Wang Yingjuan, Ji Jingfen, Bu Huaiyu, Zhao Yuwei, Xu Yao, Johnson Carl Hirschie, Kolár Jan
Shaanxi Provincial Key Laboratory of Biotechnology, College of Life Science, Northwest University, Xi'an 710069, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Jul;25(7):1014-21.
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.
芳基烷基胺N - 乙酰基转移酶（AANAT）和羟基吲哚O - 甲基转移酶（HIOMT）是哺乳动物褪黑素生物合成途径中的关键调控酶。将AANAT和HIOMT基因构建到二元植物表达载体YXu55中。以叶片条为受体，通过农杆菌介导法高效转化烟草（Nicotiana tabacum）品种秦烟95。经庆大霉素梯度筛选，再生出许多转基因植株。Southern杂交和RT - PCR分析表明，AANAT - HIOMT基因已整合到转基因植株的基因组中，且目标基因能在RNA转录水平表达。通过反相高效液相色谱法（RP - HPLC）测定转基因植株中的褪黑素含量。结果显示，YXu55（含有庆大霉素抗性基因、AANAT基因和HIOMT基因）转基因植株中的褪黑素水平远高于pZP122（仅含庆大霉素抗性基因的对照）转基因植株和非转基因植株。pZP122转基因植株中的褪黑素含量与非转基因植株几乎相同。抗氧化特性的生理测定表明：1）YXu55含AANAT - HIOMT基因的转基因植株的总抗氧化能力、2）超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性以及3）谷胱甘肽（GSH）的含量均高于对照植株（pZP122或非转基因植株）。同时，转基因植株与非转基因植株之间丙二醛（MDA）含量未出现显著差异。上述事实表明，YXu55转基因植株中褪黑素水平的提高可能有助于减轻氧化应激损伤。
