转哺乳动物cyp2e1基因烟草植株再生及其分析

Transgenic plant regeneration of tobacco (Nicotiana tabacum) haboring mammalian cyp2e1 gene

摘要:

哺乳动物肝细胞中cyp2e1基因所编码的蛋白CYP2E1在代谢异型有机物方面起着重要作用，转cyp2e1基因植物可以代谢多种小分子有机污染物；但cyp2e1基因在植物体内的表达调控和代谢机理尚不完全清楚。文中将含有cyp2e1基因的质粒pSLD50-6和对照gus基因的质粒pKH200转入根癌农杆菌GV3101，利用根癌农杆菌转基因技术将cyp2e1基因和对照gus基因成功转入烟草，分别获得了转cyp2e1和gus基因再生植株。选取PCR鉴定的再生植株进行荧光定量PCR (qRT-PCR) 分析，结果表明：在转录水平上，转cyp2e1基因烟草中，乙醇处理后cyp2e1基因的表达明显下降，苯和甲苯处理后cyp2e1基因的表达量稍有下降；而丙酮、甲醛处理和缺氧条件下cyp2e1基因的表达有不同程度的升高。此外，苯处理后，转cyp2e1基因烟草中NADPH-P450氧化还原酶和细胞色素b5酶的基因活性显著提高，说明烟草中NADPH-P450氧化还原酶和细胞色素b5酶与CYP2E1酶的解毒过程有关，可能起到哺乳动物体内的NADPH-P450氧化还原酶和细胞色素b5的功能，参与CYP2E1酶催化过程的电子传递链。

Abstract:

CYP2E1 enzyme encoded by cyp2e1 gene plays an important role in metabolism of heterogeneous organics in mammalian liver cells. The transgenic plant with cyp2e1 can metabolize various low molecular weight organic pollutants. However, it is unclear the mechanism of expression control of cyp2e1 in transgenic plant. In this study, plasmid pSLD50-6 with cyp2e1 and pKH200 with gus as control were transformed into Agrobacterium tumefaciens GV3101 separately. Then, the cyp2e1 or gus genes were transferred into tobacco (Nicotiana tabacum) and the transgenic plants were regenerated via Agrobacterium tumefaciens method. Real-time quantitative PCR (qRT-PCR) was used to analyze the cyp2e1 gene expression. The expression of cyp2e1 in transgenic tobacco with cyp2e1 decreased 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 with cyp2e1 were 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 of CYP2E1 enzyme catalytic process.