[玉米非特异性脂转移蛋白cDNA的克隆与表达及表达产物的钙调蛋白结合活性分析]

[Cloning and expression of cDNA for maize nonspecific lipid transfer protein as well as calmodulin-binding activity analysis of the expression product].

作者信息

Bai Wen-Yan, Zhao Li-Qing, Li Zhen-Peng, Xie Wan-Qin, Zhao Yu-Long, Li Cui-Feng

机构信息

Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

出版信息

Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 2006 Oct;32(5):570-6.

PMID:17075181

Abstract

Maize nonspecific lipid transfer protein (Zm-nsLTP) was cloned and expressed to investigate its CaM-binding activity. The cDNA of Zm-nsLTP was amplified using RT-PCR (Fig.1), and then inserted into the vector pET32a(+). The recombinant 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 recombinant Zm-nsLTP was bound to CaM in a Ca(2+)-independent manner, which is in accordance with the way that CaMBP-10 and Arabidopsis non-specific lipid transfer protein-1 (At-nsLTP1) are bound to CaM. 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 Protein program predicted that it has a BAA structure (Fig.4,5).

摘要

克隆并表达了玉米非特异性脂质转移蛋白（Zm-nsLTP）以研究其与钙调蛋白（CaM）的结合活性。使用逆转录聚合酶链反应（RT-PCR）扩增Zm-nsLTP的cDNA（图1），然后将其插入载体pET32a(+) 中。重组载体pET-Zm-nsLTP在大肠杆菌BL21(DE3)trxB(-) 中表达。钙调蛋白凝胶覆盖试验结果（图2）和钙调蛋白琼脂糖亲和沉淀实验结果（图3）表明，重组Zm-nsLTP以不依赖Ca(2+) 的方式与钙调蛋白结合，这与钙调蛋白结合蛋白-10（CaMBP-10）和拟南芥非特异性脂质转移蛋白-1（At-nsLTP1）与钙调蛋白的结合方式一致。Zm-nsLTP中的钙调蛋白结合结构域定位于第47至60个氨基酸区域（图3），使用Predict Protein程序进行的在线序列分析预测它具有BAA结构（图4、5）。