Isolation And Analysis Of Geraniol Synthesis Gene RcTPSs In Rosa Chinensis

Roses(Rosa sp.)belong to the Rosaceae family,are one of the most important ornamental plants in the world.Because of its rich petal colors and various fragrance,roses were widely used in landscape.Floral fragrance is an important direction of rose breeding,it also has certain economic value.Geraniol,a terpene compound,is an important component of rose fragrance.In plants,geraniol was catalyzed and synthesized by terpene synthase(TPS).However,the function of rose terpene synthase were still unknown.Thus,isolate the key genes which regulate the rose terpene biosynthesis and further investigate the function of rose terpene synthase could make great significance to comprehensive reveal the mechanism of rose aroma synthesis.In this study,two kinds of chinese ancient rose were used as experimental materials(Rosa chinensis‘Old Blush’,OB,with strong aroma;Rosa chinensis‘Old Blush’-like,OBL,with weak aroma),to comprehensive analyze rose TPS gene family、determine the rose aroma components、excavate the transcriptome data and perform prokaryotic expression experiment in vitro.The main results were given as follows:(1)InOB and OBL petals,67 kinds of aroma components were examined,including 31 significant differences metabolites.These main metabolites comprised six volatile compounds derived from fatty acid,17 terpenoids in the scent of roses,one carotenoid derived volatiles,two volatile compounds related to the shikimic acid pathway and five other compounds.Among them,16 terpenoids were more abundant in OB petals.In OB and OBL petals,2,6-Octadien-1-ol,3,7-dimethyl-,(E)-(Geraniol)and 2,6-Octadienal,3,7-dimethyl-,(E)-(Citral)were identified as the significant differential metabolites.(2)Based on transcriptome data,a total of 3,341 differential expressed genes were found,including 1,629(OB vs.OBL)down-regulated genes and 1,712(OB vs.OBL)up-regulated genes.KEGG enrichment analysis suggested that these DEGs mainly involved in “ metabolic pathways”、“biosynthesis of secondary metabolites” and “phenylpropanoid biosynthesis”.Based on previous transcriptome databases from different developmental stages and different tissues,a total of 14 DEGs which correlated with the change trend of geraniol were screened,including two terpene synthases(RcTPS37 and RcTPS52)and four transcriptional regulator which may involve in the biosynthesis of rose scent.(3)Combined with the results in(2),the gene family analysis of terpene synthase was carried out.The genome-wide identification of TPS gene family of 115 angiosperms was carried out at the genome-wide level.A total of 3,849 full-length TPS were identified and further divided into six subfamilies/subgroups: TPS-a、TPS-b、TPS-c、TPS-g and TPS-e/f.TPS-a was the largest subgroup.Synteny networks were constructed based on 1,618 syntenic relationships TPSs.The network demonstrated that TPS-a and TPS-b were widespread across 115 angiosperms;TPS-g and TPS-e/f were conversed in rosids and monocots species.(4)In rose,a total of 54 TPSs were identified,among them TPS-a contained 36 RcTPSs and these members formed two sub-clades with diversity branch lengths.The 36 RcTPSs formed large clusters on Chr1 、 Chr3 and Chr5,gene tandem duplication events were occured.In synteny networks,27 rose syntenic homologs were detected,two RcTPSs were Rosaceae specific syntelogs.On the basis of analysis of rose TPS gene family,OB and OBL were used as experimental materials,the metabolisim and transcriptome data were further analyzed.(5)Based on the analysis of rose terpene synthase gene family,the key TPSs were carried out for further experimental research.A gene positively correlated with the change trend of geraniol content was successfully cloned and named as RcTPS37 a.The similarity of the RcTPS37 a promoter sequence in OB and OBL is 97%,in promoter region,the promoter cis-acting elements analysis revealed that there were some transcription factor binding sites which may related to floral fragrance.Successfully constructed p ET30a::RcTPS37 a prokaryotic expression vector and explored the protein induced expression system.In 0.8 m M IPTG,induced at 16℃ for16 h situation,the soluble RcTPS37 a protein content is higher,and could be separated and purified by affinity chromatography.In summary,by analyzing the different components and genes of the two rose materials at metabolome and transcriptome level,combined with the analysis of rose TPS gene famiy.Here,the terpene synthase RcTPS37 and RcTPS52 which positive related to the synthesis of geraniol pathway were selected.Successfully constructed RcTPS37 a prokaryotic expression vector and established a protein induction and purification system.These results could provide references for the phylogeny and functional evolution of studies on R.chinensis terpene synthase,and also could supply useful information for the subsequent functional verification of TPS.It also could provide some useful reference for the molecular biology method to improve the scent in roses.