拟南芥HyPRP蛋白AZI1在大肠杆菌中的表达及其抗真菌活性分析

Expression of Arabidopsis HyPRP Family Member AZI1 in Escherichia coli and Antifungal Activity Analysis of the Recombinant Protein

AZI1(AZELAIC ACID INDUCED 1)基因位于拟南芥4号染色体上,编码产物是脂质转移蛋白(lipid transfer protein,LTP)家族的一个成员。该基因在系统获得抗性(systemic acquired resistance,SAR)中具有重要功能,名称来自它可以被壬二酸(azelaic acid,Az A)诱导。已有的研究结果显示,在拟南芥中由Az A和甘油-3-磷酸(glycerol-3-phosphate,G3P)诱导的SAR反应需要AZI1和DIR1,这两个脂质转移蛋白有助于G3P的积累。为了确定AZI1蛋白是否具有抗真菌活性,本工作构建了原核表达载体p ET28a-AZI1,利用大肠杆菌BL21(DE3)受体细胞制备了没有信号肽的AZI1重组蛋白。Western免疫印迹分析发现通过半乳糖苷类似物IPTG诱导表达的AZI1重组蛋白主要以包涵体的形式存在。体外抑菌实验以及激光共聚焦显微观察结果表明,用镍离子亲和层析树脂纯化的AZI1重组蛋白对灰霉菌、赤霉菌、棉花枯萎病菌和酿酒酵母细胞的生长/分裂均具有抑制作用。

AZI1（AZELAIC ACID INDUCED 1） locates on the fourth chromosome of Arabidopsis and encodes a member of lipid transfer protein（LTP） family. It possesses important functions in systemic acquired resistance（SAR） and is named after the inducible expression by azelaic acid（Az A）. Previous researches in Arabidopsis showed that AZI1 and DIR1（DEFECTIVE IN INDUCED RESISTANCE 1） were involved in SAR triggered by Az A and glycerol-3-phosphate（G3P）, and the LTPs encoded by them contributed to the accumulation of G3 P. In order to determine whether AZI1 has the antifungal activity, a prokaryotic expression vector p ET28a-AZI1 was constructed and the recombinant protein lacking the signal peptide was prepared using BL21（DE3） strain of Escherichia coli in the present work. Western blotting analysis indicated that the recombinant AZI1 produced in E. coli cells after induction with IPTG, an analogue of galactoside, mainly existed as inclusion bodies. Antimicrobial tests in vitro and observation under the laser confocal microscopy confi rmed that the recombinant AZI1 purifi ed with immobilized nickel-ion affi nity chromatography resin could inhibit the growth or division of Botrytis cinerea, Gibberella fujikuroi, Fusarium oxysporum, and Saccharomyces cerevisiae.