花生AhNCED1重组蛋白原核表达与二级结构初步分析
PROKARYOTIC EXPRESSION OF AhNCEDI RECOMBINANT PROTEIN
胡博 1李嘉怡 1游琼英1
作者信息
- 1. 华南师范大学生命科学学院,广东省植物发育工程重点实验室,广东广州,510631
- 折叠
摘要
9-顺式环氧类胡萝卜素双加氧酶(NCED)是调控ABA生物合成的关键限速酶.根据花生叶片中克隆得到基因AhNCED1,构建融合蛋白重组表达载体,将重组质粒转化到大肠杆菌DH5a中诱导表达,SDS-PAGE分析显示在0.2 mmol/L IPTG、4 h、28 ℃的条件,目的蛋白以可溶形式高效表达,分子质量在66.0 ku左右.AhNCED1重组蛋白可与制备抗体特异性结合,呈现典型的不规则卷曲结构.
Abstract
Abscisic acid (ABA) is an important hormone that mediates plant responses to abiotic stresses, including drought, salinity, and low temperature. The oxidative cleavage of cis - epoxycarotenoids catalyzed by 9 - cis -epoxycarotenoid dioxygenase (NCED) is considered to be the rate limiting step in ABA biosynthesis. We constructed a prokaryotic expression plasmid pProEXHT-AhNCEDI and expressed a soluble fusion protein of about 66 kD in E. coli BL21 (DE3) cells induced by 0. 2 mmol/L IPTG at 28 ℃ for 4 h. The recombinant protein was purified with Ni/NTA affinity chromatography. Western blot assays revealed that there was a protein band, with a relative molecular mass of 66.0 ku, indicating that antiserum could react to the native protein expressed in peanut specifically. The CD spectrum of AhNCEDI showed that it is a typical random coil protein. The proportional content of α -helixes of AhNCEDI protein increased from 20. 9％ to 82.5％ under 30％ PEG treated for 3 h and 5％ SDS made the proportional content of α - helixs increase to 41.2％. Our research give some new insights for better understanding the function of AhNCED1 protein and the regulation mechanism of ABA biosynthesis.
关键词
花生/AhNCED1重组蛋白/原核表达Key words
Arachis hypogaea/AhNCED1 recombinant protein/prokaryotic expression引用本文复制引用
出版年
2011
NETL
NSTL
维普
万方数据