| Petunia hybrida Vilm is favored by people all over the world for the big flower, varied flower color. So it was widely applied in the flower-bed arrangement, the scenic spot an ornaments, the window embellishes, and the family decoration.In China, the demand of Petunia hybrida Vilm is increasing with people's living standards improved. By seed propagation, germination rate is very low, and difficult to maintain the excellent characteristics of the parents for a hybrid species. Like other fresh cut flowers, Petunia hybrida Vilm will decay rapidly while blooming. How to prolong the florescence of flower has become one of the main subjects for studying recently. In this study ACC oxidase gene cDNA fragment was cloned from the petals senescence of Petunia hybrida Vilm, intend to construct ACC oxidase gene RNAi expression vector of Petunia hybrida Vilm, and transferred to the petunia callus, through the tissue culture regeneration system, obtain a complete regeneration.This research has taken the cotyledons of Petunia hybrida Vilm as the material. As a result, the optimum mediums of Petunia hybrida Vilm of tissue culture of every step are screened, the plant regeneration system of Petunia hybrida Vilm was established in this research; This research chose the petal senescence of Petunia hybrida Vilm to be the materials, and the total RNA of the flowers was extracted, the cDNA fragments of ACC oxidase genes of Petunia hybrida Vilm were obtained by RT-PCR, so that a solid foundation for the research of gene transferring can be made. The main results are as follows:1.Establishment of regeneration system of Petunia hybrida Vilm of tissue culture(1) The seed of Petunia hybrida Vilm sterilization of this study was based on secondary disinfection chemical reagent,75% ethanol and 0.1% mercuric chloride used in combination, these two reagents optimum disinfection sterilization time was 30s and 8min;(2) The optimum medium of callus occurrence and proliferation of Petunia hybrida Vilm was MS+1.0 mg/L 6-BA+0.5 mg/L NAA;(3) The optimal medium of the callus of Petunia hybrida Vilm induction of adventitious buds occurred was MS+0.5 mg/L 6-BA+0.2 mg/L NAA;(4) The optimum medium of adventitious bud proliferation and rooting was 1/2MS;(5) The optimum medium for transplanting is peat with perlite(1:1), the survival ratio of plantlets could be up to 66.7%(6) The optimal concentration for Hyg resistance screening was 10 mg/L.2. Improved Trizol Reagent method has been built to suit the extraction of the total RNA of the petal senescence of Petunia hybrida Vilm.In the traditional Trizol reagent law foundation, carried on the improvement to it. The results showed that OD260/OD280 value of the extracted RNA was 1.75, OD260/OD230 value of the extracted RNA was1.91, the bands of 28S and 18SrRNA were clearly discernible, brightness ratio was 1.0-2.0.3. Obtained the cDNA fragments of ACC oxidase genes in Petunia hybrida VilmTaken the petal senescence of Petunia hybrida Vilm as the material, the total RNA was extracted. Then the first-strand cDNA was synthesized by RT. The double degenerate primers were designed by the homologous sequence of the ACC oxidase genes. Through PCR, the cDNA fragments of ACC oxidase genes of Petunia hybrida Vilm had been obtained. Blast at the NCBI web site, as a result, the fragment is highly conserved, and the ACC oxidase gene family homology of Petunia hybrida Vilm of up to 100%, the lowest cost for more than 80%. The fragment of the complementary sequence encoding 173 amino acid, in the NCBI website to blast, finally found that the amino acid sequence and display the 100 ACC oxidase amino acid sequences,98 have the 100% homology, one has 99% homology, and one has 91% homology. |
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