Prokaryotic Expression And Purification Of Sucrose Synthase From Tartary Buckwheat

With a health care function and abundant dietary fiber, tartary buckwheat can contribute to control hypertension and diabetes, reduce assimilation of intestinal to cholesterol and assist nutral lipid in excreting. Sucrose synthase (SuSy) is one of the enzymes closely related to sugar metabolism, which has extraodinarily important physiological function such as influence on fruit quality, participation in growth and differentiation of cells fibers, starch synthesis, and has capability of increasing stress resistance of plants. It is reported that SuSy gene has been cloned from several kinds of plants such as solanum tuberosum, sugar beet and sugarcane which was further studied about gene expression and regulation.So far it is not reported as to cloning of SuSy from tartary buckwheat (Fagopyrumtataricum (L.)Gaertn).This study amis to lay foundation for revealation of physiological property of SuSy, relationship between its regulation effect in seed development and accumulation of free sugar in kernel and for making polyclonal antibody of SuSy .The work was conducted as follows,(1)Analysis results of SuSy sequece from tartary buckwheat indicated that the gene contained 1473bp open reading frame (ORF) encoding the protein whcich cotains 490 amino acid residues with molecular weight of 53.4kD,pI of 6.02 and a putative signal peptide. Alignment analysis showed that the amino acid sequence was 100% homologous to the reported amino acid sequence of SuSy from Beta vulgaris (acession number, AAR19769).The phylogenetic tree showed it was most closely to Beta vulgaris. Tertiary structure and secondary structure prediction results showed that the encoded protein was a concave structure containingα-helixes andβ-strands. The functional structure prediction showed it contained sucrose synthase and glycoside transfer₁ domains.(2) SuSy was amplified from one cDNA library clone and directly cloned into pET28a plasimd to construct expression vector pET28a-SuSy.The pET28a-SuSy plasmid proved by sequencing and double digestion was transformed into BL21 Star (DE3), and the expression of SuSy was indentified by SDS-PAGE. The expected protein was expressed successfully but in the form of inclusion bodies.The molecular weight of the protein was 53.4kD, and its optimal expression condition was 1mmol/L IPTG for 6h at 37℃, its expression level was 39.5% of total bacteria protein by bandscan 5.0 analysis.(3)Expressed protein purified by cobalt ions chelating affinity chromatography was identified by gelcode His-tag staining.SDS-PAGE result showed the single band was presented in gel with CBB R250 and gelcode His-tag staining.(4)Renaturation of the inclusion bodies was unsuccessful with several different methods. Sucrose Synthase cloned from tartary buckwheat was successfully overexpressed in E.coil for the first time, and purified by cobalt ions chelating afffinity chromatography.All results laid the foundation for the further study of the relationship between SuSy expression and regulation and carbohydrate metabolism.