Screening And Expression Pattern Analysis Of Triterpene Saponin PDR Transporters Of Aralia Elata(Miq.) Seem

Triterpenoid saponins are a large class of natural metabolites in plants that have important medicinal and economic value,and their biosynthesis and transport have always been the focus of research.Aralia elata is a kind of medicinal and edible plant that is rich in triterpenoid saponins and is an important material for studying the synthesis and transport of pentacyclic triterpenes.At present,there are many studies on the biosynthesis of Aralia saponins,but the research on the transport of saponins has not been reported yet.Plant multidrug resistance(Pleiotropic drug resistance,PDR)proteins belong to the ABCG subfamily of adenosine triphosphate binding cassette(ATP-binding cassette,ABC)transporters,which play an important role in the transport of secondary metabolites,especially terpenoids,across the membrane.In this study,the ABC transporter gene family was identified and systematically analyzed based on the whole genome sequencing of Aralia elata,and the PDR transporter was effectively screened.Furthermore,the key PDR genes involved in triterpenoid saponins transport were screened by bioinformatics analysis,transcriptional and metabolic association analysis,and genetic evolution analysis.This study can provide new genetic resources and theoretical support for triterpenoid saponins transport and genetic improvement of Aralia elata.The main research results are as follows:(1)The whole genome sequencing of Aralia elata identified a total of 143,143 ABC transporters,and 21 PDR transporters were screened by phylogenetic analysis,protein conserved domain analysis,and functional domain analysis.The PDR gene of Aralia elata was distributed on chromosomes 1-12,especially chromosome 6.Intraspecific and interspecific collinearity analyses showed that there was a collinear relationship among multiple genes,indicating that there were gene tandem repeats and multi-copy amplification of the Aralia elata PDR gene in genetic evolution.The results of promoter element analysis showed that PDR gene promoters mainly contained light response elements,followed by abscisic acid,methyl jasmonate,defense and stress response elements,and salicylic acid response elements;a few genes also contained MYB-light response elements,auxin,and gibberellin response elements.The specific expression pattern of the PDR gene was analyzed using the transcriptome sequencing results of roots,stems,and leaves,and the obtained results showed that the PDR gene was mainly highly expressed in the leaves and stems and low in the roots.Furthermore,21 PDR genes were screened,and the genes with too long or too short a sequence,an incomplete transmembrane structure,or an incomplete coding region were removed,and 7 triterpenoid saponin transporter candidate genes were preliminary identified.(2)The endogenous contents of total saponins and three monomer saponins(oleanolic acid,ivy sapogenin,and acanthic acid)of 3-year-old Aralia elata were measured in different plant parts(roots,stems,and leaves)at different sampling times,and the spatio-temporal expression patterns of the seven candidate genes were studied.A total of four targeted genes were identified as associated based on the Pearson correlation analysis between saponin content and gene expression.Finally,based on the homologous sequence alignment and association analysis,three PDR candidate genes(AE04G03127,AE02G00218 and AE12G01725)were identified as being involved in terpene transport.(3)We studied the induced expression patterns of candidate genes by spraying 100 umol/L methyl jasmonate(MeJA)and salicylic acid(SA)on annual seedlings of Aralia liaodongensis.The results showed that the AE04G03127,AE02G00218 and AE12G01725 genes responded to the induction of MeJA and SA.These three genes were significantly up-regulated at 6 hours of MeJA treatment as compared to the control treatment.AE12G01725 gene was significantly up-regulated at 48 h after SA treatment;AE02G00218 gene was significantly up-regulated at 6 h and 24 h after SA treatment,and AE04G03127 gene was significantly up-regulated at 6 h and 12 h after SA treatment.In addition to responding to the hormone-induced expression,the expression levels of the three genes were significantly up-regulated after mechanical injury treatment,and the highest gene expression was observed 6 h after treatment,indicating that these three PDR genes may be involved in defense and stress response.(4)Gene cloning and bioinformatics analysis showed that the AE12G01725 gene encoded1327 amino acids and belonged to hydrophilic protein.The secondary structure of the protein sequence was dominated by α helices without signal peptides,but there were 11 transmembrane helices,which was a typical ABC protein.The 3D structure of the protein was also predicted by using the PDR5 protein of yeast as a model.