Functional Study Of The Key Enzyme Gene SNAT In The Melatonin Synthesis Pathway Of Hypericum Perforatum

Hypericum perforatum L.is a perennial herbaceous plant as Hypericaceae,a kind of vital medicinal plant,the various organizational parts of it could be used as medicine.It has multiple bioactive components.Melatonin,hyperforin,and hypericin are the main active components in H.perforatum extract,which can be utilized to remedy mild to moderate depression with better tolerance and fewer side effects.Melatonin,a derivative of tryptophan known in Chinese as N-acetyl-5-methoxytryptamine,plays an essential physiological role in H.perforatum as a high content of the main active ingredient.Serotonin N-acetyl transferase（SNAT）is a crucial enzyme involved in the biosynthesis of melatonin in plants.Previous studies have shown that SNAT protease in Oryza sativa,Arabidopsis thaliana,and Malus zumi can catalyze melatonin synthesis and its precursors.Furthermore,the overexpression of its SNAT gene can increase melatonin content in plants and thus enhance the stress tolerance of plants.However,do the SNAT genes in H.perforatum participate in the regulation of melatonin synthesis?Whether the exact molecular mechanisms are involved is unclear.In this study,based on the whole genome data of H.perforatum,bioinformatics,expression patterns,and prokaryotic expression analysis were performed on the members of the SNAT family in H.perforatum,and essential enzyme genes HpSNAT1/2 involved in melatonin biosynthesis,and response to drought stress was preliminarily selected;Furthermore,the function of its genes was investigated in Arabidopsis and H.perforatum,respectively.The main research contents and results are as follows:1.A total of 48 SNAT family members were identified according to the whole genome database of H.perforatum.The phylogenetic tree,multiple sequence alignment,and conserved motif analysis of HpSNAT proteins were performed with these functionally known proteins AtSNAT1（AT1G32070）,AtSNAT2（AT1G26220）,OsSNAT1（AK059369）,and OsSNAT2（AK068156）from model plants Arabidopsis thaliana and Oryza sativa as a reference.The that evm.model.scaf₁51.204（HpSNAT1）and evm.model.scaf₆4.495（HpSNAT2）were closely related to those proteins in model plants.Multiple sequence alignment analysis showed that these genes had high homology and similar conserved motifs,which suggested that these genes might have similar functions.Therefore,we speculated that the selected HpSNAT1/2 genes might play a key role in the melatonin synthesis pathway of H.perforatum.2.The CDS sequences of HpSNAT1/2 genes in H.perforatum were cloned,respectively:HpSNAT1,516 bp,encoding 171 amino acids;HpSNAT2,1,332 bp,encoding 443 amino acids.Bioinformatics analysis showed that the two proteins did not contain signal peptides and transmembrane domains,and both belong to hydrophilic proteins.The expression pattern analysis results revealed that HpSNAT1/2 have high expression in the aboveground parts and responds to salt and drought stresses.The results of Arabidopsis thaliana protoplast transient transfection experiments showed that the two HpSNAT1/2 proteins were localized in chloroplast.3.The prokaryotic expression vectors of HpSNAT1/2 were constructed,but only one target protein of HpSNAT1 with a size of about 19 kDa was successfully induced and extracted.The results indicated that it was a soluble protein and had the highest expression level at 16℃,180 rpm for 20 h induction with the 1mM final concentration of IPTG.The concentration of extracted GST-HpSNAT1 fusion protein was about 1690 μg/mL,which laid a sound foundation for the functional study of HpSNAT1 in vitro.4.The overexpression vector of HpSNAT1/2 was constructed by Gateway recombinant technology.The Agrobacterium tumefaciens mediated transformation method was used to transfect Arabidopsis thaliana snat mutant.The three homozygous lines with high expression of HpSNAT1/2 were acquired by molecular level identification and semi-quantitative analysis.The detection results of melatonin content and drought resistance index in different Arabidopsis thaliana lines revealed that the melatonin contents in transgenic lines were 1.86-2.22 times as many as the snat,all of the leaf color,lateral root number,growth,and other phenotypes of transgenic Arabidopsis thaliana and WT were superior to the snat,the contents of ROS,MDA and H2O2 were lower than that of the snat,showing stronger drought resistance.These results suggested that the HpSNAT1/2 genes could increase the content of endogenous melatonin in Arabidopsis thaliana,which could promote the growth of plant roots and effectively scavenge the ROS and decrease the contents of MDA and H2O2 in plants,and thus enhanced the drought tolerance of Arabidopsis thaliana plants.5.Agrobacterium tumefaciens impregnated the root explant of H.perforatum,and five transgenic lines of HpSNAT1/2 were obtained by identification at the molecular level.Furthermore,the positive and highly expressed hairy root lines were selected to induce them into complete H.perforatum transgenic plants.The detected results of the metabolites contents in H.perforatum transgenic plants showed that:the melatonin and its precursors contents in OE-HpSNAT1/2 were significantly increased 2-4 times compared with wild type,which indicated that the two genes could involve in the synthesis of melatonin and its precursors and play a crucial role in their biosynthetic pathway.The expression levels of essential enzyme genes HpASMT1/3 in the melatonin synthesis pathway of H.perforatum were further detected,we found that the overexpression of HpSNAT1/2 can up-regulate the expression level of crucial enzyme genes ASMT and further increase the contents of 5-MT and melatonin,which indicated that HpSNAT1/2 genes play a vital role in the melatonin biosynthesis pathway in H.perforatum.In summary,this study was the first to identify and analyze the SNAT family members of H.perforatum,which could provide a reference for further exploring the specific functions of the SNAT genes in H.perforatum.Meanwhile,this study not only clarified the effect of HpSNATs on the melatonin synthesis of H.perforatum and Arabidopsis thaliana but also preliminarily revealed the regulatory mechanism of HpSNATs in response to drought stress in Arabidopsis thaliana,which provided an essential basis for further researching the specific functions of SNAT genes and the molecular mechanism on the regulation of melatonin in the stress response,growth and development of H.perforatum.Also,the successful establishment of the transformation system of H.perforatum could provide a sound foundation for further investigating the functions of essential enzyme genes and transcription factors in H.perforatum.