橡胶树2个胶乳转化酶基因的原核表达  

Construction of Prokaryotic Expression Vectors for Two Invertases from Hevea Latex and Optimization of their Expression Conditions

作　　者：刘术金[1,2] 李旖璠 唐朝荣[1] 

机构地区：[1]中国热带农业科学院橡胶研究所,农业部橡胶树生物学重点开放实验室,海南儋州571737 [2]海南大学农学院,海南儋州571737

出　　处：《热带作物学报》2010年第7期1091-1097,共7页Chinese Journal of Tropical Crops

基　　金：农业部基本科研业务费[XJSYWFZX2008-16、YWFZX2010-4(N)];公益性行业(农业)科研专项(NYHYZX07-033-6);天然橡胶产业技术体系育种岗,海南省自然科学基金(309045)资助

摘　　要：转化酶是控制橡胶树胶乳蔗糖代谢和影响胶乳(橡胶)产量的关键酶。将已克隆的2个橡胶树胶乳转化酶基因(HbNIN1、HbNIN2)的编码区插入到原核表达载体pET28a上,经酶切和测序鉴定,成功获得了读框准确的转化酶表达载体(pET-HbNIN1和pET-HbNIN2);再将表达载体转化大肠杆菌表达菌株BL21(DE3),对其培养温度、IPTG诱导浓度和培养时间进行优化表达。结果表明,携带2个转化酶基因表达载体(pET-HbNIN1和pET-HbNIN2)的大肠杆菌转化子分别在28℃和37℃条件下,经浓度1.0 mmol/L IPTG诱导6 h实现了目标蛋白(HbNIN1和HbNIN2)的原核高效表达,表达量大于菌体总蛋白的20%,目标蛋白主要以包涵体形式存在。该结果为进一步研究这2种转化酶蛋白的分子特性和抗体制备奠定基础。As the rate-limiting enzyme involved in sucrose metoblism in the latex of rubber tree, the regulation of invertase imposes a great impact on the latex yeild. In this work, the coding sequences of two latex invertase genes （HbNIN1 and HbNIN2） previously cloned by the authors were cloned into the E. coli expression vector pET28a. Restriction analysis and sequencing confirmed the identity and the correct translation frame of the fusion （pET-HbNIN1 and pET-HbNIN2）. The invertase expression vectors were transformed into E. coli BL21 （DE3）, and the effect of temperature, IPTG concentration and culture time on the induction of target proteins were examined to optimize the expression conditions. The result showed that the E. coli transformants harboring the expression vectors pET-HbNIN1 and pET-HbNIN2 revealed a high level expression of fusion proteins （His6：HbNIN1 and 2） , respectively, at the temperatures of 28 ~C and 37℃, when induced by 1.0 mmol/L IPTG for 6 h. The fusion proteins accounted for more than 20% of the total cell proteins, and existed mainly in the form of inclusion bodies. This result provided a solid foundation for further studying the molecular characters of the two invertases and antibody preparation.

关 键 词：橡胶树 转化酶 载体构建 原核表达 

分 类 号：Q331[生物学—遗传学]