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通过根癌农杆菌(含植物表达载体YXu55)介导的转化技术,将褪黑素生物合成酶-芳烷基胺N-乙酰转移酶(Arylalkylamine N-acetyltransferase,AANAT)与羟基吲哚O-甲基转移酶(Hydroxyindole O-methyltransferase,HIOMT)基因导入到烟草(秦烟95)中。对所获得的庆大霉素抗性烟草株系进行Southern blotting和RT-PCR分子生物学检测,结果表明,AANAT-HIOMT基因已成功地整合到烟草基因组中,并且可以在mRNA水平上进行转录。用反相高效液相色谱法(RP-HPLC)测定转化株系的褪黑素含量表明,转AANAT-HIOMT基因烟草株系的褪黑素含量均明显高于pZP122(不含AANAT和HIOMT基因的空白质粒)转基因株系和未转基因的对照植株,证明AANAT-HIOMT基因在转基因植株中的表达增强了褪黑素的合成能力。对不同株系抗氧化系统的部分指标进行了测定,并与其亲本对照植株比较,发现AANAT-HIOMT基因在转基因植物中的表达引起超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性增加,谷胱甘肽(GSH)浓度以及总抗氧化能力升高,且没有引起膜脂质过氧化物丙二醛(MDA)的变化,说明转基因烟草抗氧化潜能得到提高。
Arylalkylamine N-acetyltransferase (AANAT) was coupled with hydroxyindole O-methyl through the transformation technology mediated by Agrobacterium tumefaciens (including plant expression vector YXu55) Hydroxyindole O-methyltransferase (HIOMT) gene was introduced into tobacco (Qinyan 95). Southern blotting and RT-PCR analysis of the obtained gentamycin-resistant tobacco strains showed that the AANAT-HIOMT gene was successfully integrated into tobacco genome and transcribed at the mRNA level. Determination of melatonin content in the transformed lines by reverse-phase high performance liquid chromatography (RP-HPLC) showed that the content of melatonin in transgenic AANAT-HIOMT transgenic tobacco lines was significantly higher than that of pZP122 (AANAT and HIOMT gene-free Blank plasmid) transgenic lines and non-transgenic control plants, demonstrating that the expression of AANAT-HIOMT gene in transgenic plants enhances the melatonin synthesis ability. The results showed that the expression of AANAT-HIOMT gene in transgenic plants caused the increase of superoxide dismutase (SOD), peroxidase (POD), malondialdehyde CAT activity, glutathione (GSH) and total antioxidant capacity increased, and did not cause changes in the membrane lipid peroxides malondialdehyde (MDA), indicating that the anti-oxidation potential of transgenic tobacco Be improved.