Screening And Preliminary Functional Analysis Of Transcription Factors Interacting With CRY1in Arabidopsis

Transcription factors are proteins that can inhibit or enhance the expression oftarget genes. They can turn to be the dynamic transcription complex which has theactivity of RNA polymerase, by interacting with gene’s promoter directly or indirectly.The specific interaction between CRY2and CIB1, a transcription factor, underblue-ray has been identified recently. By promoting the expression of florescence geneFT，CIB1involves in the regulation to plant flowering in the condition of blue-ray.Because of the high homology and partial overlaps of CRY1and CRY2，we speculatedthat, there may well be some transcription factors interact with CRY1in the blue-rayphotomorphogenic mechanism mediated by CRY1. This can accommodate thephysiological responses of photomorphogenesis, as the aspects of de-etiolation of seedgermination, hypocotyl elongation and the expansion of cotyledon in Arabidopsis. Sothe following researches were conducted, and some certain innovative results wereobtained:Yeast two-hybrid system was performed to screen CRY1interaction proteins inArabidopsis transcription factors library, and six proteins were obtained.The interaction intensity between CRY1and HB22，which is one of the sixproteins, were measured by using ONPG and CPRG assay with P-galactosidase. Theresult showed that the ratio of P-galactosidase activity in blue light to in the dark is1.668or2.182respectively when irradiated with blue light (50jamol/ms) for4h. Theinteraction between CRY1and HB22in blue light response was identified.Theirinteraction irradiated with blue light for different time or with different fluence rateswere further analyzed by CPRG. It was found that that their interaction was strongestwhen incubated under50jamol/2ms blue light for3h. It was appraised that theinteraction intensity between them was related to blue light intensity.The mRNA level of HB22was then detected by Real-time fluorescent quantitativePCR. It expressed higher in cry1mutant than in col-4，and12times higher than in col-4when treated with blue light for2hour. The resjaLts demonstrated that CRY1maymediate blue light inhibition of HB22gene expression.HB22was expressed in root, stem, rosette, cauline leaf, flower and fruit pods, butmuch higher in flower and root, which indicated that HB22might mainly play roles inroot and flower. Three HB22over-expressed transgenic lines HB22oxl, HB22ox2and HB22ox3were obtained by the methods of enzyme-cut and link up and agrobacterium mediatedimmersing inflorescence.The recombinant prokaryotic expression vector pCold TF/HB22was successfjaLlyconstructed and a lot of soluble recombinant protein was gotten. The analysis ofSDS-PAGE revealed that the recombinant protein with a relative molecjaLar mass74kDa. The inducer IPTG concentration has been optimized; the results showed thatIPTG quantity change of the target protein expression had no obvious effect. A largenumber of soluble HB22proteins provide the basis for further research on activitystrength and antibody preparation of HB22protein.The interaction between HB22and CRY1was demonstrated by all the resultsabove. For the interaction mechanism between them, more experiment in vitro asGST-pull down and in vivo as chromogenic reactions using fluorescent protein wereacquired. For the biological function of HB22in Arabidopsis, some further research onHB22over-expressed transgenic plants and HB22T-DNA insertion mutant plants wererequired.