Function Analysis Of Flower-Specific Promoter Of CHS Gene From Lily

Lily is widely favored by worldwide people for the character of the big flower, varied flower color, graceful posture and strong fragrance. As one of the rare ornamental, it is suitable for use in the garden, a shrub border. These years, many researches focus on the improvement of ornamental characters including color, shape, fragrance and so on, anti-decay and anti-deceases, and have made a great progress. However, how to transform the desired characters into lily by gene technology and get the steadily genetic and novel, varieties is the problem to face for breeding scientists. This study is to construct the plant expression vector, transform it into Arabidopsis, analysis the expression function of chs promoter on the basis of the former cloning research of the chs promoter sequence. The propose is to attain the flower-specific and high-efficient expression promoter from lily, provide an efficient regulation sequence for vector construct, culture more lily varieties by transforming other genes into lily. We study focused on the later points:1. Construction of the plant expression vector pCAM-CHSPCR amplification of the chs promoter was used chsA-F and chsA-R primers, and constructed into the cloning vector pGEM-T-easy. The recombined plasmid was named pCHS-T. The binary expression vector pCAMBIA 1381 and pCHS-T were digested by BamHⅠand EcoRⅠ, and the promoter fragment was inserted in linear pCAMBIA 1381 into pCAM-CHS. Then, pCAM-CHS was introduced into Agrobacterium EHA105 by freeze-thaw method. Analysis of PCR and enzymes digestion showed pCAM-CHS was constructed successfully into EHA105 .2. Transformation by Floral Dip method and PCR analysisThe transformed Arabidopsis plants by Floral Dip method were selected by 50 mg/L hygromycin stress on MS medium. 30 positive plants were selected and the transformation rate was about 2.5%～3.0%. 9 of all plants were analyzed by PCR assay using hyg primers, gus primers and chs primers, and the result showed both of chs gene and hyg gene were isolated from all 9 plants, gus gene was isolated from only 7 of them.3. Histochemical localizaion of GUS activity GUS activity of the different tissues were analyzed by X-gluc staining and the results indicated the chs promoter could drive the gus reporter gene to express in the flower of transgenic plants, while no or very weak expression of the gus reporter gene in the other tissues. The chs promoter is supposed to be related with flower-specific expression.