The Magnetoreception Mechanism Of Cryptochrome 1 From Domestic Pigeons

Some animals in the nature have the ability to use the geomagnetic field for their orientation and navigation. Their navigation system is so precise and clever that could not be achieved by man-made systems. In the present research, we take pigeons as our research target because of their well known ability of geomagnetic navigation. We aim to study the biological mechanism under the magnetic sensing and signal transduction of the magnetic sensitive protein and many important results have been achieved.Firstly, the gene of pigeons cryptochrome was successfully cloned based on the high sequence homology of bird cryptochromes, which was named as cl CRY1(columba livia cryptochrome 1) for abbreviation and uploaded to the Genebank(KF280584). Then the cl CRY1 was successfully expressed and purified by an optimized baculovirus expression system. The relationship between the high conserved amino acid sequence and function of cl CRY1 was well discussed. The cofactor FAD bound in cryptochrome is crucial to the magnetic sensing and signal transduction. The redox state and active status was characterized. Additionally, some factors involving in the magnetic sensing process, such as the light irradiation, were induced, to test the the spectrum and photo-induced redox reactions of cl CRY1 in different conditions, and the redox status and active state of the FAD cofactor were verified.Rather to the common used transient absorption spectroscopy, we utilized the time-resolved fluorescence spectroscopy to characterize the Earth-strength magnetic field effects on photochemical reaction in cl CRY1. We observed that the fluorescence lifetimes of cl CRY1 were sensitive to the μT level magnetic field at room temperature. This magnetic sense of cl CRY1 is much more sensitive than that of plant cryptochrome. And this result strongly supports the proposal of cryptochrome magnetoreception. Furthermore, principle of magnetic sensitivity of the cl CRY1 transient fluorescence spectra was explained. Based on the discussion of the key factors in the chemical compass of cl CRY1, two possible photochemical mechanism of cl CRY1 magnetic sensing was proposed.The other important innovation of this research is that we integrated the study of magnetic signal transduction to the bird magnetoreception. In order to search for the downstream receptor of pigeon magnetic sensitive protein, a c DNA library was constructed and screened by yeast Two-Hybrid system. Eight proteins were found to interact with cl CRY1 protein, the function and property of whom was well discussed. The effects of light irradiation and magnetic field on the protein interaction were considered. According to the characteristic of bird magnetic sensing, the possibility of that these proteins were involved in the magnetic signal transduction was predicted, which is very important for the future study in this area.At last, as the cryptochrome is widely existed in various species, the Arabidopsis was used to investigate the biochemical function of cryptochromes in vivo, which could not be accomplished in pigeons. The function and activity of fused cryptochrome protein,the PHR domain and cryptochrome mutant was studied. Theses results are good references in the related researches on cryptochrome in animals.