|
|
Analysis of The Active Sites in An 5-enolpyruvy-shikimate-3-phosphate Synthase (EPSPS) Gene of AM79 aroA |
2, 3, 3, 3, 3 |
|
|
Abstract AM79 aroA (WO/2009/059485), an novel 5-enolpyruvy-shikimate-3-phosphate synthase (EPSPS) gene, is isolated from glyphosate-polluted soil using metagenomics method and showed high glyphosate resistance. However, the function mechanism of the high glyphosate resistance of AM79 aroA remains unknown. Site-directed mutagenesis to uncover the function of some specific amino acids in AM79 EPSPS was performed. Phylogenetic tree analysis showed that AM79 EPSPS was a typeⅠEPSPS. Sequence alignment indicated that the 107th Alanine (Ala), the 114th Phenylalanine (Ala), the 355th Alanine and the 356th Histidine (His) were symbols to distinguish the AM79 EPSPS from other EPSPS. To explain the function of these specific amino acids in AM79 EPSPS, point mutations were carried out via overlap extension PCR. AM79 aroA and the mutant gene were transformed into aroA defective strain ER2799, then the glyphosate resistance of the transformed strains were identified. The results showed that mutants of Phe114, Ala355 and His356, respectively, led to the decrease of glyphosate resistance, indicating that these amino acids were essential for the enzyme activity and glyphosate resistance of AM79 EPSPS. Homology modeling analysis showed that the mutation of these amino acids changed the structures of the mutated AM79 EPSPS and this might be the reason for the decreased glyphosate resistance. We hypothesized that the highlighted amino acids in AM79 EPSPS might have similar function in other typeⅠEPSPS and reverse mutations on these amino acids of Pseudomonas fluorescens G2 EPSPS and Arabidopsis thaliana EPSPS (AtEPSPS) were performed. However, the mutations of these key amino acids didn't enhance the glyphosate resistance of G2 EPSPS and AtEPSPS. This study can be useful for enhancing the glyphosate resistance of AM79 EPSPS by directed evolution process.
|
Received: 06 November 2014
Published: 17 March 2015
|
|
|
|
陈荣荣,曹高燚,刘允军. 2014. 拟南芥5-烯醇式丙酮酰-莽草酸-3-磷酸合成酶基因(EPSPS)的定点突变及抗草甘膦转基因拟南芥获得[J].农业生物技术学报,22 (4): 397-405.(Chen, R.R., Cao, G.Y., and Liu, Y.J. (2014). Site-specific Mutagenesis of the Arabidopsis Gene 5-enolpyruvy-shikimate-3-phosphate Synthase (EPSPS) to Gain Glyphosate-resistant Transgenic Arabidopsis thaliana [J].Journal of Agricultural Biotechnology 22, 397-405.)强胜, 宋小玲, 戴伟民. 2010. 抗除草剂转基因作物面临的机遇与挑战及其发展策略[J]. 农业生物技术学报, 18(1):114-125.(Qiang S, Song X L, Dai W M. 2010. The opportunity and challenge faced by transgenic herbicide resistant crops and their development strategy[J].Journal of Agricultural Biotechnology, 18(1): 114-125.)Arnold, K., Bordoli, L., Kopp, J., et al. (2006). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling [J]. Bioinformatics 22, 195-201.Boocock, M.R., and Coggins, J.R. (1983). Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate [J]. FEBS Letters 154, 127-133.Cao, G., Liu, Y., Zhang, S., et al. (2012). A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants [J]. PLoS One 7, e38718.Castle, L.A., Siehl, D.L., Gorton, R., et al. (2004). Discovery and directed evolution of a glyphosate tolerance gene [J]. Science 304, 1151-1154.Duke, S.O., Lydon, J., Koskinen, W.C., et al. (2013). Correction to Glyphosate Effects on Plant Mineral Nutrition, Crop Rhizophere Microbiota, and Plant Disease in Glyphosate-Resistant Crops [J]. Journal of Agricultural and Food Chemistry 61, 12745-12745.Dun, B.Q., Lu, W., Ping, S.Z., et al. (2006). Isolation of a glyphosate tolerance N-acetyltransferase gene and expression in E.coli [J]. High Technology Letters 16, 943-947.Franz, J.E., Mao, M.K., and Sikorski, J.A. (1997). Glyphosate:a unique global herbicide. American Chemical Society [J]. Monograph 189, 521-615.Funke, T., Han, H., Healy-Fried, M.L., et al. (2006). Molecular basis for the herbicide resistance of Roundup Ready crops [J]. Proceedings of the National Academy of Sciences of the USA 103, 13010-13015.Funke, T., Yang, Y., Han, H., et al. (2009). Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli [J]. The Journal of Biological Chemistry 284, 9854-9860.Gaines, T.A., Wright, A.A., Molin, W.T., et al. (2013). Identification of genetic elements associated with EPSPs gene amplification [J]. PLoS One 8, e65819.Gaines, T.A., Zhang, W., Wang, D., et al. (2010). Gene amplification confers glyphosate resistance in Amaranthus palmeri [J]. Proceedings of the National Academy of Sciences of the USA 107, 1029-1034.Ge, X., d'Avignon, D.A., Ackerman, J.J., et al. (2010). Rapid vacuolar sequestration: the horseweed glyphosate resistance mechanism [J]. Pest Management Science 66, 345-348.Ge, X., d'Avignon, D.A., Ackerman, J.J., et al. (2012). Vacuolar glyphosate-sequestration correlates with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe: a 31P NMR investigation [J]. Journal of Agricultural and Food Chemistry, 1243-1250.Healy-Fried, M.L., Funke, T., Priestman, M.A., et al. (2007). Structural basis of glyphosate tolerance resulting from mutations of Pro101 in Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase [J]. The Journal of Biological Chemistry 282, 32949-32955.Li, L., Lu, W., Han, Y., et al. (2009). A novel RPMXR motif among class II 5-enolpyruvylshikimate-3-phosphate synthases is required for enzymatic activity and glyphosate resistance [J]. Journal of Biotechnology 144, 330-336.Park, H., Hilsenbeck, J.L., Kim, H.J., et al. (2004). Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate-bound state and S3P-GLP-bound state [J]. Molecular Microbiology 51, 963-971.Paulsen, I.T., Press, C.M., Ravel, J., et al. (2005). Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5 [J]. Nature Biotechnology 23, 873-878.Perez-Jones, A., Park, K.W., Polge, N., et al. (2007). Investigating the mechanisms of glyphosate resistance in Lolium multiflorum [J]. Planta 226, 395-404.Salas, R.A., Dayan, F.E., Pan, Z., et al. (2012). EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas [J]. Pest Management Science 68, 1223-1230.Schonbrunn, E., Eschenburg, S., Shuttleworth, W.A., et al. (2001). Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail [J]. Proceedings of the National Academy of Sciences of the USA 98, 1376-1380.Siehl, D.L., Castle, L.A., Gorton, R., et al. (2007). The molecular basis of glyphosate resistance by an optimized microbial acetyltransferase [J]. Journal of Biological Chemistry 282, 11446-11455.Sun, Y.C., Chen, Y.C., Tian, Z.X., et al. (2005). Novel AroA with high tolerance to glyphosate, encoded by a gene of Pseudomonas putida 4G-1 isolated from an extremely polluted environment in China [J]. Applied and Environmental Microbiology 71, 4771-4776.Tan, S., Evans, R., and Singh, B. (2006). Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops [J]. Amino Acids 30, 195-204.Tian, Y.S., Xu, J., Xiong, A.S., et al. (2011). Improvement of glyphosate resistance through concurrent mutations in three amino acids of the Ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase [J]. Applied and Environmental Microbiology 77, 8409-8414.Tian, Y.S., Xu, J., Peng, R.H., et al. (2013). Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance [J]. Plant Biotechnology Journal 11, 829-838.Tzin, V., Malitsky, S., Ben Zvi, M.M., et al. (2012). Expression of a bacterial feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the shikimate pathway in Arabidopsis elucidates potential metabolic bottlenecks between primary and secondary metabolism [J]. New Phytologist 194, 430-439.Vila-Aiub, M.M., Gundel, P.E., Yu, Q., et al. (2013). Glyphosate resistance in Sorghum halepense and Lolium rigidum is reduced at suboptimal growing temperatures [J]. Pest Management Science 69, 228-232.Xu, K., Zhang, H., Blumwald, E., et al. (2010). A novel plant vacuolar Na+/H+ antiporter gene evolved by DNA shuffling confers improved salt tolerance in yeast [J]. The Journal of Biological Chemistry 285, 22999-23006.Yan, H.Q., Chang, S.H., Tian, Z.X., et al. (2011). Novel AroA from Pseudomonas putida confers tobacco plant with high tolerance to glyphosate [J]. PLoS One 6, e19732.Yu, Q., Abdallah, I., Han, H., et al. (2009). Distinct non-target site mechanisms endow resistance to glyphosate, ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant Lolium rigidum [J]. Planta 230, 713-723.Zhou, M., Xu, H., Wei, X., et al. (2006). Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy [J]. Plant Physiology 140, 184-195. |
|
|
|