转5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)基因抗草甘膦烟草和棉花的获得  

Generation of Glyphosate-tolerant Transgenic Tobacco and Cotton by Transformation with a 5-enolpyruvyl-shikimate-3-phosphate Synthase (EPSPS) Gene

作　　者：刘锡娟[1] 刘昱辉[1] 王志兴[1] 王旭静[1] 张永强[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《农业生物技术学报》2007年第6期958-963,共6页Journal of Agricultural Biotechnology

基　　金：国家转基因植物研究与产业化开发专项(No.JY03-B-06)资助。

摘　　要：以从抗草甘膦的荧光假单胞菌(Pseudomonas fluorescens)G2中克隆的、并按双子叶植物偏爱密码子改造的5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)基因aroAG2M为目的基因,用菊花Rubisco小亚基的启动子驱动,在基因的5'端加叶绿体定位信号肽,构建植物表达载体。用根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘法转化烟草(Nicotiana tabacum)获得转基因植株,PCR检测表明,外源基因已整合到烟草基因组中。转基因和非转基因植株6～8叶龄苗的叶片涂抹不同浓度的草甘膦异丙胺盐,表明转基因植株可抗4‰浓度的草甘膦,而非转基因对照植株则在2‰草甘膦时即死亡。花粉管通道法转化棉花(Gossypium hirsutum),得到3株具有草甘膦抗性的转基因植株,PCR和Southern检测显示,外源基因已整合到棉花基因组中,田间喷洒草甘膦异丙胺盐水剂,表明T1代转基因植株具有草甘膦抗性。The target gene used in this study was 5-enolpyruvyl-shikimate-3-phosphate synthase（ EPSPs ） encoding gene aroAG2M which was cloned from Pseudomonas fluorescens G2 sWain and modified with dicotyledonous plant-preferred codon usage. Plant expression vector was constructed by adding a chloroplast signal peptide at 5＇ end of the target gene and hooked with the chrysanthemum Rubisco small subunit promoter. Transgenic tobacco （Nicotiana tabacum） plants obtained via Agrobacterium-mediated leaf disc transformation were confirmed by PCR analysis. Leaves of transgenic and non-transgenic tobacco plants at 6～8 leaf stage were daubed with different concentrations of glyphosate. Results showed that the transgenic tobacco was tolerant to glyphosate up to 4‰ concentration, while the non-tmnsgenic plants died at the concentration of 2‰. The plasmid of plant expression vector was also used for cotton （Gossypium hirsutum） transformation by pollen tube pathway method. PCR and Southern blotting analysis oftransgenic cotton showed that the target gene was integrated into cotton genome. To date, three transgenic cotton plants of T1 generation have been obtained which show tolerance to the herbicide glyphosate.

关 键 词：5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS) aroAG2M基因 草甘膦 抗除草剂 烟草 棉花 

分 类 号：S188[农业科学—农业基础科学]