The Functional Study Of Arabidopsis Cryptochrome2(CRY2) Regulating FT Transcription

Cryptochromes are photolyase-like flavinproteins that show no DNA repair activity in animals and plants.Generally,cryptochromes possess two domains,the N-terminal PHR(Photolyase-Homologous Region)domain in which FAD binds,and the C-terminal extension CCE(Cryptochrome Cterminal Extension)domain that is poorly conserved and intrinsically unstructured yet essential for functions.The Arabidopsis genome encodes three cryptochromes in total,cryptochrome 1(CRY1),CRY2 and CRYDASH.CRY1 mainly mediates the blue light-dependent de-etiolation responses and inhibition of hypocotyl elongation while CRY2 mainly regulates the photoperiodic control of floral initiation.However,the functions of CRY-DASH remain controversial.Photoexcited cryptochromes will undergo a series of biophysical and biochemical changes,including circular electron transfer,phosphorylation,conformational changes and ubiquitination,resulting in blue light signals transmitting to upstream and then gene expression altered.At present,the most widely studied function of CRY2 is its regulation of floral initiation.FT is one of the most important regulators that regulate floral initiation under photoperiod in flowering plants.FT gene encodes an RAF kinase inhibitor protein that can move from leaves to shoot apex through the vascular system to promote the transformation of inflorescence meristem.So,the activation of FT transcription is the key process of photoperiodic control of flowering time for the Arabidopsis grown in long-day conditions.It has been reported that CRY2 can inhibit the degradation of transcription regulator CO or interact with transcription factor CIB1 to promote the transcription of FT.But we can't conduct a step-by-step study of this process because of the complexity of signal network mediated by CRY2 and the transient property of CRY2's photobiochemical reactions.To start the research of this project,first,we used the HEK293 T expression system to express Arabidopsis CRY2 and proved that CRY2 purified from HEK293 T possesseed the same biological functions as Arabidopsis native CRY2.We also determined the interaction between CRY2 and its signal partners with the CRY2-mediated blue light pathway reconstituted.Second,we plan to elucidate the connection between CRY2 and FT,what's more,the regulation of blue light and BIC1 need to be discovered.At last,we are going to use Dual Luciferase reporter gene assay to reveal the functions of CRY2 on regulating the transcription of FT and the functions whether regulated by blue light and BIC1.According to the above studies,we conclude that: 1?The CRY2 purified from HEK293 T cells possesses the same biological functions as Arabidopsis native CRY2 and the CRY2-mediated blue light signal transduction pathways were reconstituted.2?CRY2 interacts with several FT gene fragments directly in a blue light-enchanced manner in virto,and BIC1 inhibits the interaction between CRY2 and FT fragments.3?CRY2 binds to FT G to activate transcription of FT and blue light will enhance the transcriptional activities of CRY2 while BIC1 will inhibit it.