Cloning And Expression Analysis Of RrDXS And RrLIS Genes Related To Monoterpenes Biosynthesis In Rosa Rugosa

Rosa rugosa Thunb. is a kind of important ornament plant, and it is also a rare anatural flavor plant which belongs to genus Rosa. The deciduous shrubs have a delightful fragrance and splendid color. Monoterpenes including citronellol, geraniol, nerol, its acetic acid ester derivatives and linalool are the major fragrant component of Chinese traditional R. Rugosa and rose essential oil. Taking’Tang hong’, the leading cultivar of oil-bearing Rose as experimental material, we cloned two genes RrDXS and RrLIS which related to the monoterpene biosynthetic pathway, then expression patterns of the two genes RrDXS and RrLIS in different developmental stages and different organs of R. rugosa were researched, and the relationship between the accumulation of monoterpene constituents and the gene expression in R. rugosa were analyzed too. The main results are as follows:1. Total RNA of’Tang hong’was extracted using the CTAB method, then the full sequence of RrDXS and RrLIS gene which related to the monoterpene biosynthetic pathway were cloned by RACE method. The result for the test have shown that, the full length of RrDXS was2091bp, with the start codon, the complete open reading frame （1494bp）, termination codon, the5’ non-coding region （273bp）, the3’non-coding region （324bp） and poly （A） tail （llbp）, which encodes473amino acids, and the Genbank No. is JN675704; the full length of RrLIS was1318bp, with the start codon, the complete open reading frame （1239bp）, termination codon, the5’ non-coding region （48bp）, the3’non-coding region （31bp） and poly （A） tail （llbp）, which encodes413amino acids, and the Genbank No. is JN67570. By comparing the DNA sequence with publicly availably reference sequences in Genbank, RrDXS is most similar with Pueraia lobata（AY315652） and the similarity is85%. The deduced amino acid sequence shared averagely94%homology with Hevea brasiliensis（BAF98288）、Ricinus communis（XP₀02516843） and Lycoperisicon esculentum（NP₀01234672） DXS amino acid sequenc. RrLIS is most similar with Vitis（HM807391.1） and the similarity is75%. The deduced amino acid sequence shared averagely95%and96%homology with Populus trichocarpa（AEI52903.1）、Vitis vinifera（ADR74211.1） and Actinidia sinensis （ADD81294.1） LIS amino acid sequenc.2.’Tang hong’was used as plant material to study the expression patterns of RrDXS and RrLIS in different developmental stages and different organs of R. rugosa by RT-PCR and Realtime-PCR, the relationship of the R. rugosa volatile substances accumulation and the gene expression were analyzed also. We found that the expression level of RrDXS、RrLIS was increasing from bud to bloom stage in R. rugosa, strongest at bloom and weakest at senescence stage. The expression level of RrDXS was highest in rose receptacles, but in the petals, stamens and pistils the expression level were low; The expression level of RrLIS was high in rose petals, stamens and pistils, low in other flower organs, and the overall expression level of RrLIS was significantly less than RrDXS. Combined with GC-MS analysis, we found that the level of linalool in flower of’Tang hong’is low just as the gene expression level, while the level of citronellol, geraniol, nerol and its acetic acid ester compounds were higher and they changed in the same trend with the expression of RrDXS. But the variation in content of monoterpenes and its acetic acid ester derivatives in different parts of rose flower was not consistent to the RrDXS gene expression.,so we concluded that RrDXS gene may play a key role in biosynthesis of the aromatic components of monoterpenes in Rosa rugosa, but not a decisive key gene.