| Rose rugosa Thunb.is an important aromatic plant in the world,it's also an traditional chinese medicine and food homologous flowers.Monoterpene and its acetate compounds such as geraniol,nerol,citronellol,geranyl acetate,are the main fragrance of flowers and essential oil of rose.It is of great theoretical and practical significance that regulating the biosy nthetic pathway of monoterpenoids and promoting the accumulation of downstream metabolites by using the genetic engineering and metabolic engineering methods,so as to creates new rose cultivars with high essential oil content.Based on the preliminary research of our laboratory,we isolated the genes which related to monoterpene metabolism,cloned the promoters of related genes,and carryed out yeast single hybridization screening.This article carryed out the functional verification of the genes which related to the biosynthesis of main fragrance of rose flowers by using the Petunia hybrida as the test material.The aim of this study was to explore the key genes which regulate the metabolism of rose fragrance,and lay the foundation for culturing the new rose cultivars with high essential oil content.The main results were as follows:1.The full cDNA sequence of RrNUDXl gene which is the member of the Nudix hydrolase superfamily was cloned by RACE method using the full opening petals of Rosa rugosa'Tanghong' as the test material,and the cDNA of RrNUDX1 has a full length of 777 bp,with a complete open reading frame,which encodes 151 amino acid,and the Genbank No.is KX096710.1.Combined with the aroma components analysis of different flower development periods and different parts of the floral organs in Rosa rugosa 'Tanghong',we found that the changes of RrNUDX1 gene expression level were consistent with the changes of the main aroma components,which all increased first and then decreased.With the process of the rose flower opening,the expression level of the RrNUDX1 gene gradually increased,and the content of the main aroma components also increased.We also found the content of citronellol and citronella citrate reached the highest level in the half opening stage,and the other four components including geraniol,nerol,geraniol acetate and neryl acetate reached the highest level in the full opening stage.Take a look at the different parts of the flower organ,the aroma components of Rosa rugosa are mainly concentrated in the petals,stamens,pistils,an the content of other parts is very low,this is consistent with the expression of RrNUDX1 gene in the different parts of the flower organs.In a word this result suggested that the RrNUDXl gene was closely related to the biosynthesis of the aromatic monoterpene components of R.rugosa.2.Using the invitrogen system,a three-frame reading yeast single-hybrid library of R.rugosa was constructed by using the half opening to the bloom opening petals of R.rugosa'Tanghong'.The results showed that the library capacity was greater than 1X107CFU,the plasmid no-load rate was less than 5%and the average insert was greater than 1.2Kbp,it's fully meeting the requirements of the yeast library.This laid an important foundation for establishing a bait vector through single hybridization screening,and finding the transcription factors related to rose fragrance in the next step3.The promoter sequences of RrNUDX1 and RrDXR genes were cloned by chromosome walking method respectively.The results showed that the RrNUDX1 gene obtained 617 bp promoter sequence,it was predicted that there were two elements related to floral components'metabolism:the core element MBS(N1 TAACTG)of MYB and jasmonic acid core component(N2 TGACG).The RrDXR gene obtained 2213 bp promoter sequence,it was predicted that there were three elements related to floral components metabolism:MYBHvl binding site(D1 CAACGG),flower specific phenylpropanoids and floral formation of important cis-acting elements(D2 GACCAACC,D3 AACCTACT).The five cis-acting elements were constructed ahead of the promoter in the reporter gene,and the yeast single hybridization was screened.The results showed that these elements did not interact with RrNUDX1 and RrDXR,and could not further screen out the transcription factors that regulates the cis-acting elements.Subsequent studies need to further expand the amount of screening of the predicted fragments to obtain the corresponding transcription factor.4.The overexpression vector pCAMBIA1 304-RrNUDXl of RrNUDX1 gene was constructed and infected into Petunia hybrida by Agrobacterium tumefaciens,and we found that the Petunia plant with overexpressed RrDXR gene was not significantly different from the wild type,while the leaves of Petunia hybrida with overexpressed RrNUDX1 gene were broadened and the plants grew faster.GC-MS analysis showed that the contents of major aroma components such as methyl benzoate in Petunia plant with overexpressed RrNUDX1 gene were significantly improved,This laid a breakthrough foundation for us to use the RrNUDXl gene to improve the main floral composition of roses and to cultivate new rose cultivars with high essential oil content. |
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