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当前位置: 首页 > 文章 > 油菜乙酰乳酸合酶基因BnALS3的亚细胞定位、原核表达及其酶活分析 中国油料作物学报 2018 (3) 309-317
Position: Home > Articles > Subcellular locating,prokaryotic expression and enzymatic activity of BnALS3 gene from Brassica napus Chinese Journal of Oil Crop Sciences 2018 (3) 309-317

油菜乙酰乳酸合酶基因BnALS3的亚细胞定位、原核表达及其酶活分析

作  者：
胡茂龙;龙卫华;高建芹;陈锋;周晓婴;张维;陈松;张洁夫;浦惠明
单  位：
江苏省农业科学院经济作物研究所/农业部长江下游棉花与油菜重点实验室/国家油料作物改良中心南京分中心
关键词：
油菜;乙酰乳酸合酶;叶绿体信号肽;亚细胞定位;融合蛋白;原核表达;酶活
摘  要：
为了研究油菜乙酰乳酸合酶基因BnALS3的亚细胞定位与酶学特性,以甘蓝型油菜(Brassica napus L.)品系N131为材料,采用RT-PCR法克隆到BnALS3的c DNA序列。该序列含有一个长度为1 959bp的开放阅读框,编码蛋白为652个氨基酸,在N端含有一个由49个氨基酸残基组成的叶绿体信号肽序列。将该信号肽序列构建至植物表达载体35S::BnALS3-GFP,利用拟南芥原生质体细胞进行瞬时表达,结果显示BnALS3蛋白定位在叶绿体中。构建去除叶绿体信号肽序列的BnALS3原核表达载体p Czn1-BnALS3,转入大肠杆菌BL21(DE3)进行诱导表达。SDS-PAGE和Western Blot检测显示,在11℃条件下以终浓度为0.2mmol·L~(-1)的IPTG诱导8h后,p Czn1-BnALS3基因在大肠杆菌中成功表达,融合蛋白His-BnALS3呈部分可溶性,分子量约为67k D。酶活分析表明,原核表达的His-BnALS3最适反应p H值为7.0,最佳反应温度为37℃,酶学常数Km值和Vmax值分别为6.55mmol·L~(-1)和6.40μmol·mg~(-1)·h~(-1)。
译  名：
Subcellular locating,prokaryotic expression and enzymatic activity of BnALS3 gene from Brassica napus
作  者：
HU Mao-long;LONG Wei-hua;GAO Jian-qin;CHEN Feng;ZHOU Xiao-ying;ZHANG Wei;CHEN Song;ZHANG Jie-fu;PU Hui-ming;Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Cotton and Rapeseed ( Nanjing) ,Ministry of Agriculture/Nanjing Sub-center,National Center of Oil Crops Improvement;
关键词：
rapeseed;;acetolactate synthase;;chloroplast transit peptide;;subcellular localization;;fusion protein;;prokaryotic expression;;enzymatic activity
摘  要：
To understand the subcellular location and enzymatic characteristics,BnALS3( acetolactate synthase) from Brassica napus L. was investigated. A full length c DNA of BnALS3 gene was cloned from rapeseed line N131 by RT-PCR. The open reading frame of BnALS3 was 1 959 bp in length,which encoded a 652-amino acids sequence with a putative chloroplast transit peptide including 49 amino acid residues at N-terminal end. The transit peptide DNA was cloned for plant-expressed vector p35 S: : BnALS3-GFP construction. Transient expression from transformed protoplasts of Arabidopsis thaliana showed that BnALS3 protein was mainly located in chloroplast. Bacterial expression vector p Czn1-BnALS3( without chloroplast transit sequence) was constructed and expressed in Escherichia coli strain BL21( DE3). SDS-PAGE and Western blot showed that the recombinant protein was successfully induced to express under 0. 2 mmol ·L~(-1) of IPTG at 11℃ for 8 h. Recombinant His-BnALS3( about 67 k D) could be detected in both lysate supernate and precipitates. Enzyme activity assay indicated that under the optimal condition of p H 7. 0 and temperature of 37℃,His-BnALS3 had Km6. 55 mmol·L~(-1) and Vmax6. 40μmol·mg~(-1)·h~(-1),respectively.

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