【摘要】 用RT-PCR法克隆了成熟的玉米非特异性脂转移蛋白的cDNA,将它连接到表达质粒上并转化至大肠杆菌中表达。以钙调素凝胶覆盖法和钙调素亲和层析下拉实验对表达产物进行分析,证明它具有结合钙调素的活性,并且这种结合不依赖于Ca2+,与前期研究中钙调素结合蛋白-10和拟南芥非特异性脂转移蛋白1的结合特性相同。采用基因删除和缺失突变的方法研究玉米非特异性脂转移蛋白与钙调素结合的结构域,结果表明钙调素结合于47～60位氨基酸,预测的蛋白质二级结构为碱性双亲α-螺旋结构。更多还原

【Abstract】 Maize nonspecific lipid transfer protein(Zm-nsLTP)was cloned and expressed to investi-gate its CaM-binding activity.The cDNA of Zm-nsLTP was amplified using RT-PCR(Fig.1),and then inserted into the vector pET32a(+).The re-combinant vector pET-Zm-nsLTP was expressed in E.coli BL21(DE3)trxB-.Results of CaM-gel overlay assays(Fig.2)and CaM-sepharose pull-down experiments(Fig.3)indicated that recombi-nant Zm-nsLTP was bound to CaM in a Ca2+-inde-pendent manner,which is in accordance with the way that CaMBP-10 and Arabidopsis non-specific lipid transfer protein-1(At-nsLTP1)are bound toCaM.The CaM-binding domain in Zm-nsLTP was mapped to the region of 47–60 amino acids(Fig.3),and online sequence analysis using Predict Pro-tein program predicted that it has a BAA structure(Figs.4,5).更多还原