Identification And Functional Analysis Of Genes Controlling Biosynthesis Of Glutathione In Tetrahymena Thermophila

Glutathione(GSH)is a thiol tripeptide with antioxidant and detoxifying heavy metals,which is widely found in a variety of organisms.The content of GSH in organisms is much higher than metallothionein,phytolicin and other detoxifying heavy metal polypeptides.GSH protects organisms by chelating metal ions with sulfhydryl groups and interacting with GPX enzyme.It is of great theoretical significance and application value to improve the tolerance of organisms to harmful metal.Tetrahymena thermophila is a free-living unicellular ciliate protozoan.It has no cell wall and is sensitive to environmental pollutants.Tetrahymena has important functional gene family adapted to the environment.It is a classic model organism to explore mechanism of environmental pollutants degradation and antioxidant effect.In this study,glutamylcysteine synthase gene and glutathione synthase gene were identified in T.thermophila,and the function of the genes was analyzed.The main results were as follows:1.Bioinformatic analysis of GSH1 and GSH2 in TetrahymenaBased on the genomic database and functional genomic database of T.thermophila,?-glutathione cysteine synthase gene GSH1(TTHERM?00250970)and glutathione synthase gene GSH2(TTHERM?01049360)were identified by bioinformatics analysis.GSH1 is1881 bp,encoding 626 amino acids;GSH2 is 1938 bp,encoding 645 amino acids.GSH1 and GSH2 were expressed in vegetative growth stage,starvation stage and sexual reproduction stage,and the expression level of conjugation pair induction was significantly up-regulated.Under the stress of Cd²⁺and Pb²⁺,the expression levels of GSH1 and GSH2 in T.thermophila were significantly up-regulated.Phylogenetic analysis of Gsh1 and Gsh2 showed they had similar evolutionary branches and coevolved.Protein interaction analysis showed that both enzymes interacted with proteins related to the synthesis of cysteine and the precursor of glutathione synthesis.2.GSH1 knockout mutant is sensitive to Cd²⁺and Pb²⁺stressThe p Neo4-GSH1 plasmid was constructed and transformed into Tetrahymena by particle bombardment.The GSH1 knockout mutant KO-GSH1 was created.Under the stress of Cd²⁺and Pb²⁺,the IC50 values of KO-GSH1 cells were 26.46?M and 13.39?M,respectively,while those of wild-type cells were 30.77?M and 15.35?M,respectively.The content of glutathione in KO-GSH1 cells decreased by 85%.Under the stress of Cd²⁺and Pb²⁺,the content of glutathione in KO-GSH1 cells decreased by 95%and 94%,respectively,and that in wild-type cell lines decreased by 55%and 53%,respectively.Under the stress of Cd²⁺and Pb²⁺,expression level of MTT1,MTT3 and MTT5 and antioxidant enzyme gene SOD1,GPX1 and CAT was significantly upregulated in KO-GSH1 mutants.These results showed that KO-GSH1 mutnat is sensitive for Cd²⁺and Pb²⁺,and upregulation of metallothionein and antioxidant alleviates the sress.3.Overexpression of GSH1 and GSH2 improves tolerance of Cd²⁺and Pb²⁺in TetrahymenapXS75-GSH1 and pXS75-GSH2 plasmids were constructed and transformed into Tetrahymena by particle bombardment.OE-GSH1 and OE-GSH2 mutants were created and overexpression of GSH1 and GSH2 under 0.1ug/ml Cd²⁺induction.Immunofluorescence analysis showed that both HA-Gsh1 and HA-Gsh2 were localized in the cytoplasm.Under Cd²⁺and Pb²⁺stress,the IC50 of OE-GSH1 was 35.15?M and 18.13?M,respectively.The IC50 of OE-GSH2 was 33.98?M and 17.58?M,respectively,which indicated that the overexpression of GSH1 and GSH2 improved tolerance of Cd²⁺and Pb²⁺in OE-GSH1 and OE-GSH2 mutants.The content of glutathione was significantly increased,while the expression levels of MTT1,MTT3 and MTT5and SOD1,GPX1 and CAT were significantly decreased.The results showed that OE-GSH1 and OE-GSH2 mutans increased their tolerance to Cd²⁺and Pb²⁺by upregulating the expression levels of GSH1 and GSH2 and increasing the content of glutathione.Metallothioneins and antioxidant enzymes were also involved in Cd²⁺and Pb²⁺detoxication following GSH function.In conclusion,we first identified the?-glutathione cysteine synthase GSH1 and glutathione synthase GSH2 in Tetrahymena.GSH1 knockout mutants were created and the synthesis of GSH decreased in the KO-GSH1mutants.Under Cd²⁺and Pb²⁺treatment,upregulation of metallothionein and anti-oxidation-related genes in the KO-GSH1 mutants.In contrast,overexprssion of GSH1 and GSH2 increase tolerance of Cd²⁺and Pb²⁺in OE-GSH1 and OE-GSH2 mutants and decrease expression of metallothioneins and antioxidant enzymes.The synthesis of GSH and expression of metallothioneins and antioxidant enzymes co-regulate detoxication of heavy metal in Tetrahymena.