Functional Analysis Of OsTGAL4 And OsTGAL11 Genes In Regulating Flower Organ Development In Rice

The size and number of floral organs restrict crop yield.However,the key genes and regulatory mechanism of floral organ development in rice are still unclear.Therefore,we need to identify more functional genes of floral organ development to improve the molecular regulatory network of flower development in rice.In this paper,two novel transcription factors,OsTGAL4 and OsTGAL11,which regulate the morphology and number of floral organs in rice,were identified by reverse genetics.The single and double mutants of OsTGAL4 and OsTGAL11 genes were constructed using CRISPR/Cas9 gene editing technology,and the overexpression transgenic lines and promoter driving GUS reporter lines were constructed using transgenic technology.The functions of OsTGAL4 and OsTGAL11 in regulating rice flower development were studied in detail,and the main results are as follows:1 The tissue expression of OsTGAL4 and OsTGAL11 and the subcellular localization of the proteins they encode were clarified.OsTGAL4 and OsTGAL11 were homologous genes encoding OsTGAL proteins.RT-q PCR analysis and GUS staining of transgenic plants with promoter driving GUS reporter gene indicated that OsTGAL4 and OsTGAL11 had similar tissue expression patterns,with higher expression in young roots and panicles.OsTGAL4 was highly expressed in paleas,lemmas,anthers,filaments,stigmas,lodicules and basal parts of sterile lemmas,while OsTGAL11 was highly expressed in anthers and basal parts of sterile lemmas.Subcellular localization assay showed that OsTGAL4 and OsTGAL11 were localized in the nucleus.Yeast autoactivation assay showed that OsTGAL4 and OsTGAL11 had transcription activation activities.2 We revealed that OsTGAL4 and OsTGAL11 regulate the characteristic development of rice flower organs and have functional redundancy.Phenotypic observation and anatomical analysis showed that the flower organs in ostgal4 ostgal11 double mutants were abnormal: the body of the paleas(bop)were severely degenerated and were replaced by a pair of glume-like marginal regions of the palea(mrp)structures;sterile lemmas elongated and developed lemma-like structures containing 4 or 5vascular bundles;ostgal4 ostgal11 double mutants producd less stamens and producd multiple florets.These results suggested that OsTGAL4 and OsTGAL11 played an important role in the regulation of spikelet meristem and inflorescence meristem determinacy and organ identity.However,the single mutant ostgal4 and ostgal11 did not show obvious abnormal phenotype,indicating that OsTGAL4 and OsTGAL11 functioned redundantly in regulating floral organ development.3 OsTGAL4 and OsTGAL11 regulate the expression of characteristic genes in flower organs.We performed transcriptome sequencing of wild type and ostgal4ostgal11 #4 double mutant,and the results showed that the expression of plant stress responsive genes was up regulated in ostgal4 ostgal11 #4,including diterpenoid biosynthesis genes,cytochrome P450 genes.Results showed that the mutation of OsTGAL4 and OsTGAL11 resulted in disordered expression levels of OsMADSs,the characteristic genes of floral organs.The expression of palea regulatory genes OsMADS15 and DP1 were significantly up regulated.It is speculated that OsTGAL4 and OsTGAL11 may regulate the expression of OsMADS15 and DP1 in body of the palea development,and regulate the expression of lemma and palea specific gene OsMADS1 in lateral floret lemma degradation.In summary,this study expounded the tissue expression pattern and the subcellular localization of OsTGAL4 and OsTGAL11,and elucidated the biological functions of OsTGAL4 and OsTGAL11 in regulating the development of rice flower organ characteristics and the influence on downstream gene expression.The results are helpful to elucidate the molecular regulation mechanism of rice flower development,which has important theoretical and application significance.