Cloning Of Cardamine Hupingshanensis GST-u4 Gene And Preliminary Study On Activity Of Expressed Product

Cardamine hupingshanensis is a perennial herb of Cardamine in Cruciferae,which belongs to Se hyperaccumuLators plant.Selenium is an essential trace element for human and animals.Therefore,it is possible to supplement selenium by obtaining organic selenium which is beneficial to human beings from plants with high concentration of selenium.It was found that the expression of GST-u4 in leaves of Capsella capsici treated with 80 mg·ml^-11 selenium was significantly increased,suggesting that GST-u4 gene may be related to selenium chelating and vacuolar storage.In this paper,ChGST-u4 was cloned by RT-PCR,and bioinformatics analysis of the gene was carried out using online tools.The codon of the gene was optimized,and the expression vector was constructed,and heat shock transformed into Escherichia coli Arctic After purification,the activity of the expressed product was preliminarily studied by isothermal titration calorimetry,which laid the material foundation for the study of selenium metabolism mechanism in Cardamine capillaris.1.Chgst-u4 is a tau subfamily gene of glutathione S-transferase gene family.Its molecular weight is 672bp,encoding 224 amino acids.The molecular weight of the protein is 25.6872kDa,and the content of alanine is the most abundant,accounting for4.9%of the total amount;the theoretical isoelectric point is 6.18,the protein has no signal peptide and has 9 phosphorylation sites;it is a hydrophilic protein;the secondary structure of chgst-u4 is mainly?-spiro.There are two domains,no transmembrane region,located in the cytoplasm.2.After codon modification of ChGST-u4,the expression plasmid pCold?-ChGST-u4 was constructed.The fusion protein was expressed as inclusion body under three conditions.The final concentration of IPTG was 0.5 mm at 20?for 15 hours,and the precipitation content was higher at 220 rpm.The molecular weight of the expressed protein was about 26 kDa after renaturation and purification The purified protein was ChGST-u4.3.The results showed that the four small molecules(GSH,PC2,Seo₄^2-,Seo₃^2-)could specifically bind to ChGST-u4 by isothermal titration calorimeter,and the binding capacity was PC2<GSH<Seo₄^2-<Seo₃^2-.The four small molecules may form non covalent bonds with ChGST-u4,such as hydrogen bond,ionic bond,van der Waals force,etc.,indicating that the expressed ChGST-u4 is active,and it is possible to catalyze the chelation of selenium and glutathione to form PC2.