《植物生理学报》 2015, 51(7): 1091-1098

过表达拟南芥点突变乙酰羟酸合成酶基因改变植物对缬氨酸的抗性及增强缬氨酸合成

赵菲佚^*, 焦成瑾, 王太术, 田春芳, 谢尚强, 刘亚萍

天水师范学院生物工程与技术学院, 甘肃天水741001

通信作者：赵菲佚；E-mail: tspaulzhao@163.com；Tel: 0938-8362830

摘　要：

拟南芥乙酰羟酸合成酶(acetohydroxyacid synthase, AHAS)在支链氨基酸合成中具有重要的作用。为考察AHAS不同亚基关键位点突变对植物缬氨酸抗性与缬氨酸合成的影响, 对AHAS大小亚基上特定位点进行体外突变, 构建AHAS点突变过表达转基因植物, 研究AHAS不同亚基点突变转基因植物对缬氨酸抗性及其合成的影响。研究结果表明: AHAS小亚基G88D突变解除了终端产物对该酶的反馈抑制作用, 使转基因植物缬氨酸含量提高。大亚基E305D突变增强小亚基G88D突变效应, 而大亚基E482D突变对G88D突变具有相反的作用。AHAS全酶E305DG88D双突变转基因植物较E482DG88D具有更强的缬氨酸抗性表型和更高的缬氨酸含量。这些结果提示AHAS大小亚基间存在着相互作用, 大小亚基不同位点突变对AHAS全酶活性具有不同的影响。

关键词：乙酰羟酸合成酶; 点突变; 转基因植物; 缬氨酸抗性表型; 缬氨酸含量

收稿：2015-02-05   修定：2015-06-10

资助：国家自然科学基金(31160060和31260568)。

Overexpression of the Point Mutated Acetohydroxyacid Synthase Alters Resistance to Valine and Enhances Production of Valine in Arabidopsis

ZHAO Fei-Yi^*, JIAO Cheng-Jin, WANG Tai-Shu, TIAN Chun-Fang, XIE Shang-Qiang, LIU Ya-Ping

School of Bioengineering & Biotechnology, Tianshui Normal University, Tianshui, Gansu 741001, China

Corresponding author: ZHAO Fei-Yi; E-mail: tspaulzhao@163.com; Tel: 0938-8362830

Abstract:

Acetohydroxyacid synthase (AHAS) plays a pivotal role in the synthesis of brahched-chain amino acids (BCAAs) in Arabidopsis. To investigate effects of various specific mutated sites harboring in the large and small subunits of AHAS on resistance to valine and production of valine in Arabidopsis, transgenic plants overexpressing the point mutated AHAS were generated by site-directed mutagenesis, and the phenotype of resistance to valine and production of valine of the transgenic plants were evaluated in planta. The results showed that the G88D mutation in the small unit of AHAS abolished the feedback-resistant of valine to AHAS and this mutation resulted in increase of valine in the transgenic plants. The E305D mutation in the large unit of AHAS strengthens the effect of the G88D mutation. Interestingly, the E482D mutation in the large unit of AHAS acts antagonistically on the G88D mutation in resistance to valine and production of valine in transgenic plants. Compared with the combined double E482DG88D AHAS mutant transgenic plants, the E305DG88D AHAS transgenic plants exhibited the increased valine resistance and accumulated the more content of valine in planta. The results in this study suggested that the large unit interacts with the small unit of AHAS and different mutations in various sites of AHAS displayed distinct effects on the holoenzyme activity of AHAS.

Key words: acetohydroxyacid synthase; point mutation; transgenic plants; valine-resistance phenotype; valine content