Whole-genome Sequencing Of Hypericum Perforatum And Functional Study Of Serotonin Methyltransferase Gene

Hypericum perforatum L.(Hypericaceae)is a perennial herb,also known as St.John’ s wort,mainly used as medicine for the above-ground part of plants.Traditional Chinese medicine(TCM)believes that it has the effects of soothing liver-qi stagnation,clearing heat and dampness,reducing swelling and pain,and regulating menstruation and activating blood circulation.Modern pharmacological research shows that it also has antidepressant,antitumor,antiviral,antibacterial and analgesic effects.In western countries,its extract has been used to treat mild to moderate depression for centuries,among which hypericin,hyperforin,and melatonin are the main characteristic components.The naphthodianthrones hypericin and pseudohypericin are natural potent photosensitizers and the antiviral mechanism of H.perforatum is related to its photosensitive activity.The proved strong anti-tumor activity in vivo and in vitro upon irradiation of visible light gains recently an increased attention.And it was reported that phloroglucinol hyperforin,rather than hypericin as initially considered,is one of the most effective biomolecules of antidepressant activity.Many studies have shown that the change of melatonin level is likely to be one of the factors leading to depression,and it has been proved that exogenous melatonin can improve the Depression Behavior of chronic stress depression model rats.In plants,melatonin is considered as a new plant hormone,which can participate in plant growth regulation,scavenging reactive oxygen species and regulating photoperiod.In virtue of the existence of these metabolites,the species is listed as one of the world’s top herbal medicines and supplements by Global Industry Analysts,Inc.Despite its pharmacological importance,the biosynthetic pathways and the key enzymes are still far from being understood.The whole genome information of any species in the Hypericaceae family has been sequenced and interpreted.The genome-wide information that can be referred to is only from other distant relatives within the Malpighiales oder Linaceae family:Linum.usitatissimum,Crotonoideae subfamily:Hevea.brasiliensis,and members of the Populus genus.The lack of genomic information limits the comprehensive understanding of its genetic background and biosynthesis.Therefore,we carried out genome-wide sequencing analysis on H.perforatum and transcriptome-sequencing-annotated annotation of different tissues in order to obtain the genome sequence map of the species.At the genome level,we have studied major issues such as the growth,development,evolution,and origin of species,deepened our understanding of species,and played a huge role in discovering new genes and improving species.Therefore,research on species growth,development,evolution,origin and other major issues at the genome level will deepen our understanding of species and play a huge role in the discovery of new genes and species improvement.The main research contents and results:1.In this study,the whole genome sequencing of H.perforatum was performed for the first time.Using Illumina,Pacbio,and 10× Genomics sequencing technology,a total of 256.95 G data was obtained.Through de novo assembly,the contig N50 and the scaffold N50 reaches1.41Mb and 2.31Mb respectively.K-mer analysis showed that the genome size of H.perforatum was 393.40 Mb,and the heterozygosity was 1.13%.The combination of de novo prediction and homology-based comparisons resulted in the identification of 175.1 Mb repetitive elements in the H.perforatum genome,accounting for about 46.9%of the assembled genome.45.7%of the repeats in the H.perforatum genome were transposable elements(TE),of which 35.4%were long terminal repeats(LTR).A total of 29,150 protein-coding genes were predicted in the assembled genome,of which 22,247 genes(76.3%)were supported by RNA-sequencing data(FPKM>0.05).Through comparative genomics analysis with Arabidopsis thaliana,Oryza sativa,Solanum lycopersicum,Linum usitatissimum,Hevea brasiliensis,and Populus trichocarpa whole genome information,the expansion and contraction of H.perforatum gene families,whole-genome duplication,and positive selection analysis were studied.Finally,with the help of St.John’s Wort transcriptome database,the metabolic pathways of its three main metabolites:hypericin,hypericin and melatonin were predicted and key enzyme genes were screened.Finally,according to the transcriptome database of H.perforatum,the metabolic pathways of hypericin,hyperforin and melatonin were predicted and the key enzyme genes were screened.2.Based on the whole genome database of H.perforatum,we selected the reference genes of H.perforatum for the first time.There were 14 genes(ACT2,A CT3,ACT7,CYP1,EF1-α,GAPDH,TUB-α,TUB-β,UBC2,GSA,PKS1,PP2A,RPL13,and SAND)evaluated in four different plant tissues,four different stages of development and a variety of abiotic stresses and hormone treatments by using three programs of geNorm,NormFinder and BestKeeper.Combined with all the algorithms,ACT2 and TUB-β are the most stable combinations in different developmental stages and all the experimental samples.The results will be helpful to further improve the reliability of standardization and quantification of transcription level of H.perforatum.3.Based on the whole genome database of H.perforatum,the R2R3-MYB gene family,one of the most important plant transcription factors,was recognized and studied.A total of 109 R2R3-MYB genes were identified and clustered into 36 subfamilies.Four kinds of cis-acting elements were found in the promoter region,most of which were related to stress response and plant growth and development.The transcriptome data of different tissues(root,stem,leaf and flower)of H.perforatum showed that different R2R3-MYB genes had obvious tissue expression specificity.RT-qPCR analysis indicated that 11 stress-related R2R3-MYB genes showed specific expression patterns under different treatment conditions.At the same time,by using the Gateway technology,five subcellular localization vectors of HpMYB gene were constructed.GFP fluorescence was observed by bombarding onion epidermal cells with gene gun,and five proteins were confirmed to be expressed in the nucleus.This study provides an important basis for further study on the background and function of R2R3-MYB gene family in H.perforatum.4.As the last key enzyme gene of plant melatonin pathway,5-hydroxytryptamine methyltransferase(ASMT)was cloned by PCR to obtain the full length of 5 HpASMTl-HpASMT5 genes,which were respectively:HpASMTI,1,080 bp,encoding 359 amino acids;HpASMT2,1,062 bp,encoding 353 amino acids;HpASMT3,1,056 bp,encoding 351 amino acids;HpASMT4,1,080 bp,encoding 359 amino acids;HpASMT5.1,083 bp,encoding 360 amino acids.The results showed that these genes were highly expressed in the above ground tissues and responded to abiotic stresses such as salt and drought.By transient transfection of Arabidopsis protoplasts,it was found that HpASMT2,HpASMT3,HpASMT4 and HpASMT5 were all expressed in the nucleus and cytoplasm,except for HpASMT1,which was located in the cell membrane.It was also found that the expression of HpASMT3 in the cytoplasm was more likely to be located on the chloroplast membrane.Five GST-HpASMT fusion proteins of about 65 kDa were purified by prokaryotic expression,and finally five purfied GST free HpASMT proteins through digestion were successfully obtained.According to the enzyme activity test in vitro,it is confirmed that these five genes can all encode ASMT protein,which can transform the substrate N-acetyl-5-hydroxytryptamine into the final product melatonin.By changing the concentration of the substrate,the enzyme kinetic characteristics KM and VMax.values of HpASMT3 protein are calculated.It was also found that HpASMT3 was the most active ASMT protein in plants.5,The over-expression vectors of HpASMTl and HpASMTS genes were constructed using Gateway technology.The positive transgenic lines were screened and identified at the level of DNA and RNA by Agrobacterium tumefaciens mediated infection of Arabidopsis mutant asmt.Among them,three homozygous HpASMTl and lines HpASMT3 with high expression:OE-1,OE-3,OE-5;OE-2,OE-4,OE-5 were obtained in Arabidopsis.Compared with the mutants,the content of melatonin in the transgenic plants increased more than 2 times.Secondly,through drought and high salt stress on different Arabidopsis lines,it was found that the root system of transgenic plants was more developed under the stress,and it could more effectively eliminate the active oxygen and enhance the enzyme activity of antioxidant enzymes,thus enhancing the drought and salt tolerance of plants.At last,it was proved that the transcription factors HpMYB44 and HpMYB55 could directly bind to the promoter regions of the melatonin synthesis pathway genes HpASMT1 and HpASMT3.In conclusion,based on the construction of the whole genome map of H.perforatum,the reference gene and R2R3-MYB gene family of H.perforatum were studied and analyzed.The 5-hydroxytryptamine methyltransferase of the melatonin metabolism pathway in Hperforatum was characterized.The obtained genomic data not only provide a new reference for the analysis of the evolutionary history and model of other species in hypericidae,but also provide a basis for further research on the biosynthesis of melatonin in other plants.