《植物生理学报》 2015, 51(1): 63-72

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

王晓燕^*, 杜改亮^*, 韩瑶瑶, 刘梦欣, 马燕勤, 冯欢, 徐子勤^**

西北大学生命科学学院, 陕西省生物技术重点实验室, 西部资源生物与现代生物技术教育部重点实验室, 西安710069

通信作者：王晓燕；E-mail: ziqinxu@nwu.edu.cn；Tel: 029-88303484

摘　要：

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, AzA)诱导。已有的研究结果显示, 在拟南芥中由AzA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pET28a-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。

关键词：拟南芥; AZI1; 蛋白纯化; 系统获得抗性; Western印迹分析; 抑真菌作用

收稿：2014-11-24   修定：2014-12-28

资助：国家自然科学基金(30870194和J1210063)、陕西省重点实验室科研计划(12JS103和2010JS090)和西北大学研究生创新计划(YZZ13068)。 * 同等贡献。

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

WANG Xiao-Yan^*, DU Gai-Liang^*, HAN Yao-Yao, LIU Meng-Xin, MA Yan-Qin, FENG Huan, XU Zi-Qin^**

Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Shaanxi Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University, Xi’an 710069, China

Corresponding author: WANG Xiao-Yan; E-mail: ziqinxu@nwu.edu.cn; Tel: 029-88303484

Abstract:

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 (AzA). Previous researches in Arabidopsis showed that AZI1 and DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1) were involved in SAR triggered by AzA and glycerol-3-phosphate (G3P), and the LTPs encoded by them contributed to the accumulation of G3P. In order to determine whether AZI1 has the antifungal activity, a prokaryotic expression vector pET28a-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 confirmed that the recombinant AZI1 purified with immobilized nickel-ion affinity chromatography resin could inhibit the growth or division of Botrytis cinerea, Gibberella fujikuroi, Fusarium oxysporum, and Saccharomyces cerevisiae.

Key words: Arabidopsis thaliana; AZI1; protein purification; systemic acquired resistance (SAR); Western blotting analysis; fungistasis