Transgenic Plant Regeneration Of Tobacco(Nicotiana Tabacum) Haboring Mammalian Cyp2e1Gene And Its Analysis

CYP2E1enzyme encoded by cyp2el gene play an important role in metabolism heterogeneous organics in mammalian liver cell. The transgenic plant with cyp2el can metabolize various low molecular weight organic pollutants, such as benzene, toluene, trichloroethylene, chloroform, carbon tetrachloride, vinyl chloride and so on. In mammalian, CYP2E1enzyme was by ethanol, isopropyl alcohol, acetone, ether, benzene, toluene, formaldehyde, pyrazole, iosniazid and hypoxia. Moreover, in the metabolic process of expression control of cyp2el in transgenic plant, both of NADPH-P450oxidoreductase and cytochrome b5are two important enzymes in the electron transport chain of CYP2E1. However, it is unclear the mechanism of expression control of cyp2el in transgenic plant. In the studies, we transformed cyp2el gene into tobacco (Nicotiana tabacum) via Agrobacterium tumefaciens method, and analyzed the cyp2el gene expression and cyp2el gene separation in the first generation. At the same time, we analyzed the cyp2el gene expression on the influence of different environmental factors, and analyzed transgenic tabacco endogenous NADPH-P450oxidoreductase and cytochrome b5expression. The results were as follows:1. Plasmid pSLD50-6with cyp2el and pKH200with gus as control were transformed into Agrobacterium tumefaciens GV3101respectively. Then, the cyp2el or gus genes were transferred into tobacco(Nicotiana tabacum) and the transgenic plants were regeneration via Agrobacterium tumefaciens method. PCR detection, RT-PCR, qRT-PCR and Western Blot analysis confirmed that cyp2el gene has been transformed into tobacco successfully, and exogenous gene cyp2el was highly expressed in cyp2el transgenic plants.2. The cyp2el gene was separated in TL01, TL03and TL26lines of transgene tobacco. The separation ratio of cyp2el is51:9,48:12and34:26. It did not meet with3:1, which indicated that cyp2el gene inserted into transgene tobacco with multiple copies. qRT-PCR analysis showed the cyp2el gene expression in various organs of transgenic tobacco is different. The expression level of exogenous gene cyp2el in transgenic tobacco is leaf> root> stem> flowers. In addition, the flower variation of transgenic tobacco was observed.3. After the cyp2el trangenic plants were placed in volatile organic vials contained MS basal salt with formaldehyde50μg/mL, ethanol8mg/mL, acetone8mg/mL, benzene8μg/mL, and toluene8μg/mL, or transgenic tobacco plants immersed in sterile water for12h, qRT-PCR analysis of cyp2el gene expression was conducted. At the transcriptional level, the expression of cyp2el in transgenic tobacco with cyp2el 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. It shows that cyp2el gene in plants can be influenced by environmental factors.4. The expression of NADPH-P450oxidoreductase gene and cytochrome b5enzyme gene in wild-type tobacco, cyp2el trangenic tobacco and gus trangenic tobacco was analyzed via qRT-PCR. The the gene expression level of NADPH-P450oxidoreductase and cytochrome b5enzyme was not obviously different in wild-type tobacco, cyp2el trangenic tobacco and gus trangenic tobacco, while the gene expression in the transgenic tobacco with cyp2el were increased significantly treated by benzene. The results showed that NADPH-P450oxidoreductase and cytochrome b5enzyme in transgenic tobacco have relation with CYP2E1detoxication process. It suggested that the NADPH-P450oxidoreductase and cytochrome b5enzyme in transgenic plant formed the requirement in mammalian and participated in the electron transport chain of CYP2E1enzyme catalytic process.