Functional Study Of COI1 Gene, A Key Member Of The Jasmine Signaling Pathway In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge(Lamiaceae)is a highly valued herb whose roots and rhizomes are obtained for use in traditional Chinese medicine to treat various cerebrovascular and cardiovascular diseases.It is described as medicinal model plant due to its significant medicinal value,the clearly biosynthesis pathways of active compounds,efficient transgenic methods,and relatively small genome.Many studies have shown that jasmonates(JAs)are new phytohormones regulate plant defense and developmental process.It is a strong elicitor that is widely used to induce the production of important active compounds in S.miltiorrhiza but the regulation mechanism is still unknown.COI1 protein is a necessary member in JAs signal conduction pathway,however the mechanisms of JAs inducing tanshinones and slavianoic acids in S.miltiorrhiza are still unclear.Using molecular clone,screening two-hybrid yeast library,RNA interference and comparative transcriptone,we are trying to explore the function of SmCOI1 and its role in JAs regulated secondary metabolism via JA signaling pathway in S.mlltiorrhiza.The main results are as follows:1.The flull-length SmCOI1 cDNA sequence was cloned based on the homologous fragments with AtCOI1 by screening transcriptome database of S.miltiorrhiza and rapid amolification of cDNA ends(RACE)techniques from S.miltiorrhiza.The total length is 1,962 bp,contained opening reading frame of 1,797 bp,which could encode 599 amino acid residues.The sequence was highly homologous with COI1 of sesame and tomato,containing F-box and LRR domains.Its 3D modle is also very similar with AtCOIl.5 flanking region of SmCOI1 gene shoved several plant biotic or abiotic responsive elements by PlantCARE database analysis.The real-time quantitative PCR results indicate that SmCOI1 is constitutively expressed in the roots,stems,leaves and flowers of S.miltiorrhiza and is induced by methyl jasmonate,gibberellins,salicylic acid,mechanical damage,and low temperature.2.We constructed cDNA library for two-hybrid yeast test with 2-year-old S.miltiorrhiza.The capacity of the library is 1.1×1O7,the average size of insert fragments is 1.54 kb and the recombination rate is 100%.We also constructed the bait vector Y2H-COI1-pGBKT7 to screen the library by Mating.There were 253 clones on SD/-Trp/-Leu/-His/-Ade medium totally and 61 homologous sequences were got by BLASTX,containing SKP1,lectins,thioredoxin,CaMBPs and EPFL.It indicates that SmCOI1 could combine with SKP1 to form SCF complex and may combine with lectins and thioredoxin to participate in plant defense,while combining CaMBPs and EPFL to take part in plant hormone signal conduction.It has shown that SmCOIl may participate in defense,secondary metabolism regulation and JA signal pahthwy or crosstalk with calcium signaling pathway.3.The RNAi transgenic lines(iCOIl)which silencing the expression of SmCOI1 were obtained by transgenic method.To test the resistance to insect of iCOI1s,we fed cotton bollworms(Heliothis armigera)with leaves of iCOI1s.On the fifth day of treatment,the mean damage score of the iCOI1s was 2 fold of WT.Meanwhile,the expression of WRKY70 and NPR1 were significantly reduced in iCOIls.Furthermore,the concentrations of rosmarinic acid,salvianolic acid B,cryptotanshinone and tanshinones IIA in iCOI1s and control lines were determined by HPLC.The results showed that four kinds of metabolites were significantly reduced when compared with wild type.And the expression of key genesinvolved in tanshinones and salvianolic acid biosynthesis pathways e.g.HMGR,GGPPS,CPS,KSL,CYP76AH1,C4H,RAS and HCT were also brought down in iCOI1s using real-time PCR.The resluts indicate that SmCOI1 has closely relations with defense and secondary metabolism in S.miltiorrhiza.4.To further test the function of SmCOI1 in JAs signaling pathway,we tested and compared the root growth,the concentrations of 4 main metabolite and the expression levels of 10 JA-induced genes with iCOI1s and WT under JAs treatment.The results showed that root inhibition appeared in WT under JAs treatment whereas that was absence in iCOI1s.Similarly,the concentrations of rosmarinic acid,salvianolic acid B,cryptotanshinone and tanshinones IIA could be significantly induced by jasmonates while it did not show up in iCOI1s lines.Finally,the expression of genes induced by JAs(TAT,RAS,DXS,GGPPS,CPS and KSL)were not up-regulated by jasmonates in iCOI1 lines.All these results indicate that SmCOI1 plays an important role in jasmonates-induced physiological process.5.Using the Illumina HiSeq 2000 plamform for sequencing transcriptomes of both iCOI1s and WT under JAs treatment,55,038 Unigenes were assembled.Among them,37,200 Unigenes contain whole ORF.A total of 86.57%unigenes were annotated by Basic Local Alignment Search Tool against public sequence databases.The enriched GO term were transcription regulation,catalyze activities,and bioprocess regulation.We found 4,705 unigenes were differentially expressed between the two samples,in which 1,146 genes were up-regulated,1,231 genes were down-regulated and 2,328 genes were inconsistent.Futher KEGG pathway analysis showed that the DEGs mostly involved in 21 pathways,containing terpene biosynthesis,phenypropanoids biosynthesis and flavonoids biosynthesis.The DEGs included 36 genes involved in JAs induced secondary metabolism,among them 29 genes are firstly found.8 genes involved in JAs induced defense process and one salicylate O-methyltransferase gene may take part in JAs induced root inhibition.Those results indicated that SmCOI1 was important in JAs induced secondary metabolism,defense and morphogenesis.In addition,11 transcription factors(e.g.,WRKY,bHLH and NAC)were found response JAs dependently SmCOI1 they may paly a role as regulator in JAs induced physiological processes.In summary,SmCOI1 may combine with lectins and thioredoxin,regulating defense related genes WKRY70 and NPR1 to participate in resistance and secondary metabolism process.For another,SmCOI1 may combine with Skp1 to form SCF complex or bind with CaMBPs to involved in JAs siganl pathway or crosstalk with calcium signals.Our results will help realizing the functions of SmCOI1 and other members involved in JAs signaling,and also can help enriching the understanding of JAs signaling in S.miltiorrhiza.