Light Regulated Expression Of DBB Subfamily Genes And Functional Study On DBB1a In Arabidopsis

Light is an important environment element in plant growth and development, which plays an important role in regulating seed geimination, photomorphogenesis and flowering time. It's important to find regulator of light signaling for understanding and utilizing light signaling in plant. Zinc finger protein DBB (double B-box) gene subfamily is a series of transcriptive factor contained double B-box domain, and B-box domain is very important for light signaling. It's known that some members of DBB involve in photomorphegenesis, but the function of other members were unclear. In this paper, By the bioinformatics and real-time PCR, we studied on expression patterns of DBB gene subfamily regulated by light, the function of DBB1a gene and the relationship bwtween DBB1a and CRY-mediated signaling. The main results were as follows:(1) Through biosoft plantcare, we found that each member of DBB gene subfamily contained at least one G-box which is light cis-regulate element. The transcriptive expression of DBB subfamily in Arabidopsis under different photoperiods or light quality conditions were investigated using microarray and quantitative real-time PCR. The results showed that the transcription of six members of DBB subfamily were under the control of circadian clock:four of them were up-regulated, one was down-regulated and one wasn't regulated in responsed to light.(2) We found that the DBB1a protein located on the nuclear by using DBB1a-GFP transient expressed in the onion epidermal cells and stable expressed in the transgenic Arabidopsis plants. To detect the expression of GUS reporter gene drived by the promoter of DBB1a gene, we constructed pDBB1a-GUS transgenic plants. We found that the GUS was active in the germinating seed, cotyledon, vein, petiole and flower. The gene expression detected by RT-PCR showed similar results as in the GUS staining experiment.(3) Through the analysis of transcript levels of DBB1a of WT and phyAphyB under constitutive red light condition or WT and cry1cry2 under constitutive blue light condition by q-PCR. We had analysed the transcriptive levels of DBB1a in wild type Arabidopsis under various blue light fluence rate and various time under blue light, the result showed that the expression of DBB1a was up-regulated by fluence rate and time with blue light treatment. It indicated that the transcriptive levels of DBB1a was regulated by CRY under blue light. Besides, the analysis of the expression of DBB1a in cry1cry2 for constitutive 3 d, indicated that rhythem of DBB1a was mediated by CRY. (4) We found that DBB1a didn't interact with CRY2 in yeast, but interacted with COP1 by yeast 2 hybrid method. we hypothesesed that COP1 is partially responsible for the relationship of CRY and DBB1a.(5) By the analysis of phenotype of DBB1a over-expressed or under-expressed transgenic mutants in Arabidopsis, we found that DBB1a could regulate early photomorphegenisis. DBB1a promoted hypocotyls elongation under blue light, but didn't under red or far-red light. Besides, the content of chlorophyl was reduced in DBB1a over-expressed transgenic seedlings. Further through quantitative real-time PCR method detected that DBB1a could suppress the expression of CAB2 and CHS. We concluded that DBB1a functioned as a negative regulator of photomorphegenesis.(6) Phenotype analysis showed that the DBB1a under-expressed mutant dbb1a flowering early than the wild type under the SD, but not under the LD, and dbbla appeared abnormal flower development, for example, the petal number per flower often veried from two to thirteen. The DBB1a over-expressed mutant showed normal flowering time and floral development under both of the LD and the SD. Transcript profile analysis by q-PCR suggested that dbb1a mutant early flowering by enhancing expression of key flowering time gene FT and LFY, and folral abnormal was by reason that the expression of several floral homeotic genes in Arabidopsis were induced in dbb1a mutants, such as AP2, APS, PI, AG and miR172.(7) Responses of hypocotyl elongation of DBB1a various allele mutants to bioactive GA3 or GA biosynthesis inhibitors (pacolobutrazol) were tested, and the analysis of transcript level of GA metabolic and catabolic genes in DBB1a allele mutants under blue light, showed that DBB1a reduced the blue light-mediated inhibiton of hypocotyl elongation through causing an increase of bioactive GA levels in Arabidopsis.(8) Through screening the SCCD (suppressors of cry1cry2 dominant) mutant library in Arabidopsis, we found that SCC98-D mutant came forth earlier flowering, shorter hypocotyls and abnormal floral development. By cloned the flanking sequence of T-DNA inserted site by TAIL-PCR and RT-PCR, We demonstrated that the AT3G41768 which was inserted could mediate the expression of DBB1a. While the transcriptive level of DBB1a was upregulated in SCC98-D. This result further demonstrated that DBB1a gene involved in CRY-mediated signaling.