一种耐草甘膦的5-烯醇丙酮酰莽草酸-3-磷酸合酶的特性及植物表达

Characterization and plant expression of a glyphosate-tolerant enolpyruvylshikimate phosphate synthase.

作者信息

Vande Berg Brian J, Hammer Philip E, Chun Betty L, Schouten Laura C, Carr Brian, Guo Rong, Peters Cheryl, Hinson Todd K, Beilinson Vadim, Shekita Amy, Deter Rebekah, Chen Zhixian, Samoylov Vladimir, Bryant Charles T, Stauffer Maria E, Eberle Timothy, Moellenbeck Dan J, Carozzi Nadine B, Koziel Mike G, Duck Nicholas B

机构信息

Athenix Corp., 108 TW Alexander Drive, Research Triangle Park, NC 27709, USA.

出版信息

Pest Manag Sci. 2008 Apr;64(4):340-5. doi: 10.1002/ps.1507.

DOI:10.1002/ps.1507

PMID:18172892

Abstract

BACKGROUND

Glyphosate tolerance is a dominant trait in modern biotech crops.

RESULTS

A gene encoding a glyphosate-tolerant EPSP synthase (aroA(1398)) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA(1398) protein was expressed and purified from E. coli, and key kinetic values were determined (K(i) = 161 microM; K(m)(PEP) = 11.3 microM; k(cat) = 28.3 s(-1)). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA(1398), transgenic maize events expressing the aroA(1398) protein were generated. T(0) plants were screened for tolerance to glyphosate sprays at 1.3x commercial spray rates, and T(1) plants were selected that completely resisted glyphosate sprays at 1x, 2x and 4x recommended spray rates in field trials.

CONCLUSION

These data suggest that aroA(1398) is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants.

摘要

背景

草甘膦耐受性是现代转基因作物的一个显性性状。

结果

克隆并鉴定了来自细菌菌株ATX1398的一个编码草甘膦耐受性5-烯醇丙酮酰莽草酸-3-磷酸合酶（aroA(1398)）的基因。该蛋白由一个GTG翻译起始密码子起始，产生一种在大肠杆菌（Mig）Cast & Chalm中赋予强大草甘膦抗性的蛋白。aroA(1398)蛋白在大肠杆菌中表达并纯化，测定了关键动力学值（K(i)=161微摩尔；K(m)(PEP)=11.3微摩尔；k(cat)=28.3秒(-1)）。全长酶对草甘膦的抗性比玉米5-烯醇丙酮酰莽草酸-3-磷酸合酶高800倍，同时对底物磷酸烯醇丙酮酸保持高亲和力。为了进一步评估aroA(1398)的潜力，产生了表达aroA(1398)蛋白的转基因玉米事件。对T(0)代植株进行了1.3倍商业喷雾率的草甘膦喷雾耐受性筛选，在田间试验中选择了在1倍、2倍和4倍推荐喷雾率下完全抗草甘膦喷雾的T(1)代植株。