摘要
5-烯醇式丙酮莽草酸-3-磷酸合成酶(EPSPS)是莽草酸合成通路中关键的合成酶,也是除草剂草甘膦的靶向酶。为了研究明日叶EPSPS基因的结构及功能,基于明日叶转录组数据库信息,利用RT-PCR的方法克隆得到明日叶的EPSPS基因,命名为AgEPSPS,构建p ET-AgEPSPS原核载体,验证AgEPSPS基因功能。克隆得到明日叶AgEPSPS基因的ORF全长为1578 bp,编码525个氨基酸,分子质量为55.83 kD,等电点为7.93。它与胡萝卜的EPSPS蛋白具有高度的同源性;AgEPSPS基因在明日叶的不同组织中均有表达,其中叶片中的表达量最高。在3 mmol/L的草膦水溶液处理后,AgEPSPS基因表达量明显上调,在24 h后表达量最高。在抗性试验中,含有重组的pET-AgEPSPS质粒的大肠杆菌可以在80 mmol/L草甘膦的液体LB培养基中生长,说明其具有一定的草甘膦抗性。
5-Enolpyruvylshikimate-3-phosphate synthase(EPSPS)is a key enzyme in the biosynthetic pathway of aromatic amino acids and is a target enzyme of the herbicide glyphosate.In order to study the structure and function of EPSPS gene of Angelica keiskei,the study is based on a transcriptome database.In this study,the EPSPS gene was cloned from Angelica keiskei by RT-PCR,which is named AgEPSPS,the gene sequence and tissue specific expression were analyzed,aiming to construct a prokaryotic expression vector of p ET-AgEPSPS and verify the function of AgEPSPS gene.The AgEPSPS gene was obtained,containing a 1578 bp OFR and encoding a 525 amino acid protein,with a molecular mass of 55.83 k D and an isoelectric point of 7.93.Based on the homology alignment to other higher plants EPSP synthase gene.It has high homology with the EPSPS protein of carrot;Q PCR results showed that the AgEPSPS gene is expressed in leaves,stems and roots,with the highest expression in leaves.After treatment with 3 mmol/L glyphosate aqueous solution,we found that glyphosate can induce the up-regulation of AgEPSPS gene expression,which was highest after 24 h.In the resistance test,Escherichia coli containing the recombinant pET-AgEPSPS plasmid can be grown in 80 mmol/L glyphosate liquid LB medium,the result showed that it has glyphosate resistance.
作者
刘峰
阮盈盈
Liu Feng;Ruan Yingying(College of Landscape and Ecology,Ningbo City College of Vocational Technology,Ningbo,315100;School of Marine Sciences,Ningbo University,Ningbo,315211)
出处
《分子植物育种》
CAS
北大核心
2021年第24期8103-8108,共6页
Molecular Plant Breeding
基金
浙江省教育厅一般科研项目(Y201942657)
宁波城市职业技术学院校内科研重点专项(ZZX18123)共同资助。
