鹰嘴豆α-淀粉酶抑制剂基因CL-AI的原核表达、纯化及生物学特性  

Prokaryotic Expression,Purification and Biological Characteristics Assay in vitro of α-amylase Inhibitor CL-AI From Chickpea

作　　者：贾彦凤[1] 李林芝[1] 魏家萍 王泽[2] 阿不都克玉木.米吉提 顾爱星[3] 张桦[3] 麻浩[1] 

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室,江苏南京210095 [2]新疆农业大学干旱区荒漠研究所,新疆乌鲁木齐830052 [3]新疆农业大学农学院,新疆乌鲁木齐830052

出　　处：《核农学报》2017年第5期852-861,共10页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金(30960206;31160306)

摘　　要：为研究鹰嘴豆种子中新贮藏蛋白类型α-淀粉酶抑制剂CL-AI的抑制活性、生物活性以及三级结构,本研究利用前期克隆的α-淀粉酶抑制剂基因CL-AI及其2个亚基CL-AI-α、CL-AI-β分别构建原核表达载体p E-SUMO3,经诱导表达获得了CL-AI、CL-AI-α的可溶性融合蛋白和CL-AI-β包涵体融合蛋白,其中CL-AI-β通过构建含不同标签的表达载体,最终实现了可溶性表达。人唾液淀粉酶(HSA)抑制活性试验结果表明,CL-AI,CL-AI-α,CL-AI-β3个蛋白对HAS都有一定的抑制作用。发芽试验表明,CL-AI蛋白对小麦、玉米、水稻种子发芽过程中的发芽势、根长和芽长影响显著,与加入无菌水的处理相比,种子的简化活力指数显著下降。本研究结果为CL-AI蛋白的后续研究与应用奠定了基础。To understand the new type of alpha amylase inhibitor CL-AI protein in Chickpea seeds, its inhibitory activity, biological activity, and three-dimensional structure were studied. A new type of α-amylase inhibitor CL-AI gene and CL-AI-α, CL-AI-β subunit gene were had been cloned from Chickpea seeds in our previous studies. The gene sequence of CL-AI, CL-AI-α, and CL-AI-β were inserted into the prokaryotic expression vector pE-SUMO3, respectively. CL-AI, CL-AI-α soluble fusion protein and CL-AI-β inclusion fusion protein were obtained from induced protein expression. CL-AI-β soluble fusion protein was obtained through the construction of prokaryotic expression vectors containing different tags. Effect of inhibitory activity against human salivary amylase （HSA） showed that CL-AI, CL-AI-α, CL-AI-β could significantly inhibit HSA. The effect of CL-AI on germination of wheat, maize, and rice had significant effects on germination potential, shoot and root length, and the simplified vigor index of three seeds showed a significant decrease. These results on the study of α-amylase inhibitor CL-AI has laid the foundation for further research and application.

关 键 词：Α-淀粉酶抑制剂 原核表达 蛋白纯化 发芽 

分 类 号：S529[农业科学—作物学]