Selection and regeneration of herbicide-resistant plant cells, and resistant enzymes

抗除草剂植物细胞和抗性酶的选择和再生

• 批准号: 05660044
• 负责人: USUI Kenji
• 金额: $ 1.34万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660044/
• 关键词: Herbicide resistance; Carrot suspension cells; rice suspension cells; Sulfonylurea herbicide; Acetolactate synthase; Less-sensitive enzyme; Branched-chain amino acids; 感受性低下; 分岐鎖アミノ酸

Sulfonylurea herbicides suppress plant growth by inhibiting acetolactate synthase, a key enzyme of branched-chain amino acid biosynthesis. Carrot and rice cells resistant to 10^<-7>M and 10^<-5>M bensulfuron methyl, respectively, were selected. Their branched-chain amino acid and soluble protein levels were less reduced and ALSs were less sensitive to BSM than nonselected susceptible cells.BSM-resistant carrot cells have cross-resistance to pyrazosulfuron-ethyl (PE) and imazaqwin (IQ). ALS inhibition was non-competitive (mixed) type by BSM and PE,and un-competitive by IQ to a substrate, pyruvate. Michaelis constant to pyruvate of resistant cells was a little higher than susceptible cells. Resistant ALS showed 100 times higher I_<50> (50% inhibition concentration) to BSM,but only several times to PE and IQ,showing less cross-resistances. Feed back inhibition by branched-chain amino acids were seen to same extent in both types of cells. Crude ALS extract from the cells were separated into two active peakes by gel-filtration colum chromatography. The activity of second peak was inhibited by both BSM and branched-chain amino acid, while that of resistant cells was less inhibited by BSM.Resistant cells had 1.5-several times higher branched-chain amino acid level, suggesting that the ALS was less sensitive to feed back inhibition. The composition of soluble proteins in the cells was not affected by BSM treatment, indicationg that the reduction of branched-chain amino acids level to lead to only decrease in peotein levels.These results suggest the mutation of ALS insensitive to herbicides is due to the herbicide resistance in suspension cultured plant cells.

磺酰脲类除草剂通过抑制支链氨基酸合成的关键酶乙酰乳酸合成酶抑制植物生长。选择分别对10 μ <-7>M和10 μ <-5>M苄嘧磺隆抗性的胡萝卜和水稻细胞。与未选择的感病细胞相比，支链氨基酸和可溶性蛋白质水平降低较少，ALS对BSM的敏感性较低，抗BSM的胡萝卜细胞对苯嘧磺隆（PE）和咪草胺（IQ）具有交互抗性。BSM和PE对ALS的抑制是非竞争性的，IQ对底物丙酮酸的抑制是非竞争性的。抗性细胞对丙酮酸的米氏常数略高于敏感细胞。抗ALS的菌株对BSM的I_<50>（50%抑制浓度）比对PE和IQ的I_（50%抑制浓度）高100倍，而对PE和IQ的I_（50%抑制浓度）仅高几倍，交叉抗性较低。在两种类型的细胞中，支链氨基酸的反馈抑制程度相同。细胞的ALS粗提物经凝胶过滤柱层析分离为两个活性峰。BSM和支链氨基酸均能抑制ALS第二峰的活性，但BSM对抗性细胞的抑制作用较小，抗性细胞的支链氨基酸水平高1.5倍至数倍，说明ALS对反馈抑制的敏感性较低。BSM处理对细胞可溶性蛋白质组成没有影响，表明支链氨基酸水平的降低只导致蛋白质水平的降低，说明ALS对除草剂不敏感的突变是由于悬浮培养的植物细胞产生了除草剂抗性。