有机磷降解酶在烟草中的高效表达研究

有机磷化合物在农业生产中作为杀虫剂被广泛使用,在使用的同时也造成大面积的污染.本研究在烟草中表达细菌来源的有机磷降解酶基因ophc2,将有机磷降解酶分泌到植物体外,希望通过植物根系降解环境中的农药残留.构建了植物表达载体,包含CaMV 35S启动子序列、extensin序列、有机磷降解酶编码基因ophc2和nos终止子序列,命名为pBI-E-opphc2.胡萝卜extensin 基因的信号肽序列引导ophc2基因表达产物分泌到胞外.经过农杆菌介导的叶盘转化法转化烟草（Nicotiana tabacum cv. Xanthi）,共获得101株转化植株,PCR检测证实有80株含有目的基因片段.从80株中随机挑选60株经western杂交检测,有24株呈阳性.酶活性测定实验结果表明,转基因植株可以释放有机磷降解酶到培养基中,并且具有有机磷水解酶活性.对于转基因植物降解培养介质中和吸入植株中的有机磷农药的实际效果将在以后报道.

Expression of a Bacterial Organophosphorus Hydrolase in Transgenic Tobacco Plants

Organophosphorus(OP) compounds which widely used as pesticides in agriculture caused broad-area pollution to environment.In this study,we expressed a bacterial organophosphorus hydrolase(OPH) gene in tobacco plants in order to degrade pesticide residue in the environment by root secreted OPH.The expression vector was constructed.The final expression cassette,containing CaMV 35S promoter,the extensin sequence,ophc2 and nos termination sequence,was designated pBI-E-ophc2.The signal sequence from carrot extensin facilitated OPHC2 extracellular expression.The ophc2 gene then was transformed into tobacco(Nicotiana tabacum cv.Xanthi) leaf disks by Agrobacterium-mediated transformation.Of 101 transgenic lines,80 were shown to have the transgene by PCR analysis.24 of the randomly selected 60 transgenic plants were positive in western blot analysis.The assay of enzyme activity showed that the transgenic plants could secrete OPH into the growth medium and held the hydrolase activity.The degradation of pesticide in the culture medium and the transgenic plants tissue will be reported in the future.