水稻咖啡酰辅酶A-O-甲基转移酶基因的原核表达、抗体制备和应用  

Prokaryotic Expression,Antibody Preparation and Application of Rice Caffeoyl Coenzyme A-O-methyltransferase Gene

作　　者：索青青 吴楠 杨慧 李莉[1] 王锡锋[1] SUO Qing-qing;WU Nan;YANG Hui;LI Li;WANG Xi-feng(State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193)

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193

出　　处：《生物技术通报》2022年第8期135-141,共7页Biotechnology Bulletin

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2019YFE0108500)。

摘　　要：咖啡酰辅酶A-O-甲基转移酶(caffeoyl coenzyme A-O-methyltransferase,CCoAOMT)在植物木质素生物合成过程中发挥重要作用。目前关于水稻咖啡酰辅酶A-O-甲基转移酶的研究主要集中在基因水平,对其在蛋白水平上的功能研究却相当少。通过PCR扩增技术克隆得到OsCCoAOMT1基因,并成功构建了原核表达载体pET30a(+)-OsCCoAOMT1,转化大肠杆菌,表达出了重组蛋白。蛋白经纯化后,注射新西兰公兔,成功制备了多克隆抗体。Western blot检测表明该抗体能特异性地结合水稻不同组织的该蛋白。利用制备的抗体对水稻茎原生质体进行免疫荧光标记,激光共聚焦显微镜观察发现在Ubi:OsCCoAOMT1-GFP转基因水稻和野生型水稻原生质体里均可标记到该蛋白的信号,且转基因水稻融合蛋白的GFP信号可以与该蛋白抗体的荧光信号共定位,同时结合水稻核质蛋白分离的Western blot检测结果可知OsCCoAOMT1蛋白在主要分布在细胞质中。表明制备的抗体能用于该蛋白的特异性检测,为后续对水稻咖啡酰辅酶A-O-甲基转移酶的功能研究奠定了良好基础。Caffeoyl-coenzyme A-O-methyltransferase(CCoAOMT)plays an important role in the biosynthesis of plant lignin.At present,the research on rice CCoAOMT mainly focuses on the gene level,only few researches on its function at the protein level.In this paper,OsCCoAOMT1 gene was cloned by PCR amplification technology,and prokaryotic expression vector pET30a(+)-OsCCoAOMT1,then transformed into Escherichia coli,and the recombinant protein was expressed.Purified protein immunized New Zealand male rabbits of polyclonal antibodies were successfully prepared.Western blot analysis showed that the antibody specifically bound to the protein in different tissues of rice.Immunofluoresent labeling assay revealed that the signal of this protein can be marked in Ubi:OsCCoAOMT1-GFP transgenic rice and wild-type rice protoplasts via laser confocal microscopy.Furthermore,the GFP signal of the transgenic rice fusion protein can be colocalized with the fluorescent signal of the protein antibody.Combined with the Western blot analysis of rice nucleoplasm protein isolation,OsCCoAOMT1 protein is mainly distributed in the cytoplasm.This indicated that the prepared antibody can be used for the specific detection of the protein and laid a good foundation for the subsequent functional research of rice CCoAOMT.

关 键 词：咖啡酰辅酶A-O-甲基转移酶 原核表达 多克隆抗体 Western blot 免疫荧光标记 

分 类 号：Q943.2[生物学—植物学]