Functional Analysis Of Melatonin Biosynthetic Gene MdASMT9 In Apple Under Drought Stress

Melatonin is a kind of small molecule signal substance with various functions,which widely exists in the biological world and plays an important role in plant stress resistance,growth and rhythm regulation.A large number of previous studies showed that exogenous application of melatonin can significantly improve the resistance of apple to drought,salt stress,aging and brown spot.N-acetyl-5-hydroxytryptamine methyltransferase(ASMT)directly catalyzes the synthesis of melatonin by n-acetyl-5-hydroxytryptamine,which is the last step of melatonin synthesis.Therefore,it is considered to be the rate limiting enzyme of melatonin biosynthesis in plants.In this study,the MdASMT9 gene of Apple was identified by homologous cloning and in vitro protein catalytic test.After that,through genetic transformation,the overexpression of MdASMT9 in apple and tomato was obtained,and the function of MdASMT9 in resistance to drought stress was studied.The main results are as follows: 1.Seven candidate genes of MdASMT9 were retrieved and cloned from the apple genome database by using rice OsASMTs.In situ expression and in vitro catalytic test showed that only MdASMT9 could catalyze the synthesis of melatonin.The results showed that the expression of MdASMT9 in apple tissues was in the order of fruit > branch > bark > flower > leaf from high to low,and the expression of MdASMT9 increased during the development of fruit.In addition,MdASMT9 also responded to low temperature,drought,salt and ABA treatments.2.Through genetic transformation,two apple lines and three tomato lines with overexpression of MdASMT9 were obtained.Compared with wild-type gl-3,the expression level of MdASMT9 in transgenic apple plants was significantly increased,and the melatonin content was also significantly increased.3.Under natural drought,compared with wild type,the overexpression of MdASMT9 in apple and tomato plants alleviated the damage of drought stress on the light system and cell membrane structure by improving antioxidant capacity and accumulating more osmotic regulators,and enhanced the drought tolerance of plants.4.Under the condition of long-term drought,the overexpression of MdASMT9 can regulat stomatal opening,improve the photosynthetic capacity and water use efficiency of apple and promote the growth of plant shoot.At the same time,under the condition of long-term drought,overexpression of MdASMT9 also increased the activity of antioxidant enzymes,cleared the accumulation of reactive oxygen species,and improved the resistance to long-term drought stress.