Construction Of Over-expression Vector Of RrDXR And RrAAT And Genetic Transformation Study In Rosa Rugosa

Rose (Rosa rugosa Thunb.) is the traditional flower that used to extract essential oil. The main components of rose essential oil are citronellol, geraniol monoterpene alcohols and its ethyl ester derivatives Citronellyl acetate, geranyl acetate act..Previous research findings that l-deoxy-D-xylulose-5-phosphate Reductoisomerase (RrDXR) and alcohol acyl(RrAAT) transferase genes are the key genes for the synthesis of monoterpenes flower fragrance in Rosa rugosa. This study construct the over expression vectors of these two genes,establish Petunia EAGLE regeneration system and the genetic transformation system. Lay the foundation for validation the two genes’function through the method that Agrobacterium tumefaciens mediated transformation of Petunia. At the same time the main results are as follows:1. The full opening petal of’Tang hong’ was used to extract total RNA by the CTAB method.After obtain the target gene,we successfully constructed the two gene’s over expression vector for pCAMBIA1304-DXR and pCAMBIA1304-AAT. Then introduced into Agrobacterium tumefaciens EHA105 and preserved. To lay the foundation for the next study:the target genes Transformate into petunia and verify its function.2. We got the Petunia EAGLE sterile material via direct seeding of sterile seeds into MS medium.Through using different sterilization methods, adding different concentration of hormone combination. Selected the best sterilization method and culture mediuminduced which induced Petunia callus differentiation. Establish the regeneration system of Petunia.The experimental results showed that the best sterilization method was 75% alcohol disinfection, the highest germination rate was 93.33%, the best callus induction medium was MS+1.5mg/L 6-BA+0.2mg/L NAA+30g/L sucrose+7g/L agar, pH5.8; the optimal rooting medium was 1/2MS +30g/L sucrose+7g/L agar, pH5.8.3. The test was on the basis of Petunia EAGLE regeneration system. The best formula was selected from the genetic transformation system. Hyg concentration of antibiotics for differentiation of callus induction in leaves was 5mg/L; Hyg concentration of antibiotics for the rooting medium was 3mg/L; Cef concentration of antibiotics for antibacterial was 300mg/L. In the experiment, the transformation method of the leaf disc mediated by Agrobacterium tumefaciens was used to transform over expression vector for pCAMBIA1304-DXR and pCAMBIA1304-AAT into petunia, the callus and adventitious bud was successfully induced on the basis of the selection of the transformed leaf discs.