木薯MeGBSSI与MeSSV的蛋白互作验证OA

Granule-bound starch synthase I(MeGBSSI)is the key enzyme for amylose synthesis in cassava,primarily responsible for catalyzing the formation of amylose through α-1,4-glucosidic linkages on the surface of starch granules.However,how MeGBSSI cooperates with other starch-synthetic enzymes to orchestrate starch biosynthesis remains unclear.In the previous stage of this research group,through yeast two-hybrid technology,it was hypothesized that the soluble starch synthase MeSSV was a potential binding protein of MeGBSSI.In this study,the MeGBSSI and MeSSV genes were cloned using the main cultivated variety of cassava,Huanshan 8(SC8),as the material.Yeast two-hybrid vectors pGBKT7-MeGBSSI and pGADT7-MeSSV were constructed.Through yeast two-hybrid point-to-point experi-ments,the interaction relationship between MeGBSSI and MeSSV was preliminarily verified.The luciferase comple-mentation(LCA)and double-molecule fluorescence complementation(BiFC)techniques were used to further clarify the direct protein interaction between the two.The results showed that the protein sequences encoded by the MeGBSSI and MeSSV genes,compared with the sequenced variety AM560,respectively,had 4 and 2 amino acid substitutions;the MeGBSSI protein was non-toxic to yeast cells and had no self-activation activity;this protein interacted with the soluble starch synthase MeSSV in yeast;the LCA experiment showed that the chemical signals significantly enhanced after co-expression of MeGBSSI and MeSSV;the BiFC experiment further proved that the two could form a complex in plant cells,and the interaction signal was located in the chloroplast.The findings would provide new insight into the syner-gistic regulation among cassava starch-synthetic enzymes.