Cloning And Functional Analysis Of Circadian Clock Gene OsLUX1 In Rice

Flowering represents the pivotal transition from vegetative to reproductive growth stage in plants,which is a critical process for plant successful reproduction.Rice production plays a critical role in ensuring food security in China.Proper timing of flowering is essential for determining regional adaptability and optimal yields in rice cultivation.Early flowering may result in a shortened carbon assimilation period,leading to decreased crop yield.On the other hand,delayed flowering can lead to decreased seed production towards the end of the growing season,resulting in yield losses.Meanwhile,photoperiod-sensitive materials may exhibit divergent flowering times across different latitudes,thereby impacting the success of cultivation.Therefore,investigating the genes that control flowering time and elucidating their biological functions can provide valuable insights into the molecular regulatory network of flowering,as well as furnish gene resources and research directions for molecular breeding.A significant late flowering mutant was identified from a mutant library of rice cultivar ZH11.Subsequently,the candidate gene OsLUX1 was identified as a component of the Evening Complex（EC）circadian clock that regulates photoperiodic flowering in rice.Hence,the mutant was designated as oslux1.The OsLUX1 gene was isolated through a map-based strategy,and its rhythmic expression patterns as well as involvement in the photoperiodic flowering pathway were investigated.The primary findings and conclusions of this investigation are presented below:1、Under natural long day（NLD）and natural short day（NSD）conditions,the oslux1 mutant showed a significant later flowering compared to the wildtype ZH11,with a delay of more than 45 days.Furthermore,the oslux1 mutant exhibited changes in other agronomic traits,such as an increase in height and tiller,changes in grain shape,and a significant decrease in thousand-grain weight.2、It was found that the late-heading phenotype of oslux1 is controlled by a single recessive gene.Using F₂population obtained from crossing oslux1 with indica cultivar Dular based on Bulk Segregation Analysis（BSA）,the OsLUX1 gene was initially mapped to the region between RM3362 and RM5310 on rice chromosome 1.Further Indel markers were designed and OsLUX1 was eventually mapped between S40 and S15 markers,with a physical distance of 43 kb.There were 9 genes in this region,four of which are transposons,and the sequencing of the remaining five genes is analyzed revealed that one of them had a one-base deletion in one of the genes,which caused a frameshift mutation and resulting in a premature stop in amino acid.A search of the database revealed that this mutant gene encodes a MYB transcription factor OsLUX1,which is a component of the Evening Complex（EC）in the circadian clock.3、To verify that a single base deletion in the OsLUX1 gene was responsible for the oslux1 mutant phenotype,the functional complementary vector p OsLUX1:OsLUX1 was constructed,which contained.The complete CDS of the OsLUX1 gene,2.0 kb upstream sequence,1.4 kb downstream sequence.The total of4.8 kb genome sequence was inserted into the p CAMBIA1300 vector.With the p CAMBIA1300 empty vector as the control,the oslux1 mutant was transformed by Agrobacterium tumefaciens mediated method,and the p OsLUX1:OsLUX1 transgenic positive plants were obtained.Investigations found that the flowering time of all transgenic-positive plants was recovered to that of the wild type.4、To determine the expression pattern of OsLUX1,real-time quantitative PCR（q RT-PCR）was used to detect the rhythm expression of OsLUX1 at 48 h in WT leaves under SD and LD conditions.It was found that OsLUX1 was a dark expression gene with the lowest expression level at light opening,and then the expression level increased gradually until 12 h after light opening,and then the expression level reached the peak.The expression pattern was not affected by day length.The expression of OsLUX1 gene in different tissues of rice was also analyzed by q RT-PCR.It was found that OsLUX1 was expressed in all tissues,with the highest expression level in leaves and relatively lowest expression level in roots.To determine the tissue expression pattern of OsLUX1 promoter,a p OsLUX1::GUS vector was constructed and transformed into wild-type ZH11.GUS staining showed that OsLUX1 gene was expressed in different tissues,and the highest expression level was found in leaves,which verified the results of q RT-PCR.To identify the subcellular localization of OsLUX1,the N-terminal of full-length ORF of OsLUX1 was fused with the GFP gene in p35s:GFP-C-BIN and obtained the 35s:OsLUX1-GFP fusion expression vector.35s:OsLUX1-GFP vector was transformed into wild type ZH11 using Agrobacterium tumefaciens mediated method,the yonng root of transgenic plants were selected and sliced,then observed by confocal.The results showed that OsLUX1-GFP fluorescence signals and DAPI nuclear localization signals were colocalized in the nucleus,indicating that OsLUX1 was localized in the nucleus.5、Bi FC experiment was used to verify the assembly mode of rice EC.The Os ELF3-1-n YFP,Os ELF4a-c YFP and OsLUX1-c YFP vectors were constructed and co-transformed into rice protoplasts,respectively.Os ELF3-1 was found to interact with Os ELF4a and OsLUX1,respectively,but Os ELF4a can’t interact with OsLUX1.The results suggested that the EC complex is relatively conserved between rice and Arabidopsis.6、To determine the regulatory pathway of OsLUX1 involved in photoperiodic flowering in rice,q RT-PCR was used to analyze the expression of key genes Hd3a,RFT,Hd1,Ehd1,Ghd7,Os GI and important circadian clock genes Os PRRs and OsLHY in both WT and oslux1.The expression levels of Hd3a,RFT,Ehd1 and OsLHY genes were significantly reduced in the oslux1 mutant under both LD and SD conditions,while Hd1 and Ghd7 genes showed a slight increase.In addition,the expression of other regulatory genes was not significantly different between oslux1and WT,suggesting that OsLUX1 may participate in the circadian clock cycle and probably promote flowering through the Ehd1-Hd3a/RFT1 pathway.To verify OsLUX1 regulate the Ehd1-Hd3a/RFT1 pathway,the Ch IP-q PCR was carried out using 35s:OsLUX1-GFP transformed oslux1 plants.The results showed that OsLUX1can direct binding to the promoter region of Hd3a and RFT1,while can’t binding the promoter region of Ehd1.These results suggest that OsLUX1 is involved in rice flowering by directly regulating Hd3a and RFT1.To further verify the role of OsLUX1 in the regulation of photoperiodic flowering,the RFT-OX and Hd3a-OX overexpression vectors were constructed and transformed them into oslux1.The heading time of RFT-OX and Hd3a-OX transgenic plants was significantly earlier than that of the empty vector control.Our results suggested that OsLUX1 is involved in the Hd3a/RFT1 flowering pathway.