耐草甘膦菜豆耐性分子学机理研究  

Studies on Molecular Mechanism of Glyphosate Tolerance in Bean

作　　者：向文胜[1] 李孱[1] 陶波[1] 王相晶[1] 李景鹏[1] 苏少泉[1] 张文吉[2] 

机构地区：[1]东北农业大学生命科学与生物技术研究中心,哈尔滨150030 [2]农业部农药化学及农药使用技术重点开放实验室中国农业大学基础学院应用化学系,北京100094

出　　处：《农药学学报》2001年第1期23-29,共7页Chinese Journal of Pesticide Science

基　　金：国家自然科学基金!(39670498)资助项目

摘　　要：用耐性菜豆根尖为材料，以λgt10为载体获得了4.8×105个重组噬菌体的cDNA文库。以植物EPSP合成酶基因保守序列合成二段探针，经噬菌体原位杂交筛选出了阳性克隆，并进行了酶切鉴定。测定 cDNA 序列长度为2024个核苷酸，其中编码长1569个核苷酸，共编码523个氨基酸和一个终止密码子，与已发表的其它植物成熟EPSP合成酶氨基酸序列相比具有很高的同源性（≥84.8%），但进入叶绿体运输肽的氨基酸同源性低。以耐性菜豆EPSP合成酶的cDNA序列为模板，用RT-PCR方法扩增感性菜豆EPSP合成酶cDNA片段，并克隆于pUC18质粒上，经测定序列并比较发现：感性菜豆EPSP合成酶cDNA核苷酸序列1737位碱基为G，而耐性的为C，从而表达出的513位氨基酸残基感性的为E，耐性的为Q，表明两种菜豆EPSP合成酶cDNA核苷酸序列一位点的差异是它们对草甘膦耐性不同的原因。To reveal molecular basis of glyphosate tolerant bean, the cDN A library of tolerant bean root tip was constructed with λ gt10 as vector. The number of recombinant phages is 4.8×105 .Using two synthetic fragments as pro be, positive clone was obtained by in situ hybridization of λ plaques,and a lso identified by restrict enzyme digestion and Southern hybridization. Sequ ence of cDNA of bean EPSP synthase contains 2 024bp, and the coding region o f 1 569bp codes for a polypeptide of 523 amino acids and one te rmination codon. Comparison of the amino acid sequence homology among many pla nt EPSP synthases indicate that the mature EPSP synthases are highly conserved proteins (more than 84.8%). But there are lower homologous in the chloroplast t ransi t peptide portion. Using the tolerant bean EPSP synthase cDNA sequence informa tion, cDNA fragment of susceptible bean EPSP synthase was amplified by RT-PC R. PCR products cloned into pUC18 and sequenced. The nucleotide sequence ana lysis of cD NAs of the two EPSP synthase revealed a single-point difference, which acco mpanied with one animo acid difference. From these results, it was clarified tha t this single-point difference of nucleotide was the cause of glyphosate-toler ance in the tolerant bean.

关 键 词：菜豆 草甘膦 EPSP合成酶 CDNA克隆 耐性机理 核苷酸序列 

分 类 号：S436.43[农业科学—农业昆虫与害虫防治] S482[农业科学—植物保护]