轉哺乳動物cyp2e1基因煙草植株再生及其分析

李佩菡，向太和，謝軍，馮婷，陸文怡

杭州師范大學生命與環境科學學院，浙江 杭州 310036

農業生物技術

轉哺乳動物cyp2e1基因煙草植株再生及其分析

李佩菡，向太和，謝軍，馮婷，陸文怡

杭州師范大學生命與環境科學學院，浙江 杭州 310036

哺乳動物肝細胞中cyp2e1基因所編碼的蛋白CYP2E1在代謝異型有機物方面起著重要作用，轉cyp2e1基因植物可以代謝多種小分子有機污染物；但cyp2e1基因在植物體內的表達調控和代謝機理尚不完全清楚。文中將含有cyp2e1基因的質粒pSLD50-6和對照gus基因的質粒pKH200轉入根癌農桿菌GV3101，利用根癌農桿菌轉基因技術將cyp2e1基因和對照gus基因成功轉入煙草，分別獲得了轉cyp2e1和gus基因再生植株。選取PCR鑒定的再生植株進行熒光定量PCR (qRT-PCR) 分析，結果表明：在轉錄水平上，轉cyp2e1基因煙草中，乙醇處理后cyp2e1基因的表達明顯下降，苯和甲苯處理后cyp2e1基因的表達量稍有下降；而丙酮、甲醛處理和缺氧條件下cyp2e1基因的表達有不同程度的升高。此外，苯處理后，轉cyp2e1基因煙草中NADPH-P450氧化還原酶和細胞色素b5酶的基因活性顯著提高，說明煙草中NADPH-P450氧化還原酶和細胞色素b5酶與CYP2E1酶的解毒過程有關，可能起到哺乳動物體內的NADPH-P450氧化還原酶和細胞色素b5的功能，參與CYP2E1酶催化過程的電子傳遞鏈。

煙草，cyp2e1基因，NADPH-P450酶，細胞色素b5，表達

Abstract:CYP2E1 enzyme encoded bycyp2e1gene plays an important role in metabolism of heterogeneous organics inmammalian liver cells. The transgenic plant withcyp2e1can metabolize various low molecular weight organic pollutants.However, it is unclear the mechanism of expression control ofcyp2e1in transgenic plant. In this study, plasmid pSLD50-6 withcyp2e1and pKH200 withgusas control were transformed intoAgrobacterium tumefaciensGV3101 separately. Then,thecyp2e1orgusgenes were transferred into tobacco (Nicotiana tabacum) and the transgenic plants were regenerated viaAgrobacterium tumefaciensmethod. Real-time quantitative PCR (qRT-PCR) was used to analyze thecyp2e1gene expression. The expression ofcyp2e1in transgenic tobacco withcyp2e1decreased obviously treated by ethyl alcohol and reduced slightly by benzene and toluene, while it enhanced by acetone, formaldehyde and oxygen deficit in different levels.In addition, the gene expression of NADPH-P450 oxidoreductase and cytochrome b5 enzyme in the transgenic tobacco withcyp2e1were increased significantly treated by benzene, which showed that NADPH-P450 oxidoreductase and cytochrome b5 enzyme in transgenic tobacco have relation with CYP2E1 detoxication process. It suggested that the NADPH-P450 oxidoreductase and cytochrome b5 enzyme in transgenic plant formed the requirement in mammalian and participated in the electron transport chain ofCYP2E1 enzyme catalytic process.

Keywords:tobacco (Nicotiana tabacum),cyp2e1gene, NADPH-P450 oxidoreductase, cytochrome b5, expression

細胞色素P450是一類與重金屬結合的血紅蛋白，在代謝異型有機物方面起著重要作用；其中，CYP2E1作為 P450酶家族中的成員之一，主要分布在哺乳動物肝臟中，具有非常重要的解毒功能[1-3]。美國華盛頓大學Doty課題……