狗尾草转酮醇酶抑制剂筛选方法的构建与应用  

Construction and application of screening method for inhibitors against Transketolase from Setaria viridis

作　　者：郑宇 王帅 栗猛超 杨冬臣 王彦恩[2] 张金林[1] ZHENG Yu;WANG Shuai;LI Mengchao;YANG Dongchen;WANG Yanen;ZHANG Jinlin(College of Plant Protection,Hebei Agricultural University,Baoding 071000,China;College of Science,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学植物保护学院,河北保定071000 [2]河北农业大学理学院,河北保定071000

出　　处：《河北农业大学学报》2023年第4期98-103,117,共7页Journal of Hebei Agricultural University

基　　金：国家自然科学基金资助项目(31871981).

摘　　要：为开发以转酮醇酶为靶标的新型除草剂,本研究以转酮醇酶为研究对象,通过PCR扩增技术,克隆狗尾草转酮醇酶基因,经酶切,连接等方法构建原核表达载体,琼脂糖凝胶电泳检测蛋白大小符合理论预测数值,经测序验证序列正确。通过原核表达技术获得狗尾草转酮醇酶蛋白,经SDS-PAGE电泳验证蛋白无误。采用pH-based法进行酶活测定,评价不同药剂处理下酶活抑制率,筛选得到RD194、ZBW3、ZBW39、ZBW34和ZBW115种药剂,酶活抑制率分别为81.25%、93.75%、81.25%、96.88%和87.50%,优于其它药剂;通过荧光定量PCR实验验证筛选,结果显示,除RD194外,其余4种药剂均使转酮醇酶基因转录水平显著上调,与预期一致。本研究构建了1种快速筛选转酮醇酶潜在抑制剂的方法,为靶向开发新型除草剂奠定重要基础。In order to develop new herbicides against a new target Transketolase,the Transketolase gene of Setaria viridis was cloned through PCR amplification and expressed with the prokaryotic expression system.The protein size was consistent with the expected value in the agarose gel of electrophoresis,and the sequence was confirmed by sequencing.The Transketolase protein of Setaria viridis was obtained by prokaryotic expression technology,and was verified by SDS-PAGE electrophoresis.The pH based method was used for enzyme activity determination to evaluate the enzyme activity inhibition rate under different drug treatments.Five drugs RD194,ZBW3,ZBW39,ZBW34,and ZBW11 were selected with enzyme activity inhibition rates of 81.25%,93.75%,81.25%,96.88%and 87.50%,respectively.The screening results were verified by fluorescence quantitative PCR experiment.The data showed that except RD194,four agents significantly increased the transcription level of Transketolase gene,which was consistent with expectations.In this paper,a rapid screening method for potential inhibitors of Transketolase was established,which laid an important foundation for targeted development of new herbicides.

关 键 词：转酮醇酶 原核表达 荧光定量PCR 药剂筛选 

分 类 号：S482.1[农业科学—农药学]