耐草甘膦菜豆耐性酶学机理的研究

感性、耐性菜豆从萌芽到24d的EPSP合成酶比活性是不同的，但变化规律相似。出苗时比活性较高，10d时降至最低点，随后上升，16d时达到最高点，然后缓慢下降；两种菜豆在生长的同一天酶比活性差异小，最大倍数仅为1.1；用草甘膦处理两种三出复叶时的菜豆1d后酶比活性上升15%～25%。实验测定了经4步纯化，比活性达6219.4nmol· min-1· mg-1蛋白以上的两种菜豆EPSP合成酶的一些酶学性质。PEP是酶的最适底物，感性、耐性 Km(PEP)分别为3.5和7.1μmol· L-1,两者相差2倍,亲和力Ki(草甘膦)分别是6.2和 16.7μmol· L-1,两者相差2.7倍。两种菜豆对草甘膦耐性不同的酶学机理在于耐性菜豆的EPSP合成酶与草甘膦的亲和力小，与酶底物亲和力大；而感性菜豆EPSP酶则与草甘膦的亲和力大，与其底物亲和力小。

Studies on Enzymology Mechanism of Glyphosate Tolerance in Bean

During the seedling period, EPSP synthase specific activities were different between susceptible and tolerant beans. While the developmental patterns were similar, the two beans EPSP synthase specific activities declined during the first days after germination, then increased to reach a maximum 16 days later, and thereafter began to slowly decrease again. During the same growing stage, enzyme level maximum difference was only 1.1 times between the two beans. After treatment with glyphosate for 24 hours, the two beans enzyme activities increase 15%～20%. Some properties of EPSP synthase purified from susceptible and tolerant beans were examined. The enzyme's specific activity reached more than 6219 nmol·min-1 ·mg-1 protein by a four-step purification. The optimum substrate of susceptible and tolerant bean EPSP synthase Km(PEP) values were 3.5 and 7.1 μmol·L-1 ,respectively. Difference was 2 times between two kinds of beans.Susceptible K i(glyphoste) values were 6.2 and 16.7 μmol·L-1 ,respectively. Difference was 2.7 times between the two kinds beans. It has shown that affinity of tolerant bean EPSP synthase to glyphosate is weaker, to PEP is stronger, while affinity of susceptible bean EPSP synthase to glyphosate is stronger, to PEP is weaker. That's the reason that they are different in glyphosate tolerance