Studies On The Fertility Alternation Mechanism Of TGMS Lines And The Function Of OsLHY In Regulating Photoperiodic Flowering In Rice

Rice(Oryza sativa)is not only one of the most important food crops,but also the model plant of monocots for functional genomics.The photoperiod-or thermos-sensitive genic male sterility(P/TGMS)lines are crucial for two-line hybrid rice breeding,which made a great contribution to global food security.So it is of great theoretical and practical significance to elucidate the molecular mechanism of fertility alteration in rice TGMS lines.Till now,a number of sterile genes that control P/TGMS have been mapped,and a few of them were cloned in rice,while the molecular mechanism of the fertility alteration is still rarely studied.Zheda13S is a new rice TGMS material generated by our laboratory.In this study,we identified many candidate genes related to fertility alteration by transcriptome analysis in young panicle of Zheda13S under restrictive and permissive temperatures.Among them,a circadian clock gene OsLHY is lowly expressed under restrictive temperature compared with permissive temperature in both Microspore Mother Cell stage(MMC)and Meiosis stage(MEI).Then,the function of OsLHY was analyzed systematically through combined methods of CRISPR/Cas9 editing technology,transgenic technology and molecular biology.And the main results are as follows:1.RNA-Seq was performed to compare the expression profiles of young panicle from Zheda 13S at MMC and MEI under restrictive and permissive temperatures.A total of 1070 differentially expressed genes(DEGs)were identified in the two stages,of which 398 genes were up-regulated and 672 genes were down-regulated.The GO and KEGG enrichment analyses revealed that DEGs were enriched in protein folding,protein binding,regulation of transcription,transcription factor activity and metabolic related processes.Furthermore,protein-protein interaction network prediction and topological analyses indicated that Ub _L40s,DNA-directed RNA polymerase subunit,and HSPs were hub genes,and Ub _L40s were predicted to interact with proteolysis related genes and DNA-directed RNA polymerase subunit,and HSPs interacted with kinases to play important roles in regulating fertility alteration.It suggested that besides Ub _L40s,DNA-directed RNA polymerase subunit,kinases and HSPs might also involve in fertility alteration in rice TGMS lines.2.Compared to permissive temperature,the expression of circadian clock gene OsLHY was decreased in MMC and MEI of Zheda13S young panicle under restrictive temperature temperature.A total of 9 T₀ generation oslhy mutants were generated by using CRISPR/Cas9 editing technology.oslhy mutants caused delayed heading date in rice under long-day(LD)conditions,but had no direct effect on fertility.Analysis of OsLHY expression patterns in different tissues showed that it exhibited the highest expression level in leaf blades.The results of subcellular localization showed that OsLHY is a nuclear localized protein.Diurnal rhythm of circadian clock genes analysis showed that their expression profiles were all altered in the oslhy mutant.Among them,the OsELF3 was down-regulated which act as a flowering activator under LD conditions.Further investigations showed that the expression of the LD-flowering repressor genes Hd1 and Ghd7 was up-regulated in the oslhy mutant,while the expression of the LD-flowering activator genes Ehd1,Hd3a and RFT1 was also down-regulated.Dual-luciferase assays showed that OsLHY could repress the transcription of OsGI,Hd1,Ghd7,Hd3a,RFT1 and OsELF3,and activate the transcription of Ehd1.Moreover,the yeast one hybrid assay confirmed that OsLHY could directly repress OsGI,RFT1 and OsELF3 by binding to their promoters,which is consistent with that in Arabidopsis.These results suggest that OsLHY promotes rice flowering mainly through regulating Hd1-and Ehd1-mediated photoperiodic pathways under LD conditions.In conclusion,in this study,many DEGs were obtained by comparing the expression profiles of young panicle from Zheda 13S under restrictive and permissive temperatures,which may be involved in the fertility alteration process of TGMS lines.In addition,the function of the DEG OsLHY was systematically analyzed,and it was found that OsLHY acts as a rice flowering activator under LD conditions.Then we further explored how OsLHY regulated the heading date in rice.These findings provided a new perspective and foundation to elucidate molecular mechanism of fertility alteration in rice TGMS lines.Meanwhile,it enriches the understanding of the relationship of rice circadian clock and heading date regulatory network,which provides a new material and theoretical basis for artificial regulation of rice heading date,and provides an important reference for further study of the function of circadian clock gene LHY.