Exogenous toluene gas removal improvement in recombinant rabbit cytochrome P450 2E1 (CYP2E1)-transgenic Ardisia pusilla DC

The cytochrome P450 2E1 (CYP2E1) enzyme encoded by CYP2E1 plays an important role in the metabolism of organic compounds in mammalian liver cells. In this study, the plasmid pCAMBIA2300 harboring the rabbit CYP2E1 was transferred into Agrobacterium tumefaciens; this was then transferred into internodes of Ardisia pusilla. Shoot primordia, whose appearance resembles that of the in vitro protocorm-like body of orchids, regenerated from 121 of 332 internodes that were cultured in selection medium supplemented with 5 mg·L−1 kanamycin. PCR analysis confirmed that 25 of 89 putative transgenic plants (28.0%) stably harbored the CYP2E1 and NPTII genes, and Southern blot analysis revealed the presence of one to three copies of CYP2E1 within the genome of the 25 transgenic plants. The mRNA expression of the transgene was confirmed in 12 of 25 CYP2E1-transgenic plants by Northern blot analysis. Western blot analysis indicated the presence of a 53-kDa recombinant rabbit CYP2E1 protein in the 12 CYP2E1-transgenic A. pusilla plants. The aniline 4-hydroxylase activity of rabbit CYP2E1 in nine CYP2E1-transgenic A. pusilla plants was higher than that in non-transgenic A. pusilla plants. There was a statistically significant increase (5.1–6.1-fold) in the toluene removal ability of the transgenic line CYP2E1-2–2 compared to that observed in wild-type Ardisia plants at different times after exogenous toluene exposure.