Screening And Identification Of Protein Kinases Interacting With Blue Light Receptor CRY2in Arabidopsis

Plants need sunlight when they carry out photosynthesis and sense the externalenvironment signal. Photoreceptors mediate light regulation of gene expressioninvolved in multiple processes such as photomorphogenesis, photoperiodic floweringand biological rhythm. Blue light receptor cryptochrome2(CRY2) undergoes bluelight-dependent phosphorylation that is one of the most important biochemicalreactions, which is closely related to its function and blue light dependent degradation.In order to reveal the mechanism of the initial blue light signal transduction mediatedby cryptochromes, the effort to seek the kinases and the phosphorylation relatedproteins interacting with CRY2specifically under blue light is becoming more andmore important. So far, it has been found that CIB1and SPA1can interact withcryptochrome specifically under blue light, but blue light-dependent cryptochromeinteractive kinases or phosphorylation related proteins have not been reported. Themolecular mechanism of cryptochrome phosphorylation under blue light and themechanism how to start blue light signal remain unkown. A putative protein kinaseCIK1(Cryptochrome2Interacting Kinase1) interacting with CRY2was found throughyeast two hybrid screening in this study. The protein structure analysis revealed thatCIK1contains the conservative serine/threonine kinase site. The protein-proteininteraction was verified by further yeast two hybrid and BiFC experiments. Theco-localization experiments in protoplast transient expression system were conductedand the T2transgenic Arabidopsis lines overexpressing CIK1were obtained in thecurrent study. The detailed research results are as follows:(1) CIK1can interact with CRY2in a blue light dependent way. The X-galassays were utilized to rule out the false positives. The bioinformatic analysissuggested that CIK1is a member of the serine/threonine kinase family.(2) The liquid assays were performed to tested the interaction between the CIK1(full length), CIK1CT(CIK1C terminal), CIK1K(CIK1kinase domain), CIK1NT(CIK1N terminal) and CRY2respectively in yeast cell under blue light anddark. The results showed that CRY2interact with CIK1and CIK1CT dependent onblue light, not with CIK1NT and CIK1K in the yeast cell.(3) The carrier fusing YFP reporter gene was constructed and was transformedinto the Arabidopsis protoplast. The results showed that CRY2and CIK1areco-localized in the nucleus.(4) The results of BiFC assays suggested the in vivo interaction between CIK1and CRY2. The interaction lies in a blue light dependent way.(5) Infusion and Gateway technology were used to construct plant expressionvectors. The vectors were transformed into wild type Arabidopsis by flowers dippingmethod. Basta and hygromycin screening were performed in order to get the positivetransgenic seedlings, Western blot was used to identify transgenic lines with CIK1overexpressing. These CIK1transgenic lines obtained in this study laid a foundationfor further study such as protein biochemical analysis, CO-IP assays and genefunction researches.