The Function Analysis Of Eugenol Synthase Gene RcEGS1 In Rose

Rose is not only one of the most important cut flowers in worldwide,but it is also economically valued as a source of natural fragrance and flavour.Due to the limit of traditional cross-breeding and the complexity of scent-related metabolic pathway,fragrance seems to have been largely lost during the later stages of breeding process.Yunnan province is one of main origin for Rosa,and one of main production area of cut and garden roses.However,due to most of garden roses lacking of fragrance,edible and oil roses cultivars lacking of diversity,and lagging behind in basic research problems of rose scent,are the "bottleneck,of limitation to rose industry.So,it is not only of great theoretical significance to reveal the pathway of biosynthesis and metabolism and its regulation mechanism of rose scent,but also can lay a foundation for the development and application of essential oils and edible varieties.In this study,the volatile compounds of main Rosa resources were firstly analyzed,and the aim is to understand the different compounds among different rose species.Using the high-throughput transcriptome sequencing technology,the scent-related genes were obtained from Chinensis Old garden rose cultivar Rosa chinensis ’Old Blush’.The full-length of cDNA RcEGS1 is cloned,which is specially expressed in stamens and petals of R.chinensis ’Old Blush’,and performed the protein expression and function analysis.Furthermore,the promoter of RcEGS1 was isolated from ’Old Blush’.Regulation gene of RcEGS1 were identified by using yeast one-hybrid system.The main results and conclusion in the study are listed as follows:1.Using one group chinensis roses:R.odorata var.gigatea,R.odorata var.erubescens,and R.chinensis ’Old Blush’,the other group European roses:R.gallica,R.damascene and R.centifolia,the volatile compounds of these species were analyzed and compared by SPME-GS/MS.The results showed that the significant different compounds are showed between the two group roses.The typical volatile of Chinense roses is 3,5-Dimethoxytoluene and 1,3,5-trimethoxybenzene.The dominant compounds of European roses are 2-phenethyl alcohol and geraniol.In addition,the significant different compounds were showed between petal and stamen in these species.Eugenol is specifically showed in stamens of these species,and is the main compounds in the European species.2.The transcriptome of R.chinensis ’Old blush’ at different developmental stages,from flower buds to senescent flowers,was investigated using Illumina sequencing technology.De novo assembly generated 89,614 unigenes with an average length of 428 bp.Based on sequence similarity search with known proteins,62.9%of total clusters were annotated(E-value<10-5).Out of these annotated transcripts,25,705 and 37,159 sequences were assigned to gene ontology and clusters of orthologous groups,respectively.The dataset provides information on transcripts putatively associated with known scent metabolic pathways.It is found that 307 Unigenes were invovled in scent biosynthesis,including RcEGS1,RcAAT,RcOOMT2 and so on.Digital gene expression(DGE)was obtained using RNA samples from the stage of flower buds,open flowers,and senescent flowers.It is found that RcOOMT2 and RcEGSl were specially expressed in petals of ’Old Blush,.RcAAT,cytochrome RcP450 and RcR2R3 MYB were higher expression in petals than that of leaves.Comparative DGE and quantitative real time PCR permitted the identification of five transcripts encoding proteins putatively associated with scent biosynthesis in roses.The study provides a foundation for scent-related genes discovery in roses.3.Using qRT-PCR,RcEGS1 were found specific expressed in stamens and petals of R.chinensis ’Old Blush’.A low levels of RcEGS1 transcript were detected at the stage 1 of R.chinensis ’Old Blush5,and RcEGS1 mRNA accumulation increased continuously for the duration of flower development with a maximum expression level attained at the stage 4,when scent emission was at its peak,followed by a gradual decrease of transcripts to the background level at the stage 7.when volatile emission was at its lowest level.Furtherly,the full-length cDNA of RcEGS1 were obtained by RACE technology.Then,prokaryotic expression vector pDEST17-RcEGS1 was constructed by Gateway cloning technology to get heterologous recombinant protein expression in E.coli and purification.The expression of predicted 35kD recombinant protein was obtained.The expressions of RcEGS1 encoding genes were successfully silenced in rose flowers by virus-induced gene silencing(VIGS)and grafting.The results showed that eugenol emissions were significantly reduced.The relative content of eugenol was 11.916%of total volatiles in the control non-transformed plants and only 4.336%of total volatiles in the 3 silenced rose lines.These results can not only lay a foundation for the research of synthesis pathway of key synthase genes in rose,but also provide candidate genes for rose-scent breeding.4.RcEGS1 promoter was cloned from R.chirnensis ’Old Blush’.The sequences of RcEGS1 promoter were 1776bp,contained the conserved elements,and predicted to be with several cis,regulatory elements:MBS-box(TAACTG)、G-box(CACGTA)、TGACG-motif(TGACG)and MYB core(CNGTTR),which are related to secondary metabolism of plants.Yeast one-hybrid library of R.chinensis ’Old Blush’ at flower opening was constructed with the three-frame reading.The library capacity was more than 1.12×107CFU.The average insert of the library was 1Kb to 3Kb,which is fully meeting the demand of selecting the yeast library.Furthermore,the elements bait yeast vectors of with cis-transcription elements MBS-box,G-box and one fragment bait yeast vectors were constructed,respectively.Using yeast one-hybrid system,the transcript factor MYB was screened with the domain of R2R3,six the synthase genes and unknown functions of four genes were found in this study.These results can not only lay a foundation for the research of synthesis pathway of key synthase genes in rose,but also lay a foundation for understanding the upstream gene involved in regulation eugenol,and provide new idea for rose-scent breeding through regulation the metabolic pathway.