Cloning And Function Analysis Of A Full-length CDNA Encoding Brassiaca Napus L. Constitutively Photomotphogenic 1

Light plays an important role in plant growth and development. There is also a very elaborately light signal-regulated network perceiving the intensity, orientation, property and period length of light to mediate photomorphogenesis, flower induction, biothythm and so on in plants. Thorough researchs on model plant Arabidopsis thaliana show that AtCOP1, as a molecular switch, plays a key factor in light signal transduction network. However, the research involved photomorphogenesis in oil-bearing crop, such as rape, is lagging behind. As well as Arabidopsis thaliana, whether there is COP1 in rape, if there, what is the structure, functions and role of the mechanism is still unknown. So in this study, started with cloning COP1 gene from Brassica napus L., a series of researches on it were conducted. The results of detailed research are as follows:(1) By analysis of the cDNA sequence of COP1 from Arabidopsis thaliana, Pisum sativum, Lycopersicon esculentum and Oryza sativa, adopting techniques RT-PCR and modified genome walking and combining the known sequence in rape database, full-length cDNA in encoding region of BnCOP1 with 2034bp was first cloned from Brassica napus, which coding for 677 amino acids. Sequence homology analysis of amino acid deduced from the cDNA showed that BnCOP1 shared high homology (94%) with AtCOP1.(2) Amino acid sequence deduced from the cDNA of BnCOP1 was analyzed by using the bioinformatics methods. The results showed that BnCOP1 protein, with nuclear location, possesses the similar spacial structure with AtCOP1 both in two- and three-dimensional level. Importantly, it also contains three conserved domains: one RING, one coiled coil and seven WD40 domains.(3) Semi-quantitative RT-PCR and real-time quantitative PCR analysis demonstrated that though BnCOP1 gene expression was detected in all analyzed tissues, the relative expression in flower was significantly higher than that in root, leave, stem, silique, cotyledon and hyposotyl.(4) The recombinant prokaryotic expression vector pCold TF/BnCOP1 was successfully constructed using pCold TF vector and purified soluble recombinant protein was gotten. In addition, fusion protein of BnCOP1 was induced to express with IPTG at low temperature. The analysis of SDS-PAGE showed that the recombinant protein with a relative molecular mass 128 kD was highly expressed in E.coli and the fusion protein was present both in the supernatant and the pellet part of E.coli lysates. The supernatant was further purified by afinity chromatography and detected by western blotting, which demonstrated that high quality recombinant protein was obtained.(5) By constructing 35S::BnCOP1-GFP plasmid, stable transgenic Arabidopsis lines overexpressing BnCOP1 were generated through Agrobacterium-mediated transformation. Interestingly, the plants overexpressing BnCOP1 appeared slimmer phenotype in plant height, stem diameter length and leaves size than WT.(6) Hypocotyls length analysis of these transgenic seedlings showed that over expressed BnCOP1 enhanced photomorphogenesis only under red light, which demonstrated that BnCOP1 was sensitive to red light but not to blue or far-red light. All these results verified that there was some difference between BnCOP1 and AtCOP1 on the light-mediated repression of hypocotyls elongation.