牛筋草PFK蛋白多克隆抗体的制备及其在草甘膦抗性鉴定上的应用  

Preparation of polyclonal antibody against PFK protein and application in identification of glyphosate resistance in Eleusine indica

作　　者：陈景超[1] 李志玲 崔海兰[1] 于海燕 马宇菲 李香菊[1] CHEN Jingchao;LI Zhiling;CUI Hailan;YU Haiyan;MA Yufei;LI Xiangju(State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Jining Normal University,Ulanqab 012000,China)

机构地区：[1]中国农业科学院植物保护研究所,植物病虫害综合治理全国重点实验室,北京100193 [2]集宁师范学院,乌兰察布012000

出　　处：《植物保护》2024年第5期199-204,共6页Plant Protection

基　　金：科技创新2030重大项目(2022ZD04021);中国农业科学院国家南繁研究院“南繁专项”(SWAQ03);国家西甜瓜产业技术体系(CARS-25)。

摘　　要：抗草甘膦牛筋草中磷酸果糖激酶(PFK)与靶标酶5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)的含量存在线性关系,是牛筋草对草甘膦产生抗性的指示蛋白之一。为实现抗草甘膦牛筋草的快速鉴定,本研究克隆了牛筋草PFK全长基因,构建原核表达载体,并免疫大兔最终获得多克隆抗体,进一步对抗体的特异性和灵敏性进行了检测,并用该抗体对抗性牛筋草进行了检测鉴定。结果发现,PFK基因全长1656 bp,编码551个氨基酸,该基因的原核表达载体pGEX-4T-1-PFK经诱导后表达的蛋白大小为45 kD,蛋白纯化后浓度为3.5 mg/mL。制备的多克隆抗体能特异地识别牛筋草PFK蛋白,稀释512000倍后仍可检测到抗原,并能够准确鉴定PFK不同表达量的抗性牛筋草。结果表明:本研究制备的PFK多克隆抗体具有特异性强、灵敏度高的特点,可鉴定EPSPS过表达的抗草甘膦牛筋草,这为牛筋草对草甘膦抗药性的快速鉴定提供了实用工具。Phosphofructokinase(PFK)is one of the indicator proteins for glyphosate resistance in goosegrass Eleusine indica as the content of PFK is positive correlation with the target 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).In this study,the full-length of PFK gene was cloned from E.indica,the prokaryotic expression vector pGEX-4T-1-PFK was constructed and polyclonal antibodies were obtained by immunizing rabbits.Then,the specificity and sensitivity of the antibody were detected.Results showed that the full length of PFK gene is 1656 bp,encoding 551 amino acids.A 45 kD recombinant protein with the concentration of 3.5 mg/mL was obtained after IPTG induction and purification.The antibody could only specifically recognize the PFK protein of E.indica,and the antigen can still be detected when antibody was diluted by 512000 times.The antibody could quickly identify the PFK protein and accurately identify the resistant goosegrass with different levels of PFK expression.These results suggested the PFK polyclonal antibody could identify glyphosate-resistant E.indica caused by EPSPS overexpression.This provides a practical tool for rapid identification of resistance to glyphosate for E.indica.

关 键 词：磷酸果糖激酶 牛筋草 多克隆抗体 草甘膦 抗药性 

分 类 号：S451.1[农业科学—植物保护]