Substitution of Ala-183 to Thr in 5-enolpyruvylshikimste 3-phoshpate synthase of E. coli (k12) and Transformation of Rapeseed (Brassica napus) with altered gene to tolerance of plant to Glyphosate
Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimste 3-phoshpate synthase (EPSPS),a key enzyme in the aromatic amino acid biosynthesis in microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity to glyphosate and transformation of it to plants is one of the most effective methods for production of glyphosate tolerant plants. In this research, we study on alanine183 of E. coli (k12) EPSPS enzyme. This amino acid is an important residue for EPSPS-Glyphosate configuration. We used site directed mutagenesis (SDM) method to inducing a point mutation in E. coli EPSPS gene to convert alanine183 to threonine (Ala183Thr). The manipulated EPSPS gene was cloned in pUC18 as a universal cloning vector and pBI121 as a plant expression vector. The molecular analysis and sequencing showed that the manipulated gene has been correctly changed and cloned in correct orientation in both plasmids. Recombinant pBI121 containing altered EPSPS gene was transferred to rapeseed (B. napus) via Agrobacterium tumeifaciens mediated transformation method. The transformed explants were screened in 25 mgL-1 kanamycin containing shoot induction medium. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there is a significant different between transgenic and control plants. Presence and copy numbers of construct in transgenic plants were confirmed with PCR and southern blotting analysis respectively.
Rapeseed (Brassica napus L.) Glyphosate resistance SDM Manipulated EPSPS and Gene transformation
Danial Kahrizi Ali Hatef Salmanian Amir Mousavi
Razi University, Faculty of Agriculture, Kermanshah, Iran National Institute for Genetic Engineering and Biotechnology, P.O.BOX: 14155-6343, Tehran, Iran
国际会议
第十二届国际油菜大会( The 12th International Rapeseed Congress)
武汉
英文
690-697
2007-03-26（万方平台首次上网日期，不代表论文的发表时间）