Exploring And Functional Analysis Of Important Genes In Rice Panicle Development

The number of spikelet per panicle is one of the most important traits in rice productivity determination.The development process of spikelet number per panicle is affected both by genetic and environmental factors,and its mechanism has always been a research hotspot.Scientists have studied and obtained a lot of important panicle development genes by mutant library screening,QTL and GWAS methods.These achievements have laid great foundation for understanding and improving rice panicle development and production.However,typical gene location method is limited by genetic materials,and part function genes is hard to be screened.With sequencing technology and gene editing technology greatly developed,limitation of genetic material is getting to be broken out.That will help scientists find and develop new research methods for discovering and studying rice panicle development gene.Our study works on building rice development transcriptome by next-generation sequencing and filtering candidate genes accounting for rice panicle development,which was highly expressed in rice young panicle.Then,mutant library was created by CRISPR/Cas9 gene editing technology.Important candidate genes involving panicle development were selected by observing and identifying mutant phenotype.Last,based on phenotype identifying,OsNAM and OsCUC3 was chosen to be studied.By phenotype screening,expression analyzing and transcriptome,our study deciphers and reveals biology function and molecular mechanism of OsNAM in regulating rice panicle development.Main conclusions are summarized as follows:1.Constructing a transcriptome database covering different organs across rice whole growth stage,summarizing gene expression pattern and screening candidate genes for panicle development.Gene expression database of different organs across rice whole life was constructed by next-generation sequencing.Four gene expression patterns across panicle development process were summarized by our database,including genes expression decrease and increase by panicle development,genes differently express in one or two stages,and genes express irregularly.Lots of important genes,such as Os PLT14,RFL,FZP,were clustered in one expression pattern,in which gene expression decrease by panicle development proceeding ahead.Screening standard was set based on first expression pattern.A list of genes was selected as candidate genes,which were considered to be related with panicle development.2.Creating mutant library and identifying 13 important panicle development gene.In our study,mutant library was created by CRISPR/Cas9 gene editing technology based on candidate genes.By observing and analyzing phenotype of mutant lines,panicle phenotype characteristics of mutant lines were preliminarily recorded and confirmed.Base on phenotype of mutant lines,mutant library could be divided into three groups.Mutant lines showing significant panicle structure change were classified into group one and two.Correspondingly,there were 13 genes in two groups,including OsNAM、OsCUC3、Os PIN1c、Os PIN1d、Os MP、Os SPL7、Os PLT7、Os PLT8、Os PLT9、Os HOX3、Os GRF7、Os GRF9 and Os GRF12.These genes were considered as important candidate genes regulating rice panicle development.3.OsNAM and OsCUC3 participate in rice plant and panicle architecture.OsNAM and OsCUC3 belongs to homology genes of CUCs genes in Arabidopsis.OsNAM encodes a typical NAC transcription activation factor,which is located in the nucleus.OsNAM functioned by interacting with itself to form homology dimer.Osnam-1 mutant line showed twisted leaf blade,fused leaf,smaller panicle,aberrant floret and sterity,while OsCUC3 mutant lines showed no obvious phenotype change.However,OsCUC3 can interact with OsNAM and osnamoscuc3 double mutant lines appeared severer phenotype change.Overall,OsNAM and OsCUC3 regulate rice shoot apex and leaf development during embryo and seedling stage coordinately and redundantly.5.Osmi R164-OsNAM module regulates meristem development precisely.OsNAM mRNA accumulated at young tissue,including coleoptile,young panicle,root and floral organ.OsNAM protein was distributed broadly at shoot apex and inflorescence meristem.Among them,distribution of OsNAM protein showed specific features that the reporter signal was highly accumulated in boundary zone,which is between shoot apex and initiated young leaf.And,its distribution was directly regulated by Osmi R164.RT-PCR result showed that expression level of OsNAM in r OsNAM(Osmi R164-resistant version OsNAM)related materials was significantly increased at shoot apex and young panicle.Meanwhile,r OsNAM related materials(r OsNAM-14,p OsNAM::r OsNAMGFP)appeared inhibited growth,including short leaf,dwarf architecture,small panicle,confused floral organ and so on.It was proven that precise regulation of Osmi R164-OsNAM module is necessary for rice growth and development,by controlling not only expression level but also distribution pattern of OsNAM.5.OsNAM participates in rice plant and panicle architecture.RNA-Seq analysis was applied in our study to provide insight into biology function and regulation mechanism of OsNAM in shoot apex and young panicle development process.Differently expressed genes between WT and osnam-1 in both shoot apex and young panicle included well known function genes,such as OSH1,Os PLT14,RFL,Os MADSs.Comparing shoot apex and young panicle of WT and osnam-1 by GO enrichment analysis,our study suggested that OsNAM plays important roles in shoot apex and young panicle development process,including pathway of cell proliferation and development,reproductive stage transition,cell wall building,hormone response and so on.Among them,stress response and chitin metabolic and response pathway was enriched significantly.It revealed that OsNAM was involved in regulating and coordinating growth and stress response in plant.In summary,our study analyzed dynamic transcriptome characteristics during panicle development and screened candidate genes which were highly expressed in panicle,by constructing transcriptome.Several important candidate genes were selected and validated by mutant library construction and phenotype observation.Among them,OsNAM and OsCUC3 is studied well and proven to playing an important role in rice panicle and architecture development.Additionally,our result confirmes the effectiveness of our screening method.The find of 13 important panicle genes complements the existing molecular regulation network of rice panicle development,and provides an important theoretical basis for breeding research.