Molecular Cloning And Functional Characterization Of Apple Cryptochrom Genes MdCRYl And MdCRY2

The cryptochromes belong to a class of photoreceptors. They specially receive blue lightand regulate growth and development in higher plants. Previous studies indicate thatcryptochromes exist in plants and animals and function as photoreceptors. Their functionshave been intensively studied in model plants. Light is one of the most important environmentfactors for fruit tree growth and development. However, it is largely unknown how fruit treesrepond to light signals. In this study, two cryptochrome genes MdCRY1and MdCRY2wereisolated from apple. Subsenquently, their gene structural features, expression profiles andfuctions were analized characterized. The main results were shown as follows.1. The full length of cDNAs of MdCRY1and MdCRY2were obtained by using RT-PCR andRACE amplification. Two predicted MdCRY1and MdCRY2proteins contain N-terminalphotolyase-related domain, the FAD binding domain and C-terminal DQXVP-acidic-STAES(DAS) domain, which are highly conserved among CRY1and CRY1proteins in other plantspecies.(1) The full length cDNA of MdCRY1is2310bp in length. Its ORF is2040bp and encods apolypeptide containing680amino acid residues. The predicted protein is5.3in PI and76.6kDa in molecular weight.(2) The full length cDNA of MdCRY2is2102bp in length. Its ORF is1944bp and encods apolypeptide containing648amino acid residues. The predicted protein is5.7in PI and73.5kDa in molecular weight.2. RT-PCR analysis showed that MdCRY1and MdCRY2constitutively expressed in all tissuestested. In addition, the expression patterns of MdCRY1and MdCRY2in response to light werealso tested using bagged apple fruits and apple callus. The result indicated that the expressionof two genes was noticeably induced in reponse to light. These results suggested thatMdCRY1and MdCRY2are involved in light responses in apple.3. Functional complementation test showed that the ectopic expression of MdCRY1andMdCRY2recovered the developmental defects in photomorphogenesis in Arabidopsis mutantcry1and cry2seedlings to a certain degree, respectively. Meanwhile, overexpression of twogenes in WT enhanced photomorphogenesis phenotypes, respectively. These results indicatedthat MdCRY1and MdCRY2regulate plant photomorphogenesis in response light signal. Inaddition, ectopic expressions of MdCRY1and MdCRY2also promote anthocyaninsaccumulation in transgenic Arabidopsis, suggesting their involvement in the regulation of fruitcoloration in apple. 4. Yeast-two-hybrid and BiFC (bimolecular fluorescent complementation) assays indicatedthat both MdCRY1and MdCRY2interact with Arabidopsis AtCOP1protein, furtherconfirming that both proteins are components in light signaling pathway in apple.