Molecular Cloning And Characterization Of The RmCs And RmMDH Genes In Rhododendron Micranthum Turcz

Rhododendron, one of ten traditional famous flowers in China, is widely cultivatedthroughout the world for its high ornamental value. It is fairly sensitive to the change in pH,especially in the alkaline condition, and most of them can not normal growth and developmentunder alkaline environment in the Northern China. Therefore, how to cultivate Rhododendronin alkaline Northern China is the focal point of the research. At present, some acid fertilizerswere added into the soil to enhance its adaptability. The results indicated that this was not aneffective ways to solve the problem. Although the stress resistance of some species wasenhanced with transgenic technology, there is no report on Rhododendron which is aneconomically important ornamental.In this study, two full gene-length cDNAs of RmCS and RmMDH from leaves were clonedusing RT-PCR and RACE. Furthermore, the expression patterns of RmCS and RmMDH genesin different tissues and the same tissue at different developmental stages were assessed byrelative real-time PCR. As the accuracy of qRT-PCR mainly relies on the selection of properreference genes, we performed the gene stability analysis of6candidate reference genes in Rh.micranthum Turcz among different samples including different developmental stages, varioustissues. Besides, an efficient regeneration and transformation system of Rh. micranthum Turczwas established and optimized to transform Rh. micranthum Turcz by Agrobacterium-mediatedmethod. In order to test the function of these two genes, the sense and antisense plantexpression vectors were constructed and transformed into tobacco and Rhododendron via theAgrobactria-mediated leaf-disc transformation method. Parts of transformed plants wereobtained after genomic PCR detection. The homozygote T2generation of transgenic tobaccowas obtained. Some physiological parameters of the transgenic tobacco were tested understress conditions. The main results are as follows:1. In this study, we isolated two genes, RmCS (GenBank number: JQ412747) and RmMDH (GenBank number: JQ412742) from leaves of Rh. micranthum Turcz by RT-PCR.Homologous comparison analysis revealed that RmCS showed high homologous with the Vitisvinifera and the Populus trichocarpa, the RmMDH shared high homologous with the Camelliasinensis and the Populus trichocarpa.2. Selection of reliable reference genes is a prerequisite for accurate normalization of geneexpression in quantitative real-time polymerase chain reaction (qRT-PCR). The expressionstabilities of6candidate reference genes in different tissues and the same tissue at differentdevelopmental stages were assessed in Rh. micranthum Turcz. The results indicated that EF1αand UBQ were the most stable reference genes for different tissues (leaf, root, stem and flower)at the same developmental stage, whereas18S should be avoided. During leaf development,EF1α and18S can be used as the most reliable reference genes, but CYP was the most variableone. During flower development, the most stable genes were CYP and EF1α, while18S wasthe least stable one.3. The results revealed that RhMDH was expressed in all the tissues, but the flowerpresented the most abundant transcript among all the tissues with the peak at the fully openedand post-anthesis flower stages. The similar expression level was found in root, stem and leaf.It showed highest expression in October during leaf development. RmCS exhibited the highestexpression in stem, the lowest expression levels in root. The highest expression level at thepre-anthesis flowers stages was exhibited during flower development. During leaf development,RmCS exhibited the highest expression in August and September, subsequent to April andOctober, and the least abundant transcript was May, June and July.4. The stems of plantlet were used as explants to study the effects of different hormonecombinations on explant induction, propagation of clustering shoots and the rooting. Theresults indicated that the most suitable medium for shoots induction was Read+ZT4.0mg/L+NAA0.05mg/L+sugar (3%), the inductivity rate reached92.41%; the most suitable mediumfor multiplication was Read+ZT3.0mg/L+NAA0.1mg/L+sugar(3%), and the multiplicationcoefficient was7.56; Read+IAA0.5mg/L+sugar (2%) was the best rooting medium, and therooting rate was up to92.5%. The results showed that400mg/L Carb was suiltable de-bacterium concentration for Rh. micranthum Turcz callus. Km70mg/L could restrain stemsfrom differentiating adventitious buds. The suitable selction concentration for adventitiousbuds rooting was10mg/L Km.5. The sense and antisense plant expression vectors of these genes (RmMDH and RmCS)were constructed, the two sense expression vectors were transformed into tobacco via theAgrobactria-mediated leaf-disc transformation method. The sense and antisense plantexpression vectors were transformed into Rhododendron via Agrobacterium-mediated. ThePCR and Southern blot results showed that these genes were integrated into the genome oftobacco. Compared to the wild-type tobacco, transgenic tobacco with over-expression ofRmMDH and RmCS presented smaller leaves. Under NaCl, PEG and AlCl3stresses, transgenictobacco with over-expression of RmMDH and RmCS showed lower MDA contents and higherproline contents than the wild type. These results indicated over expression of RmMDH andRmCS in tobacco could enhance the ability to endure stresses.