Regulation Of CCA1 Gene Expression By The Interaction Of Arabidopsis Blue Light Receptor CRY2 And LWDs

The circadian clock is a timing mechanism inside plant cells,which mainly includes input pathways,core oscillators,and signal output pathways.Under the stimulation of external environmental factors(mainly light and temperature),the internal timing mechanism of the system takes about 24 h as a oscillating dynamic rhythm.This system is of great significance in coordinating plant growth and development,metabolism and physiological responses,and endowed plants with adaptability to the living environment.The core oscillator of the plant circadian clock system is involved in regulating plant growth and development and adaptability to biotic and abiotic stress through multi-level regulation of complex signal feedback regulatory loop networks.CCA1 is the first circadian clock core oscillator gene to be identified.Further experimental results have demonstrated that Arabidopsis exhibits late flowering and inhibits the expression of endogenous CCA1 when CCA1 is overexpressed.Based on this,the first feedback-regulated signaling pathway mechanism of the biological clock in Arabidopsis thaliana was confirmed: CCA1 / LHY has an inhibitory effect on TOC1 expression in the early morning,while TOC1 has a promoting effect on CCA1 / LHY expression at night,which lays a foundation The basis for studying the transcription-translation feedback system of the plant circadian clock.However,the regulation of the plant biological clock by Cryptochrome2(Cryptochrome2,CRY2)is still unclear.LWDs can regulate the expression of CCA1 by binding to the promoter of CCA1 to promote the oscillation of the plant biological clock.Therefore,we further investigated whether the regulation of CCA1 by LWDs depends on CRY2.The specific research results are as follows:1.The preliminary analysis in vitro of yeast bis-histidine auxotrophic experiments proved the interaction between CRY2 and LWDs,and found that their interaction was induced by blue light;HEK293T(human embryonic kidney cells)expressed CRY2 and LWDs proteins for Co-The IP experiment further confirmed the interaction between CRY2 and LWDs.The interaction between CRY2 and LWDs in plants was proved by the plant bimolecular fluorescence complementation and Co-IP experiments in vivo.2.By comparing plant phenotypes of Col4,cry1cry2,LWD1 / col4,and LWD1 / cry1cry2,the statistical results of rosette leaf number and flowering time indicate that the regulation of LWDs on flowering time of Arabidopsis depends on CRYs.3.CCA1 :: LUC was used to detect biological phenotypes in wild-type and cry1cry2 mutants,and it was found that the amplitude of CCA1 became smaller in cry1cry2 and the cycle became longer.4.The pCAMBIA3301 CCA1 984 :: LUC was transferred into cry1cry2,Col4,LWDs-cry1cry2,and LWD1 by Agrobest,which proved that LWD1 regulates the expression of CCA1 in a CRYs-dependent manner.