【摘要】 5-烯醇式丙酮莽草酸-3-磷酸合成酶(EPSPS)是莽草酸途径中的一个重要酶,它是非选择性除草剂草甘膦的靶标酶,在高等植物中定位于叶绿体质膜上。根据EST拼接的序列设计引物,从陆地棉品种珂字棉312中获得了全长为1834 bp的cDNA序列,其最大可读框为1565bp,编码521个氨基酸。陆地棉EPSPS基因与其它植物中同类酶在氨基酸水平上有广泛的同源性。通过与已知的其它高等植物叶绿体转运肽剪切位点比对,推断棉花EPSPS基因含有74个氨基酸叶绿体运输肽和447个氨基酸组成的熟蛋白。该酶具有保守的PEP结合位点及催化位点的特征序列。半定量分析表明,该基因产物广泛存在于棉花根、茎和叶等各组织中,在叶片中表达量较高。进一步扩增棉花核基因组获得了3344 bp的DNA序列,它包含8个内含子和7个外显子。棉花EPSPS基因的克隆为抗草甘膦棉花种质资源的创制提供了理论基础。更多还原

【Abstract】 5-Enolpyruvylshikimate-3-phosphate(EPSP) synthase is an enzyme of the shikimate pathway.It is the target enzyme of nonselective herbicide glyphosate.This enzyme is located in the chloroplasts of the higher plants.Primers were designed according to the assembled ESTs of Gossypium hirsutum,and a 1834 bp cDNA fragment of G.hirsutum cv.Coker 312 was obtained in this experiment.The frafment contains an ORF comprising of 1565 nucleotides which encodes 521 amino acid.A comparison of the deduced amino acid sequences of cotton and other higher plants genes reveals highly homology over the length of the entire mature protein.The site of transit peptides cleavage is deduced from a comparison to the known cleavage site in other plants.The enzyme,including 74 amino acid chloroplast transit peptides and 447 amino acid mature proteins,comprises conserved region of PEP binding sites and the characteristic sequence of catalytic activity sites.Semi-quantitative analysis of EPSP synthase expression showed that the gene expressed extensively in cotton roots,stems,leaves,and other organs,and leaf is the most highly expressed tissue.A genomic DNA sequence with 3344 bp clone containing an EPSP synthase was isolated from the upland cotton in this experiment,and the EPSPS gene contains eight introns and seven exons.更多还原