橡胶树中碱性转化酶HbNIN9表达及酶学特性分析  

Prokaryotic Expression and Biochemical Properties of a Neutral/Alkaline Invertase Gene(HbNIN9) from Hevea brasiliensis

作　　者：王佳 龙翔宇[2] 阳江华[2] 方永军[2] 秦云霞[2] 白蓓蓓 卢基来 唐朝荣 Wang Jia;Long Xiangyu;Yang Jianghua;Fang Yongjun;Qin Yunxia;Bai Beibei;Lu Jilai;Tang Chaorong(Institute of Tropical Agriculture and Forestry,Hainan University,Haikou,570228;Rubber Research Institute,Chinese Academy of Tropical AgriculturalSciences,Haikou,570228)

机构地区：[1]海南大学热带农林学院,海口570228 [2]中国热带农业科学院橡胶研究所,海口570228

出　　处：《分子植物育种》2019年第6期1827-1833,共7页Molecular Plant Breeding

基　　金：国家自然科学基金资助项目(31570672)资助

摘　　要：转化酶作为蔗糖分解代谢的关键酶,在植物生长发育、生物与非生物胁迫响应等生理过程中发挥重要作用。基于对橡胶树不同组织转录组数据库的分析,本研究克隆了一个在橡胶树种子中有较高丰度表达的中碱性转化酶基因HbNIN9;系统进化分析和在线软件预测均显示其亚细胞定位为线粒体,属于中碱性转化酶α亚族成员;利用原核表达体系成功获得Hb NIN9优化表达的重组蛋白,优化表达的诱导条件为0.5 mmol/L IPTG,25℃培养6 h;酶学特性分析表明,HbNIN9蛋白酶活反应的最适温度为45℃,最适pH值为7.5,米氏常数Km值为18.09 mmol/L。本研究从蛋白水平证实了Hb NIN9为典型的中碱性转化酶,有助于深入揭示线粒体型转化酶在橡胶树种子糖代谢中生理功能,丰富橡胶树中碱性转化酶家族成员的生理生化研究。Invertases, as a kind of key enzymes of sucrose metabolism in higher plants, play an important role in plants growth, development and stress response. Basing on transcriptome data of tissues from Hevea brasiliensis,HbNIN9, a new neutral/alkaline invertase gene mainly expressed in seeds, was cloned. Phylogenetic analysis and subcellular location prediction showed that Hb NIN9 was located in mitochondria and belonged to a member of αgroup of neutral/alkaline invertases. HbNIN9 was successfully expressed in E. coli, and the expression conditions were optimized as 6 h induction with 0.5 mmol/L IPTG at 25℃. The biochemical properties of HbNIN9 enzymatic activity were investigated. HbNIN9 showed the highest enzymatic activity at pH 7.5 and 45℃, and had a Km of18.09 mmol/L for its substrate of sucrose. The study shows that Hb NIN9 is a typical neural/alkaline invertase in the protein level, which can help to reveal the physiological function of the mitochondrial-type invertase in the seed sugar metabolism of the rubber tree, and enriched the physiological and biochemical study of alkaline transforming enzyme family members in Hevea brasiliensis.

关 键 词：巴西橡胶树 蔗糖转化酶 原核表达 线粒体型中碱性转化酶 

分 类 号：S794.1[农业科学—林木遗传育种]