Researches About Interaction And Genetic Transformation On Light Regulator Of COP1, HY5

Anthocyanin biosynthesis in higher plants is regulated by light.Photoreceptors receive light and are involved in light signal transduction.In this study,Brassica rapa 'Tsuda',which has a characteristic of the light-dependent anthocyanin biosynthesis,was used as materials. Light signal transduction genes COP1 and HY5 were cloned,their expression and their interaction were studied.The main results were obtained as follows.1 The cDNA cloning and the expression character of putative COP1,HY5 in 'Tsuda' plants.COP1 and HY5 homologous to those cDNAs of Arabidopsis were isolated from 'Tsuda'.The amino acid sequences showed over 90%sequence identity to those of Arabidopsis. Ring and WD-40 domains in COP1,and bZIP domain in HY5 were separately found in putative COP1 and HY5 of 'Tsuda'.The expression of COP1 and HY5 was observed in seedlings of Tsuda" plants exposed to UV-A(352 nm),blue light,red light,far-red light,and UV-B,but no obvious differences were found among different lights.It suggested that COP1 and HY5 might participate in light signal transduction induced by above-mentioned kinds of light.In mature plants,the expression of COP1,HY5 was induced by UV-A.2 The interaction of COP1 and HY5To test the interaction by GAL4 yeast two-hybrid system,COP1 and HY5 genes were subcloned into an expression vector.The insertion of the genes were confirmed by sequencing. COP1 and HY5 expression vectors were screened in the selection medium and reporter gene analysis.The assay showed the interaction of COP1 and HY5.It was suggested that COP1 and HY5 isolated from Tsuda'had a similar role in Arabidopsis.3 Construction of vector HY5-dsRNAi and prelimimary study of its genetic transformationHY5-dsRNAi expression vector of Gateway cloning system was constructed with primers designed according to the sequence of HY5.To see the function of HY5 by RNAi,the factors influening the regeneration of turnip 'Tsuda' were studied.Cotyledonary petiole explants showed the best the regeneration frequencies of turnip "Tsuda' cultivars.To establish a high-frequency regeneration system,the hormone combinations of thioidiazuron(TDZ) and naphthaleneacetic acid(NAA) and shoot regeneration efficiency was compared for combinations of benzyladenine(BA) and NAA.The results showed that Murashige and Skoog(MS) medium containing 7.0 mg/L TDZ and 1.0 mg/L NAA was suitable recipe to achieve high-frequency shoot regeneration.The petiolate cotyledon cultured in MS medium containing TDZ 7.0 mg/L + NAA 1.0 mg/L + AgNO₃ 5.0 mg/L can be induced highest-frequency regeneration.The highest shoot formation rate was about 80%.