Study On The Molecular Mechanisms Of Rice CCT Family Genes OsPRR73 And OSCO3 In Regulating Heading Date

Heading date is an important agronomic trait that affects rice yield and determines the seasonal and regional adaptability of rice planting.Rice heading marks the achievement from vegetative to reproductive phase.This physiological process is mainly affected by external environmental factors,including light and temperature,as well as some inherent genetic characteristics of plants.As an important internal factor,the biological clock(circadian)is an endogenous regulatory system of plants,which responds to the external environment change and coordinates the internal signals of plants to make corresponding changes in physiological processes,so as to make them more adaptive.Photoperiod is the important external factor affecting rice heading.In this study,the genetic materials of OsPRR73 encoding a homolog to Arabidopsis circadian clock protein PSEUDORESPONSE REGULATOR 7(PRR7)and CONSTANS-like gene OsCO3 were constructed.The functions of OsPRR73 associated with clock and heading regulation were characterized with multiple techniques.And the molecular mechanisms of OsCO3 associated with rice heading and stress-resistance regulations were elucidated.The main results in this study are as follows.1.Os PRR73 regulates rice heading and circadian rhythm.(1)Os PRR73 overexpressed plants Os PRR73 OX delayed rice heading on both long-day(LD)and short-day(SD)conditions.Osprr73 mutant headed 3 days earlier compared with the wild-type under LD condition,while no significant difference was observed compared with the wild-type under SD condition.Thus,OsPRR73 participates in the regulation of rice heading by responding to the photoperiod.(2)The amplitudes or phases of free-running expressions of circadian clock associated genes OsPRR73,Os LHY,OsTOC1,OsGI and OsELF3 were altered in osprr73 and OsPRR73 OX compared with the wild-type under constant light and dark conditions.And osprr73 mutants had a slight shorten rhythm period compared with the wild-type.Therefor,OsPRR73 participates in the regulation of rice circadian clock.2.This study through a variety of molecular biology methods,such as the subcellular localization,binary Luciferase enzyme activity analysis,yeast one hybridization analysis,Chromatin Immunoprecipitation enrichment analysis and EMSA,verified the OsPRR73 as a transcription repressor repressed Ehd1,a key gene for heading promotion,resulting late flowering time,and the preliminary molecular mechanism was characterized that OsPRR73 formed a feedback loop with the rice core circadian clock protein Os LHY.3.osprr73/ehd1 and osprr73/oslhy doubke mutants and oslhy single mutants were constructed respectively in this study,and heading dates were recorded and related genetic relationships were analyzied: Ehd1 has an epistatic heading effect to OsPRR73 in osprr73/ehd1 mutant under SD condition,while osprr73/ehd1 showed a later heading date than ehd1 under LD condition which indicates that OsPRR73 has other pathways in regulation of rice heading besides of Ehd1 pathway;Os LHY regulates rice heading under NLD condition,oslhy and osprr73/oslhy mutants had a same delaying heading phenotype,Os LHY was epistasis to OsPRR73.The position of OsPRR73 in the molecular network of rice heading control was preliminarily clarified.4.Os CO3-overexpressing plants delayed rice heading under both LD and SD conditions;Ehd1 was significant repressed under both LD and SD conditions.Thus,OsCO3 participates in the molecular mechanism of rice heading by repressing Ehd1.5.In this study,RNA-Seq analysis was performed on Os PRR73 OX,Os CO3 OX and wildtype: Os LEA3,a gene induced by Abscisic Acid(ABA),and Os MADS1,a gene related to panicle development,were significantly down-regulated both in OsPRR73 OX and OsCO3 OX compared with the wild-type.And compared with wild-type,OsCO3OX and OsPRR73 OX inhibited rice seed germination,and OsCO3 overexpressed seedlings were sensitive to drought.In addition,OsPRR73 OX and OsCO3 OX had a larger panicle phenotype compared with the wild-type type;yeast one hybridization analysis and binary Luciferase enzyme activity analysis in protoplast showed that OsCO3 and OsPRR73 directly bound the OsMADS1 promoter.Therefore,OsPRR73 and OsCO3 have similar functions,both are suppressors of rice heading,and involved in the regulation of panicle structure and rice stress response.In this study,the molecular mechanism of Os PRR73 was analyzed for the first time,and the effect of rice clock gene on rice yield was clarified.At the same time,OsCO3 was found to be involved in the regulation of rice heading,panicle development and stress tolerance of rice.The further study of their functions and molecular mechanisms can provide theoretical basis for the rice molecular design breeding.