Study On The Interaction Mechanism Between Plant UV-B Photoreceptor UVR8 And RUP2

Light not only provides energy for plants,but also acts as a signal to regulate plant photomorphogenesis.UV-B（Ultraviolet light-B）is a part of sunlight that affects almost all stages of plant life cycle（from seed germination to flowering and fruiting）.The UV-B photoreceptor UVR8（UV RESISTANCE LOCUS 8）exists as a inactive homodimer in the ground state that instantly monomerises upon UV-B absorption.Photoactivated UVR8 cooperates with several signaling factors to modulate plant responses to UV-B light and regulate plant growth and development.UVR8-mediated signaling pathway mainly includes the UVR8-COP1（CONSTITUTIVELY PHOTOMORPHOGENIC 1）core signaling pathway and other signaling pathways mediated by UVR8 with other transcription factors.In addition,UVR8 signaling is also negatively regulated by RUP1/2（REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1/2）.At present,the molecular basis of UVR8-COP1-mediated signaling pathway has been characterised,but the molecular mechanism underlying signaling pathway mediated by UVR8 and transcription factors,or negative feedback regulation of UVR8 signaling remains largely elusive.In this project,we verified the interaction of UVR8 with transcription factors and negative feedback regulators in vitro using methods of structural biology and molecular biology,screened the interaction proteins that form stable complexes with UVR8,and analyze the structure of the RUP2-UVR8^W285Acomplex.The main results of this study are shown as follows:1.We verified the interaction between the constitutionally activated mutants UVR8^W285A/UVR8^R338Aand the signal factors described abovein vitro.The results showed that UVR8^W285A/UVR8^R338Ainteracts with RUP1/RUP2.The yield of UVR8^W285A-RUP2 protein is higher than other combinations.2.We optimized the expression of UVR8^W285A-RUP2 complex by screening of different expression systems,fusion tags and protein engeneering.We finally obtained several full length or truncated forms of UVR8^W285A-RUP2 complex in Expi293F^TMcells.3.According to the protein optimization,we performed crystal screening and optimization of the full length and truncated UVR8^W285A-RUP2 and finally obtained good crystal and high-resolution diffraction data.These results will help us to understand the interaction mode of UVR8^W285A-RUP2 and provide useful information for elucidating the molecular mechanism underlying UVR8 redimerization facilitated by RUPs.This study also provides reference information for the researches on negative feedback regulation of other photoreceptors.