Functional Analysis Of The Circadian Clock Gene OsLHY In Oryza Sativa

The circadian clock,which is an endogenous,stable,complicated and subtle time control network system,extensively exists in various organisms and makes organisms adapt to local environments better.The circadian clock is more significant for plants which cannot gain advantages or avoid disadvantages through movement.Plants can precisely predict the changes of environment and resist stresses by regulating circadian clock.Therefore,it is possible to adapt to various climates by manipulating circadian clock genes of crops.By using molecular genetic approaches,many cultivated crops have been shown to adapt to new environment by regulating biological clock,which enhances their abilities of acclimatization.Mechanism of circadian clock in arabidopsis has been largely understood.At CCA1,the core oscillator gene of the circadian clock in arabidopsis,has been researched for over 20 years,but OsLHY,a homologous gene of Arabidopsis CCA1,is still largely unknown.My work mainly focous on the following points:(1)Research on OsLHY by the reverse genetic approach;(2)The functional analysis of two alternatively spliced transcripts of OsLHY through introducing them into Arabidopsis;(3)Transient transformation assay for OsLHY’s transcriptional activity;(4)Construction of the OsLHY overexpression line;(5)Construction of the OsLHY gene knock-out strain through CRISPR/Cas9 and detect the influence of OsLHY deficiency on other circadian clock genes;(6)Analysis of the promoter region of OsLHY and construction of the OsLHY overexpression lines with GFP tag.The results are as follows:(1)There are two alternatively spliced transcripts of OsLHY.(2)There was extremely tiny difference between the 2 types alternatively spliced transcripts,but phenotypic complementation assay showed that the 2 alternatively spliced transcripts affected different aspects of the circadian clock and maybe performed different regulatory functions.There has been no similar report on this phenomenon.(3)Result of transient transformation assay showed OsLHY represses the target genes consistent with At CCA1,which indicated function of CCA1/LHY is conserved in plants.(4)The expression of LHY is not elevated in LHY-overexpression lines,on the contrary,the expression of endogenous OsLHY is strongly repressed in transgenic plants.Phenotype analysis showed the LHY-OX plants displayed developmental retardation and the plant height is lower than wild type.The OsLHY overexpression lines in Arabidopsis display decreased fertility,even sterility.(5)OsLHY-KO Lines showed increased GI and TOC1 expression,shortened period,and shifted phase;the growth became obviously worse in growth chamber.(6)The transcription of OsLHY started at 5800 bp upstream of ATG,the whole 5’UTR exhibited similar expression pattern to OsLHY(7)OsLHY affected the expression of other biological clock genes and the growth of rice.lots of transgenic plants,such as estradiolinducible OsLHY lines,estradiol-inducible OsLHY RNAi-knockdown lines of rice and GR-inducible OsLHY plasmid,are constructed for further research,which laid foundation for understanding OsLHY regulation and interaction network of the circadian clock in rice.