花生AhNCED1重组蛋白原核表达与二级结构初步分析  

PROKARYOTIC EXPRESSION OF AhNCED1 RECOMBINANT PROTEIN

作　　者：胡博[1] 李嘉怡[1] 游琼英[1] 

机构地区：[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

出　　处：《华南师范大学学报（自然科学版）》2011年第1期87-92,共6页Journal of South China Normal University(Natural Science Edition)

基　　金：广东省自然科学基金项目(06025049)

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.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-AhNCED1 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 AhNCED1 showed that it is a typical random coil protein.The proportional content of α-helixes of AhNCED1 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 

分 类 号：Q78[生物学—分子生物学]