| In order to obtain novel color of Rose successfully and regulate anthocyanin metabolic pathway effectively, it is imperative to select efficient tissue-specific promoters to improve the expression of exogenous genes in the transgenic rose. Meanwhile, for the purpose of transgenic research followed by more in-depth and expression regulation of exogenous gene precisely, it should be tried to avoid repeating the same sequence of the promoter fragment to reduce the risk caused by gene silencing. This study was designed to clone rose tissue-specific promoters to specifically start gene expressing in flower tissue. Furthermore, we also improved the expression activity of the promoter by fusing enhancer with core region of Rose flower specific promoter RCHS to become chimeric promoter, which can be well established as effective regulatory elements for the multi-gene expression vector and can be achieved the goal of enhancing the expression activity of tissue-specific promoter. The main results were obtained as follows:1. Establishment of Rapid Propagation System of Chinese RoseWe take the shoots carrying axillaries buds in the middle of the branches in the "Carola" rose stem segment(semi-lignified) as explants, using MS as the basic medium with different hormone combinations of NAA and 6-BA to induced buds, proliferation on adventitious buds, and roots. After acclimatization of plantlets, it can become a robust rose plant within 5 to 6 months. The best germination rate was 96.67% when the basic medium was MS supplemented with 2mg/L 6-BA+0.1mg/L NAA. The best medium for the shoot multiplication was MS + 2mg/L 6-BA + 0.05mg/L NAA, and the rate increased up to the fold of 3.36 in comparison with the control. The best rooting medium for "Carola" rose was 1/2MS+0.5mg/L NAA, and rooting rate can be up to 100%. The survival rate of sterile seedlings cultivated in the open can be up to 90% by domestication and transplanting.2. Optimization of the Transformation System for Rose Petals via Particle BombardmentAccording to the result of reporter gene detection, it is determined that only the appropriate technical parameters can be ensured to establish a stable and efficient gene gun transient transformation system to the receptor material of "snow" rose petals. In this test, we use the same amount of pCAMBIA 1305.1 plasmid 0.5μg per bombardment which contains gus gene to test the optimum distance of bombardment and acceleration pressures. It shows that the highest transient gus expression was obtained when scuttles were bombarded at 1150 psi, with 9 cm target distance.3. Specific Rose Chimeric Promoter Cloning and Functional AnalysisAccording to the rose chalcone synthase gene sequence of CHS, we take the use of Genome walking method to amplify 1.5kb upstream regulatory sequences. After the core promoter regulatory elements were analyzed, three fragments were obtained, 275 bp, 720 bp, and 1357 bp, respectively, as controls, where each of them contains the core region named RCHS of the promoter sequences. As the comparison, we take some heterologous enhancer fragments such as OCSenhancer and ?-element combined with the core region of rose flower specific promoter sequence RCHS to unidirectional designed chimeric promoters about 530 bp by artificial designation and synthesization.4. Construction of plant expression vector and transient transforming analysis.Plant expression vectors contain one unidirectional chimeric promoter, among which three control promoter fragments successively extended and GUS report genes are constructed. Transient Transforming the four plants expression vector constructed to the rose petals, staining the transgenic petals and detecting the GUS activity. It is indicated that the core fragment of 275 bp was able to drive the GUS gene expression in the petals specifically. The extended length before the 5 ’ terminal sequence had no significant effect on driving GUS gene expression activity, but the unidirectional chimeric promoters significantly improved the GUS gene expression levels.The above results verified the rose CHS promoter are concerned with flower organ tissue-specific expression promoter, and the unidirectional chimeric promoters artificially designed and synthesized can significantly enhanced the activity of the promoter, and significantly improved the expression level of exogenous genes in transgenic plants. In order to build a stronger expression construction, it can provide the necessary regulatory element to the floral tissue specific promoter when plant expression vector of the multi-gene co-expression constructed. |
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