红假单胞菌立体选择性降解菊酯类农药机理及降解酯酶基因克隆

There are 1-3 chiral carbon atom(s) in molecular structure of pyrethroids, and correspondingly, 2 n isomers are concurrence in pyrethriods, but the insecticidal activity is only existing in one or few isomer(s). Lots of no insecticidal activity isomer(s) pour into entironment because of using of racemic pyrethroids in agricultural production process, resulted to severe threatening to entironment. So it is very important to uncover how to reduce the no insecticidal activity of isomer(s) of pyrethroids, for alleviating damage to entironment and human besing self and raising the profit of agricultural production. One of very important approach of cleaning pesiticides in entironment is microbial degradation, and is also enantioselective to chiral pesicides. This research project aimed to clarify enantioselective degradation of main pyrethroid by Rhodopseudomonas sp. PSB07-21, which screened in our former studies. In addition, the trails of cloning of the degradation enzyme gene and 3-D structure analysis of enzyme will perform, and then, muton(s) of this gene with function of degrading different isomer(s) of pyrethroids will construct by direct evolution strategy. The congruent relationship between 3-D structure of degradaion enzyme and enantioselective degradation should be deduced. The conclusions of this research project should be importantly for yielding insecticidal isomer(s) of pyrethroids by enantioselective degradation enzyme.

菊酯类农药分子结构中含有1-3个手性中心，有2个至多个异构体，而其杀虫活性成分只存在于1个或少数几个异构体。目前使用的菊酯类农药绝大多数是外消旋体，大量无活性的菊酯类农药异构体投入到环境中，给生态环境、人类自身健康带来严重威胁。因此，研究减少菊酯类农药无活性异构体的投入方法，既能减轻对生态环境的危害，又能增加防治效果、降低生产成本。微生物降解是生态环境中清除农药残留的一种重要途径，且微生物对手性污染物具有立体选择性降解作用。因此，本项目在前期研究红假单胞菌降解菊酯类农药残留物的基础上，研究红假单胞菌立体选择性降解菊酯类农药特性与机理，克隆降解酯酶基因、并构建筛选降解不同异构体的突变子，分析突变子酯酶三维构型，推导降解酯酶基因预测蛋白的构型与菊酯类农药分子构型之间的对应关系。本研究为利用立体选择性降解酶生产富含活性异构体的高效菊酯类农药提供理论依据。

以菊酯类农药甲氰菊酯作为选择压，筛选并鉴定高效降解微生物菌株6个（沼泽红假单胞菌）；测定降解微生物菌株PSB-S的全基因组序列（GenBank登录号JHAA00000000）。在菌株PSB-S全基因组序列分析基础上，从菌株PSB-S中克隆高效降解菊酯类农药的降解酯酶基因3个，采用原核表达方式，获得3个降解酯酶，研究了3个降解酯酶降解菊酯类农药的特性。采用同源建模方式（SWISS-MODEL），构建了3个降解酯酶的分子构型，分析了降解酯酶结构域菊酯类农药降解对应关系。建立了菊酯类农药立体选择性异构体高效检测方法，筛选获得选择性降解微生物红假单胞菌菌株1个。