Accumulation Of Melatonin And Functional Verification Of Melatonin Synthase Genes In Tea Plant(Camellia Sinensis)

Melatonin is a well-known chemical which possesses many beneficial effects on human health.It is widespread in different organisms,which regulates the responses to circadian rhythms and functions as a reactive oxygen species(ROS)and reactive nitrogen species(RNS)scavenger.It plays a critical role in plant abiotic stresses for its strong potential in antioxidation.Reports illustrated that Serotonin N-acetyltransferase and N-acetylserotonin methyltransferase are the key genes in the biosynthetic pathway of melatonin.However,in tea plant,the melatonin biosynthesis genes encoding the key enzymes in the pathways have not yet been reported.In current report,Melatonin was extracted and separateed in the tea plant.A rapid-amplification of c DNA ends(RACE)was applied to clone the full-length c DNA sequences of CsASMT and CsSNAT,which are two important genes in the biosynthetic pathway.Molecular characterization and function identification of the genes were performed using prokaryotic expression analysis.In addition,the expression patterns of these two genes together with some other known stress resistant genes under different biotic and abiotic stresses were also clarified using real-time RCR(q RT-PCR).The main results are summarized as follows:1.Melatonin was extracted and separated in the different organs of tea plant,including leaves,flowers and seeds.We found that melatonin of leaves have the highest content(5.12ng/g),while the melatonin content in flowers and seeds are 1.25ng/g and1.59ng/g,respectively.Interestingly,we haven’t found melatonin in the dried tea samples.The metabolic vigorous of seeds usually in high level during July and August giving rise to an increase of ROS.Thus,the melatonin was generated more to scavenging the ROS and easing the damage of plant.In one day,the melatonin content of tea plant seeds have two high level periods,that are 0.869ng/g at 5 o ’clock and 1.59ng/g at 14 o ’clock.2.Based on 5’-RACE and 3’-RACE the full-length c DNA sequences of CsASMT and CsSNAT genes were cloned.Consequently CsASMT and CsSNAT have 1282 bp and 1014 bp full-length c DNA sequences,respectively.The ORF length are 1065 bp and 744 bp,respectively,encoding 354 and 247 amino acids.3.Multiple Alignment and phylogenetic tree analysis showed that CsASMT and Malus zumi ASMT9 have the highest homology level,which is 57 %.CsSNAT and Vitis vinifera SNAT have the highest homology,which is 66.19%.Conserved domain analysis revealed S-adenosyl-L-methionine(SAM)binding sites and the putative N-acetylserotonin(NAS)binding domains in CsASMT,while a acetyl-Co A binding motif in CsSNAT.4.ORF sequences of CsASMT and CsSNAT genes were inserted into the expression vector p Mal-c2 X,and then mobilized into BL21(DE3)strain for inducible expression.A mass of soluble protein were observed and induced by 0.5 mg/ml IPTG at 28 ℃for 6h with molecular weight of about 50 k D and 70 k D in CsASMT and CsSNAT,respectively.By means of HPLC,which show that fusion protein of CsASMT have no enzyme activity,fusion protein of CsSNAT have a weaker enzyme activity.5.Expression patterns of CsASMT and CsSNAT and several other stress-resistand genes were investigated under different biotic and abiotic stresses.The stress-resistant genes displayed the same expression tendency after treated with 100μmol/L melatonin at low temperature(4℃).The Mn-SOD,APX and CsASMT were up-regulated and reached to maximum in the fifth day,while CAT、dhn2 and CsSNAT up-regulated to maximum in the third day under treatment.Among them,the dhn2 up-regulated 5.3 times compared with control.After treatment with Ectropic oblique attack,CsASMT and CsSNAT had varying degrees of expression but with low level.CsASMT up-regulated to the maximum level at24 h after treatment,which was 1.4 times than control.CsSNAT had the highest expression level at 9h after treatment,which was 1.8 times than control.After Me JA treatment,CsASMT and CsSNAT were both up-regulated and had the highest expression level in 3h,which were 1.3 times and 2.3 times compared with control,respectively.After ABA treatment,CsASMT were all up-regulated at each point in the treatment,which exhibited a highest expression level 12h(4.2 times than control).However,CsSNAT expression were not significantly altered between induction and control.In case of SA treatment,CsASMT up-regulated significantly at each time point with a highest regulation multiple in12 h,which was 7.8 times of control.Nevertheless,CsSNAT has the opposite orientation,which demonstrated down-regulation under SA treatment.According to the results,melatonin may play a vital role in resisting cold stress of tea leaves.In addition,the CsASMT is more sensitive than CsSNAT after ABA and SA treatment.