Arylalkylamine N-acetyltransferase (AANAT) and Hydroxyindole O-methyltransferase(HIOMT) are the key regulation enzymes in the melatonin biosynthesis pathway in mammals. The AANAT and HIOMT genes were constructed into a binary plant expression vector YXu55. Using leaf strips as the recipiences, we efficiently transformed tobacco (Nicotiana tabacum) variety qinyan 95 by the Agrobacterium mediated method. After gradient selection with gentamycin, a number of transgenic plants were regenerated. Southern blot and RT-PCR analyses showed that the AANAT-HIOMT genes were integrated into the genome of the transgenic plants and the target genes could express at the level of RNA transcription. By RP-HPLC, we measured the melatonin contents in transgenic plants. The results showed that the melatonin level in YXu55 (containing the gentamycin-resistance gene, the AANAT gene and HIOMT gene) transgenic plants were much higher than those in pZP122 (control containing only the gentamycin-resistance gene) transgenic plants and nontransgenic plants. The content of melatonin in pZP122 transgenic plants was nearly the same as that in nontransgenic plants. Physiological determination of antioxidative characteristics demonstrated that 1) the capacity of total antioxidation, 2) the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and 3) the content of glutathione (GSH) were increased in YXu55 transgenic plants containing the AANAT-HIOMT genes as compared to the control plants (pZP122 or nontransgenic plants). At the same time, malonaldehyde (MDA) content did not appear remarkably difference between transgenic plants and nontransgenic plants. The above mentioned facts indicate enhancement of melatonin levels in YXu55 transgenic plants might help to reduce damage by oxidative stress.
Acetylserotonin O-Methyltransferase ; genetics ; Agrobacterium tumefaciens ; genetics ; Arylalkylamine N-Acetyltransferase ; genetics ; Catalase ; metabolism ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Melatonin ; biosynthesis ; Peroxidase ; metabolism ; Plants, Genetically Modified ; enzymology ; genetics ; Superoxide Dismutase ; metabolism ; Tobacco ; enzymology ; genetics ; Transduction, Genetic ; methods