木薯MeGalt1基因克隆、在采后生理腐烂过程中表达分析及载体构建

Cloning and expression of MeGalt1 and vector construction in cassava during physiological deterioration after harvesting

β-1,3-半乳糖基转移酶(Galt)是形成糖蛋白中Lewis a结构的关键酶,从木薯块根中克隆MeGalt1基因,分析其在木薯块根不同采后腐烂程度中的表达,旨在揭示MeGalt1基因在木薯块根采后生理腐烂中的作用.进化分析表明,木薯中存在3个MeGalt1基因并成功克隆MeGalt1-1、MeGalt1-2和MeGalt1-3,分别具有1890、1893和1902 bp的CDS区,编码629、630和633个氨基酸.Western Blot显示木薯块根采后生理腐烂过程受到蛋白质N-糖基化修饰调控.定量分析表明MeGalt1-1和MeGalt1-3随着腐烂程度加深其表达量也随之升高.在此基础上,成功构建MeGalt1基因的植物过表达载体pCAMBIA1300r-MeGalt1-1和pCAMBIA1300r-MeGalt1-3,为进一步解析MeGalt1在木薯块根采后生理腐烂中的分子作用机制提供材料.

β-1,3-galactosyltransferase(Galt)is a key enzyme in the formation of Lewis a structure in glycoproteins.To reveal the role of MeGalt1 gene in the post-harvest physiological deterioration(PPD)of Manihot esculenta Crantz,the MeGalt1 gene was cloned from cassava tuberous roots.Then the expression of the MeGalt1 gene was characterized under different degrees of PPD.Evo-lution analysis showed that 3 MeGalt1 genes were existed in cassava,named MeGalt1-1,MeGalt1-2 and MeGalt1-3,and they were further successfully cloned from cassava tuberous roots.The sizes of the coding sequence regions of the 3 genes were 1890,1893 and 1902 bp,encoding 629,630 and 633 amino acids,respectively.Western blotting showed that PPD was regulated by N-glycosy-lation.RT-PCR revealed that the expressions of MeGalt1-1 and MeGalt1-3 were increased with the severity of PPD.On this basis,the plant overexpression vectors pCAMBIA1300r-MeGalt1-1 and pCAMBIA1300r-MeGalt1-3 were successfully constructed,which provides materials for the further analysis of MeGalt1 gene in the PPD of cassava tuberous roots.