Changes Of Melatonin Content And Prokaryotic Expression Analysis Of SNAT Gene In Grapevine

Melatonin is an indoleamine,it is ubiquitous in most organisms.As an antioxidant and free radical scavenger,melatonin can regulate a variety of physiological functions in animals and plants.The levels of melatonin and the antioxidant capacity of human plasma will be increased by eating fruits rich in melatonin.As a kind of economically cultivated fruit tree,the grape is rich in nutritional value.However,there is not much study on endogenous melatonin content changes in grape.This study measured the effects of salt stress and drought stress on the content of endogenous melatonin in grape.Additionally,in order to make further study on the correlation between melatonin content and melatonin synthesis genes in grape,the changes of melatonin content and the expression patterns of melatonin synthesis genes during fruit development were investigated.We also cloned the CDS sequence of VvSNAT gene,and induced the gene to produce soluble protein in Escherichia coli by prokaryotic expression technology.The characteristics and enzymology functions of SNAT gene in grape were preliminarily studied.The main findings are as follows:1.The treatment of drought and salt stress significantly increased the melatonin content in leaves and roots of grape seedlings,and the increase range of melatonin content was enlarged with the increasing degree of stress treatment.After the seedlings were treated with 120 mmol/l NaCl,the melatonin content of grape roots and leaves reached 627.25 and 3220.42 pg/g,respectively,which was about 7 times higher than that of control seedlings.When treated with 10 % PEG 6000,the content of melatonin in roots and leaves was much higher than that in the control group.1.The presence of melatonin in grape was detected at different developmental stages.With the turning and ripening of the fruit,the melatonin content increased at first and reached the peak then decreased,and finally increased,and the melatonin content was 1.32 ± 0.06 ng/g FW at the initial coloring period.During fruit development,the changes of tryptamine and serotonin were coincided with melatonin,which reached the peak at the initial coloring period.2.Using the melatonin synthesis gene of rice as template,four VvTDC genes,seven VvT5 H genes,three VvSNAT genes and five VvASMT genes were screened from grape genome as candidate genes in the synthesis pathway of melatonin by homologous alignment.The expression patterns of VvT5H1,VvT5H5,VvT5H7,VvSNAT1,VvSNAT2,VvSANT3,VvASMT1 and VvASMT2 were consistent with melatonin content,while the expressions of VvTDC1 and VvASMT5 gene were not consistent with melatonin.3.In this study,we cloned the full CDS sequence of VvSNAT gene,with 741 bp,encoding 246 amino acids,which had a high similarity with those amino acid sequence of other plants.The recombinant protein of SNAT was induced by prokaryotic expression.In vitro activity test showed that the purified recombinant SNAT protein had the activity of N-acetyltransferase,which could transfer acetyl from Acetyl coenzyme A to serotonin.The phylogenetic tree of SNAT showed that the relationship between grape and sheep was farther,and the relationship of SNAT between cyanobacteriaand rice was closer.