玉米淀粉分支酶基因SBEⅡb高效沉默表达载体的构建及转化表达

摘要

淀粉分支酶（ starch branching enzyme， SBE）是淀粉合成的限速酶。为了进一步研究SBEⅡb沉默对玉米生长及直链淀粉合成的影响，克隆了玉米（ Zea mays）淀粉分支酶SBEⅡb基因片段，构建了SBEⅡb的发卡结构表达载体pTFU－SBEⅡb hairpin，用农杆菌介导法将其导入玉米HiⅡ幼胚中，经除草剂筛选获得了194株转化再生植株，其中4株结实，获得转基因种子35粒。 T1代植株经PCR及试纸条检测获得3株阳性材料，半定量RT－PCR结果得出SBEⅡb的表达量降低，推断出基因表达水平降低对直链淀粉的合成具有正效应。%Starch branching enzyme ( SBE) is a rate-limiting enzyme in the synthesis of starch.To declare the effect of SBEⅡb silencing on maize growth and amylase synthesis, SBEⅡb gene from Zea mays was cloned, and its expression vector pTFU-SBEⅡb containing hairpin structure was constructed.The construct was transformed into maize HiII immature embryos by Agrobacterium-mediated method.194 regenerated plants were obtained through herbicide screening, 4 of them produced 35 seeds. Three positive plants were detected by PCR and BAR strip from T1 generation plants.Semi-quantitative RT-PCR results showed that the expression of SBEⅡb was reduced.And then, we deduced that the reducing level of SBEⅡb has positive effect on amylose synthesis.