| Melatonin,a pineal secretory product,was firstly discovered in the bovine pineal gland in 1958.In 1995,Dubbels and Hattori et al.identified melatonin from higher plants such as tomato and morning glory.Scientists pay more attention to the function of melatonin in animals and plants.In plants,melatonin plays important roles in the regulation of many physiological processes and functions as a growth regulator to regulate plant growth and development.Melatonin is a well-known hormone that regulates various biological processes in animal,including the circadian rhythm,antioxidant activity,immunological enhancement,seasonal reproduction,emotional status,and physical conditions.Therefore,melatonin is widely used in daily life as a sleep-regulating drug.It is often used to adjust for circadian rhythm disorder,which caused by jet lag or other sleep disorders,and to improve a person's sleep-wake cycle and circadian rhythm.With the globalization and rapid development of China's economy,frequent time difference and high-intensity shift work and other irregular life rhythms led to a circadian rhythm disorder phenomenon is very common in society.It can effectively supplement melatonin content and maintain melatonin metabolism homeostasis to improve circadian rhythm disorder by regularly eating animal and plant medicinal materials with high melatonin content.Therefore,it is of great practical value to search for and study the high melatonin content of food and medicine of plant and animal,especially the high melatonin content of public plant food and medicine.Mulberry(Morus alba L.)is originally produced in China and has been widely cultivated as the feed of silkworms,so it has been one of the main cash crops in China since ancient times.Previous research mainly focused on mulberry variety breeding,mulberry cultivation and pest control,aiming at providing high yield and high quality mulberry leaf to silkworm.With the deepening of the study of mulberry,it was found that mulberry leaves contain rich nutrients and a variety of active substances,making its nutritional value and medicinal value more and more attracted people's attention,so it was listed as "medicine and food homologous" plant resources by the Ministry of Health.Mulberry leaves and fruit contained higher melatonin content than other tissues.Therefore,mulberry leaves and fruit may be promising sources of melatonin.The melatonin contents among different varieties of the same species of plant could be significantly different.China is one of the countries with the most abundant mulberry varieties.Therefore,the selection of mulberry varieties with high melatonin content can not only be used as suitable plant materials for melatonin supplement in People's Daily life,but also provide materials for analysis of the molecular mechanism underlying the melatonin biosynthesis,which has important practical and scientific significance.At present,the research on melatonin in mulberry mainly focuses on identification and quantitative analysis.Systemic identification and quantitative analysis,selection of high-content variety and molecular mechanism of melatonin and its isomers biosynthesis have not been reported in mulberry,which greatly hinder the application,deep development and multiple utilization of mulberry as a public raw material for efficient supplement of human melatonin.In this study,UPLC-MS/MS was used to identify melatonin in different mulberry varieties and quantify melatonin of 50 different mulberry varieties to select the mulberry variety with the highest melatonin content.Bioinformatics analysis was carried out to identify the genes involved in melatonin biosynthesis from M.notabilis database,which the highest melatonin content was detected in Morus notabilis C.K.We conducted an expression analysis of melatonin biosynthetic genes and in vitro enzymatic synthesis of melatonin to clarify the molecular mechanism underlying the efficient biosynthesis of melatonin in M.notabilis.Identification of melatonin identification,natural melatonin isomers were accidentally identified in mulberry and the melatonin isomers of different varieties of mulberry were quantitatively analyzed.To uncover the effects of daily environmental conditions on the production of melatonin and its isomers in mulberry leaves,we used UPLC-MS/MS to detect the content of melatonin and isomers in mulberry leaves with non-genetic factors.The natural melatonin isomers were identified in plant tissues for the first time,and the biosynthesis of melatonin isomers has never been reported.We used the same mulberry varieties of different tissues and the same extraction and detection methods to carry out the identification and quantitative analysis of melatonin and its isomers.The target genes that may be involved in the biosynthesis of melatonin isomers were screened by qRT-PCR and enzyme functions to clarify the biosynthetic pathway of melatonin isomers in mulberry.The results provided insights into the molecular mechanism underlying melatonin biosynthesis in mulberry and expand our knowledge of melatonin isomer biosynthesis.The main results are as follows:1.Identification and quantitative analysis of melatonin and isomers in different mulberry varieties and selection of varieties with high content of melatonin and isomersWe used UPLC-MS/MS to obtain high melatonin-content varieties for immune regulation.Melatonin and two novel isoforms(MI-1 and MI-2)were found in the Morus plants.MI-2 was only present in four mulberry varieties,and the low content made it impossible to quantify.We analyzed the content of melatonin of 50 different mulberry varieties.The results showed that the content of melatonin ranged from 0.26 to 0.83 ng/g,with a difference of 3-fold.The five mulberry varieties with higher melatonin content were Chuansang,Xinong 6071,Shennongjia Changsuisang,Jialing-NO.16 and Jialing-NO.20 in order.The results showed that Chuansang,Xinong 6071 and Shennongjia Changsuisang could be used as the best raw materials to supplement melatonin.The total content of melatonin and its isomers ranged from 0.23 to 20.58 ng/g,with a difference of89-fold.The five mulberry varieties with higher content of melatonin and its isomer were Cesha,Shennongjia Changsuimula,Xiaohuayepisang,India mulberry and Ganluo-NO.6 in order.To uncover the effects of daily environmental conditions on the production of melatonin and its isomers in mulberry leaves,we used UPLC-MS/MS to detect the content of melatonin and isomers in mulberry leaves with different time and different maturity.The melatonin and its isomer contents in mulberry leaves at the same developmental stage from different varieties increased from April 28 th to June 28 th,but showed the opposite trend from June 28 th to August 28 th.The melatonin isomer contents in the leaves of three different mulberry varieties increased with leaf position.The results showed that high content of melatonin and its isomers could be obtained by picking leaves of Cesha,Shennongjia Changsuisang and Xiaohuayepisang at the 20 th leaf position on June 28.2.Analysis of the molecular mechanism underlying the efficient biosynthesis of melatonin in M.notabilisWe identified 37 candidate genes involved in melatonin biosynthesis in M.notabilis by bioinformatics methods.Based on the CDS sequences of genes involved in melatonin biosynthesis,37 candidate genes were successfully cloned and sequenced,including one tryptophan decarboxylase gene(MnTDC),seven tryptophan 5-hydroxylase genes(MnT5H1–7),six serotonin N-acetyltransferase genes(MnSNAT1–6),and 20 N-acetylserotonin methyltransferase genes(MnASMT1–20)and three caffeic acid O-methyltransferase genes(MnCOMT1–3).Bioinformatics analysis showed that the structural characteristics of candidate genes involved in melatonin biosynthesis in M.notabilis were basically consistent with the reported genes involved in melatonin biosynthesis in other species.Domain-prediction and multi-sequence alignment analysis showed that the catalytic domains and active sites were conserved in these mulberry proteins.The expression levels of the 37 candidate genes involved in melatonin biosynthesis in different tissues(root,stem,leaves and bark)was detected by qRT-PCR.The five gene families had diverse expression profiles in the different tissues.MnTDC was expressed at higher levels in leaves.Compared with the other five MnT5 H genes,the expression levels of MnT5H1 and MnT5H2 were high in all four tissues.The expression level of MnT5H2 was 20-fold that of MnT5H1 in the stem.MnT5H7 was not expressed in the four tissues.The expression patterns of all the SNATs except MnSNAT6 were similar among the four tissues,and the expression levels in the stems were much higher than in other tissues.In the four tissues,the expression level of MnSNAT5 was higher than those of the other five MnSNATs.MnSNAT6 was not expressed in any of the four tissues.For 20 MnASMT genes,all the genes were broadly expressed in four tissues,except the MnASMT3.The expression of MnASMT12 was highest in root and bark.The expression patterns of the three MnCOMTs were different in mulberry.The expression level of MnCOMT1 was high in the four tissues,among which the expression levels were the lowest in bark and the highest in stem,respectively.Combined with results of phylogenetic tree,sequence alignment and expression level analysis,MnTDC,MnT5H2,MnSNAT5,MnASMT12 and MnCOMT1 were used to as target genes to verify the mechanism of melatonin biosynthesis in mulberry.MnTDC,MnT5H2,MnSNAT5,MnASMT12,MnASMT16,MnASMT20,and MnCOMT1full-length c DNAs were independently cloned into pCold TF vectors for the expression of recombinant proteins in E.coli.Then,the overexpression proteins were purified.The obtained proteins was consistent with the predicted molecular weights,respectively,in the SDS-PAGE analysis.Then,L-tryptophan and MnTDC were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnTDC had the ability to convert L-tryptophan to tryptamine.Then,tryptamine and MnT5H2 were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnT5H2 had the ability to convert tryptamine to serotonin.Then,serotonin,acetyl-Co A and MnSNAT5 were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnSNAT5 had the ability to convert serotonin to N-acetylserotonin.5-methoxytryptamine,acetyl-Co A and MnSNAT5 were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnSNAT5 had the ability to convert 5-methoxytryptamine to melatonin.N-acetylserotonin,S-adenosyl-L-methionine and MnASMT12(MnCOMT1)were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnASMT12(MnCOMT1)had the ability to convert N-acetylserotonin to melatonin.Therefore,serotonin,S-adenosyl-L-methionine and MnASMT12(MnCOMT1)were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnASMT12(MnCOMT1)had the ability to convert serotonin to 5-methoxytryptamine.Finally,N-acetylserotonin,S-adenosyl-L-methionine and MnCOMT1 were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnCOMT1 had the ability to convert N-acetylserotonin to melatonin.Therefore,serotonin,S-adenosyl-L-methionine and MnCOMT1 were co-incubated in the reaction buffer and analyzed the products using UPLC-MS/MS.The result showed that MnCOMT1 had the ability to convert serotonin to5-methoxytryptamine.MnASMT12,a class I group member of ASMT,catalyzed not only N-acetylserotonin to melatonin but also serotonin to 5-methoxytryptamine.To verify the functions of classes II and III members of the MnASMT genes family,we selected class II ASMT,MnASMT16,and class III ASMT,MnASMT20,for further study based on their higher expression levels.MnASMT16 and MnASMT20 were expressed in E.coli BL21(DE3)and obtained their corresponding recombinant proteins.Then,N-acetylserotonin and S-adenosyl-L-methionine were co-incubated in the reaction buffers containing respective MnASMT16 and MnASMT20,and analyzed the products using UPLC-MS/MS.Results showed that MnASMT16 and MnASMT20 had the ability to convert N-acetylserotonin to melatonin.Consequently,serotonin and S-adenosyl-L-methionine were co-incubated in the reaction buffers containing respective MnASMT16 and MnASMT20 proteins,and analyzed the products using UPLC-MS/MS.However,only MnASMT20 protein had the ability to convert serotonin to5-methoxytryptamine.In summary,enzymatic assays indicated that MnTDC,MnT5H2,MnSNAT5,MnASMT12 and MnCOMT1 were efficiently converted their corresponding substrates to the key melatonin-related intermediates or melatonin.Enzymatic assays indicated the presence of multiple biosynthetic pathways M.notabilis.The proteins encoded by the genes of three ASMT gene subfamily members all showed ASMT protein activity.All the results lay a foundation for the efficient biosynthesis of melatonin.3.Molecular pathway analysis of melatonin isomer biosynthesis in mulberry We used the same mulberry varieties of different tissues and the same extraction and detection methods to carry out the identification and quantitative analysis of melatonin and its isomers.The results showed that the types of melatonin isomers were different in four mulberry cultivars.The results showed that four mulberry varieties leaves contained melatonin and two melatonin isomers(MI-2 and MI-3),and four mulberry varieties fruits only contained melatonin and isomer(MI-2).We found that MI-3 was only present in the leaves and not in the fruit of the four different mulberry varieties.The total contents of melatonin and melatonin isomers in leaves and fruits of "Dashi" were the highest among the four mulberry cultivars.The qRT-PCR was used to detect the expression levels of candidate genes involved in the biosynthesis of melatonin in leaves and fruit of mulberry varieties ‘Dashi' and ‘Baiyuhuang'.All the genes involved in the biosynthesis of melatonin,except MaASMT4 and MaASMT20,had different expression patterns in both the leaves and fruit of the two varieties.The MaASMT4 gene was abundantly expressed in the leaves of ‘Dashi' and ‘Baiyuhuang',but was almost not expressed in the fruit of the two mulberry varieties.The expression level of the MaASMT20 gene in the leaves of the two mulberry varieties was over 25-fold greater than in their fruit.A similar result was confirmed in the other two mulberry varieties(‘Jialing NO.30'and ‘Zhongsang 5801').These results suggested that MaASMT4 and MaASMT20 may be involved in the synthesis of MI-3,which was only detected in mulberry leaves.Therefore,the two genes,MaASMT4 and MaASMT20,were selected as target genes for biosynthesis of melatonin isomers.We successfully cloned MaASMT4 and MaASMT20 from ‘Dashi'.MaASMT4 and MaASMT20full-length c DNAs were independently cloned into pCold TF vectors for the expression of recombinant proteins in E.coli BL21(DE3).Then,the overexpression proteins were purified.The obtained proteins was consistent with the predicted molecular weights,respectively,in the SDS-PAGE analysis.We co-incubated S-adenosyl-L-methionine and N-acetylserotonin separately with MaASMT4 and MaASMT20 in the reaction buffer and analyzed the products by UPLC-MS/MS.MaASMT4 and MaASMT20,a class III group member of ASMT,were able to convert N-acetylserotonin to MI-3.In addition,we successfully cloned MaASMT9 of class I ASMT and MaASMT16 of class II ASMT from ‘Dashi'.MaASMT9 and MaASMT16 full-length c DNAs were independently cloned into pCold TF vectors for the expression of recombinant proteins in E.coli.MaASMT9 and MaASMT16 were expressed in E.coli BL21(DE3)and obtained their corresponding recombinant proteins.We co-incubated S-adenosyl-L-methionine and N-acetylserotonin separately with MaASMT9 and MaASMT16 in the reaction buffer and analyzed the products by UPLC-MS/MS.The recombinant proteins of the two MaASMTs(classes I and II)were only able to convert N-acetylserotonin to melatonin.These results indicate that the molecular pathway of melatonin isomer biosynthesis is similar to that of melatonin.ASTM members of a specific subgroup were able to biosynthesize melatonin isomers in mulberry(Morus alba).The present study lay a foundation for further molecular mechanism analysis of melatonin isomer biosynthesis in plant. |
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