is up to the highest, sothe fittest induction time was8h. The molecular weights of the GST-μ and GST-σ protein are24kDa and23kDa respectively. The molecular weights of the MT fusion protein is35kDa(The molecular weights of the GST tag protein is26kDa. The molecular weights of the MTprotein is9kDa. The molecular weights of both GST and MT is35kDa).