水稻中类受体蛋白激酶FERONIA-like Receptor 1的表达纯化及抗体制备  

Expression, Purification and Antibody Preparation of Rice FERONIA-like Receptor 1

作　　者：吴秀秀 蒋顺 于峰[2] 曹建中 汪龙 WU Xiu-xiu;JIANG Shun;YU Feng;CAO Jian-zhong;WANG Long(College of Food Science and Engineering/National Engineering Laboratory for Deep Processing of Rice and By-products,Central South University of Forestry and Technology,Changsha 410004,Hunan,China;College of Biology,Hunan University,Changsha 410082,Hunan,China)

机构地区：[1]中南林业科技大学食品科学与工程学院/稻谷及副产物深加工国家工程实验室,中国湖南长沙410004 [2]湖南大学生物学院,中国湖南长沙410082

出　　处：《生命科学研究》2019年第5期393-400,416,共9页Life Science Research

基　　金：湖南省研究生创新基金资助项目(CX2016B343);中南林业科技大学研究生创新基金资助项目(CX2016A03)

摘　　要：类受体蛋白激酶参与调控植物的器官发育和抗逆性,是一种重要的蛋白激酶。通过对水稻中类受体蛋白激酶家族成员FERONIA-like receptor 1 (FLR1)进行蛋白质结构预测分析,发现FLR1蛋白具有跨膜结构,其N端在细胞膜外(FLR1-BW, 1~450 aa), C端在细胞膜内(FLR1-BN, 474~892 aa)。为了得到FLR1抗体用于其功能分析,将FLR1的N端和C端分别构建到原核表达载体pET-32a、pGEX-4T-1、pET-22b、pTriEx-4上,比较FLR1-BW和FLR1-BN在不同载体和不同条件下的表达效果。结果显示:FLR1-BW在pET-32a及pGEX-4T-1中能够有效表达,最优条件为16℃/16 h、0.5 mmol/L IPTG;而FLR1-BN蛋白表达后会对大肠杆菌产生细胞毒性,但在293细胞中能够表达。进一步利用纯化的FLR1-BW蛋白制备FLR1多克隆抗体,结果显示该抗体特异识别FLR1蛋白。本研究为类受体蛋白激酶的抗体制备提供了良好的思路并为研究FLR1功能奠定了基础。The rice FERONIA-like receptor 1(FLR1) is a member of receptor-like kinase involving in rice development and stress resistance. Bioinformatics predicted that FLR1 protein can be divided into N-terminal(FLR1-BW, 1~450 aa) and C-terminal(FLR1-BN, 474~892 aa) domains. To elucidate the function of FLR1,its N-terminal and C-terminal domains were cloned into expression vectors pET-32 a, pGEX-4 T-1, pET-22 b,and pTriEx-4, respectively. The plasmids were transformed into BL21(DE3) and proteins were expressed under different conditions. The results showed that FLR1-BW was successfully expressed in E. coli from pET-32 a and pGEX-4 T-1 and the optimal conditions were 16 ℃/16 h with 0.5 mmol/L IPTG. The recombinant FLR1-BW was purified to homogeneity and used for antibody production in rats. However, FLR1-BN protein was cytotoxic to E. coli, but was expressed successfully in 293 cells. The research put forward a new strategy for receptor-like kinase antibody preparation and laid a foundation for studying the functions of FLR1 in the future.

关 键 词：水稻 FLR1 蛋白质表达纯化 抗体制备 

分 类 号：Q946.1[生物学—植物学] S511.22[农业科学—作物学]