Identification And Functional Analysis Of Root-specific Promoters In Panax Ginseng Mey.

Ginseng,Panax ginseng Mey.,is a medicinal plant species belonging to Panax genus,the Araliaceae family,known as the king of herbs in oriental medicine,and has been renowned domestically and abroad since ancient times.The major pharmacological components of ginseng are triterpenoid saponins,i.e.,ginsenosides,that are abundant in roots.Recent development of the ginseng genome sequence and various transcriptomes has made it possible to deeply study functional genes and decipher the molecular regulation mechanism underlying ginsenoside biosynthesis.Promoters are the key elements regulating the expressions and biological functions of functional genes.They are in the immediate upstream of the genes that are specified with cis-acting elements as binding targets for proteins necessary to initiate gene transcription,thus playing a role of "switch" to turn on and off the activities of genes.There are three types of promoters,constitutive,inducible,and tissue specific.Constitutive promoters consistently regulate gene expression,and thus,function under all circumstances;inducible promoters regulate gene expression and function only when they receive a stimulus such as an environmental stress;tissue-specific promoters regulate genes to express only in a specific tissue or organ.A variety of tissue-specific promoters have been isolated and widely used for genetic improvement in crops,such as rice,wheat,and maize,but no tissue-specific promoter has been reported in ginseng yet.The aims of this study were to identify,isolate,and functionally analyze root-specific promoters for ginseng,thereby facilitating research and application of functional genes in ginseng and its genetic improvement,especially the biosynthesis of ginsenosides.Summarized below are the major findings of this study.1.Identification of root-specific promoters.(1)The transcriptomes of 14 tissues of a four-year-old ginseng plant were analyzed using an expression level of TPM(transcripts per million)=0 as the threshold value.Seventy-one root-specific candidate genes that were expressed in all ginseng root tissues were obtained.Gene ontology(GO)annotation showed that 44 of them were root-specifically expressed.(2)Alignment and comparative analysis with ginseng genome sequence identified that 10 of these 44 genes had perfect identities to those in ginseng genome and therefore,selected as the targeted genes for identification of root-specific promoters.(3)Analysis of the 2.0-kb sequences immediately upstreaming the 10 root specifically expressed genes identified three promoters that contained cis-acting elements,ROOTMOTIFTAPOX1 and OSE1ROOTNODULE,regulating gene expression specifically in roots.Therefore,these three promoters were selected as candidates for root-specific promoters and designated PgRoot17-pro,PgRoot53-pro and PgRoot49-12-pro.Correspondingly,the three root specifically expressed genes were designated PgRoot17,PgRoot53,and PgRoot49-12.2.Validation of the three root-specific promoter candidates,PgRoot17-pro,PgRoot53-pro and PgRoot49-12-pro,in regulating of gene expression specifically in roots.(1)Genetic transformation into Arabidopsis.Promoters,PgRoot17-pro,PgRoot53-pro and PgRoot49-12-pro,were transformed into Arabidopsis and analyzed by GFP(green fluorescent protein).GFPs were observed only in the roots of the promoter transgenics under blue light,indicating that these three promoters are all root specific.(2)Genetic transformation into ginseng adventitious roots.Similarly,PgRoot17-pro,PgRoot53-pro and PgRoot49-12-pro were transformed into ginseng adventitious roots and analyzed for GFP assay.The GFP of the PgRoot17-pro transgenics was higher than that of PgRoot53-pro and the PgRoot49-12-pro transgenics had the least GFP.The result verified that these promoters all expressed in ginseng adventitious roots and functionated in in-vitro cultured roots.3.Analysis of PgRoot17-pro,PgRoot53-pro and PgRoot49-12-pro for elements necessary for their function.Bioinformatic analysis showed that all three promoters,PgRoot17-pro,PgRoot53-pro and PgRoot49-12-pro,have the core regulatory elements,TATA-BOX and CAAT-BOX,and cis-acting elements,ROOTMOTIFTAPOX1 and OSE1ROOTNODULE,that are associated with root-specific expression.In addition,they also contain several abiotic stress-responding elements,including phytohormone response elements such as defense and stress response elements,methyl j asmonate response elements,and gibberellin response elements.4.Roles of PgRoot17,PgRoot53,and PgRoot49-12 in ginsenoside biosynthesis and their response to MeJA(methyl jasmonate)regulation.(1)The three root-specific promoters-containing genes were further subjected to the analyses of expression correlation with ginsenoside contents,co-expression network with 16 cloned genes involved in ginsenoside synthesis,and impacts of gene mutations on ginsenoside contents in four-year-old plant roots of 42 ginseng famers’ cultivars.PgRoot17 and PgRoot53 were shown to be involved in the biosynthesis of ginsenosides Re and Rg1,respectively,and PgRoot49-12 was involved in the biosynthesis of ginsenosides Rb1 and Rd.(2)Expressions of PgRoot17,PgRoot53,and PgRoot49-12 were induced with MeJA in ginseng adventitious roots.The adventitious roots treated with MeJA had significant different contents of Re,Rg1,Rbl,and Rd at different times after MeJA treatment compared to those not treated with MeJA.Similarly,the expressions of these genes were also significantly different in the adventitious roots treated with MeJA from those not treated with MeJA.These results further indicated that these genes were involved in the biosynthesis of ginsenosides Re,Rg1,Rb1,and Rd.In summary,the present study,for the first time,identified three root-specific promoters and validated their functions in gene transcription in ginseng adventitious roots.It was also found that the genes containing these promoters were involved in biosynthesis of several ginsenosides.These root-specific promoters and their genes provide not only promoters and genes necessary for genetic improvement of ginseng adventitious roots and ginsenoside production through adventitious root culture,but also tools necessary for advanced research,development,and application of functional genes in ginseng.