The Production Of Transgenic Tomato Cultivar Micro-Tom Plants Expressing Melatonin Synthetase Genes And The Studies On Antioxidative Capacity In Transgenic Plants Under Salt Stress

Melatonin (N-acetyl-5-methoxytryptamine) is a kind of indolehormone synthesized and secreted by the pineal gland of vertebrates with simple structure and low molecular weight, including roles in circadian rhythm adjustments, sleep modulation, cancer inhibition, anti-aging and immunomodulation. Melatonin was first discovered in plants in1995, since then, numerous studies have reported its presence in a wide range of plants. In recent years, most evidences suggests that melatonin in plants is involved in regulation of plant development, promotes the branching of the root system, and protects plants against environmental stress by exogenous melatonin treatment, but there is few reports about its function in plants by transgenic means. The enzyme genes in melatonin synthesis pathway could be transferred into certain plant genomes by genetic transformation, and the level of endogenous melatonin would be expected to improve. Furthermore, the transgenic plants and the wild type will be compared to analyze their physiological responses to environmental stress, and it will be a new way to study the function of melatonin in plants.Melatonin synthetase genes of both AANAT (Arylalkylamine N-acetyltransferase)and HIOMT (Hydroxyindole O-methyltransferase) were transferred into the miniature tomato cultivar Micro-Tom mediated by Agrobacterium tumefaciens, and transgenic tomatoes were obtained. The content of melatonin in transgenic tomatoes were measured. Meanwhile, the physiological changes under salt stress were compared between transgenic and non-transgenic tomatoes in order to evaluate antioxidative protection of melatonin to plants that under stress. The study obtained the following results:(1) In tissue culture experiments, we studied the the effects of different concentrations of hormone combinations on callus and adventitious buds induction using cotyledons and hypocotyls as explants. The results showed that cotyledon was more suitable as explants to induce callus and adventitious buds, and the best medium was MS+6-BA2.0mg/L+IAA0.2mg/L. In addition, the best medium for adventitious buds elongation was MS+6-BA1.0mg/L+GA31.0mg/L, the best root induction medium was1/2MS+IBA0.3mg/L.(2) Some factors such as the sensitivity of cotyledon to gentamicin, the pre-culture and co-culture time of explants, and the concentration and infection duration of Agrobacterium influencing the efficiency of Agrobacterium mediated transformation were discussed. The results indicated that the cotyledons were pro-culture for2days, infected for10min with concentration of Agrobacterium OD6000.4～0.6, co-cultured for2days, and then cutured on selected medium with100mg/L gentamicin could obtain more positive transgenic clones of tomato. PCR and RT-PCR analysises of the transgenic plants confirmed AANAT and HIOMT genes was successfully integrated into the tomato genome and transcripted properly.(3) The level of melatonin in transgenic tomatoes was determined using high performance liquid chromatography procedure. The datas showed that the level of melatonin in pYXU55transgenic plants were higher than that in non-transgenic plant and pZP122transgenic plants. It indicated that the content of endogenous melatonin in tomatoes incresed due to the transformation of key enzyme gene regulating melatonin synthesis.(4) The antioxidative capacity of transgenic and non-transgenic plants was compared under slat stress. The results showed that transgenic tomato has a relatively low content of MDA, higher activities of SOD, POD and CAT compared with non-transgenic plants under normal culture conditions, but the difference was not significant. While the indicators between the two were different significantly under slat stress. There were small increase on content of MDA and gentle decrease on activities of SOD, POD and CAT of transgenic tomatoes compared with non-transgenic tomatoes. It was confirmed that transgenic tomato can significantly reduce the oxidation of membrane lipid, improve the activities of antioxidant enzymes, and reduce damage on plants caused by salt stress.