Cloning And Functional Analysis Of Tow Rice Panicle Development Gene LAX2-4 And PAA7

Rice（Oryza sativa L.）is one of the most important food crops in the world and feeds more than half of the world’s population.High and stable yield has been the primary pursued for food security.As one of the key components of rice yield,the grain number per panicle is composed of branch and spikelet number per panicle,the rate of spikelet degeneration and seed setting.In this study,two natural panicle mutants lax2-4 and paa7isolated from Egyptian variety“Giza159”were used for genetic analysis,gene mapping,cloning and functional studies.This study provides the genetic and theoretical basis for further research on the network of rice young panicle and material for rice high yield breeding.The main results of this study were summarized as following:1.lax2-4 mutant mainly showed lax panicle phenotype,decreased lateral spikelet number and grain number per panicle.Genetic analysis indicated that the lax panicle phenotype of lax2-4 mutant was controlled by a recessive nuclear gene.Further genetic mapping and cloning indicated that the fourth exon of LAX2 was lost caused by an approximately 2-Mb inversion in lax2-4,four type transcripts were formed due to the recombination of chromosome fragments.The level of wild type transcripts was significantly reduced in lax2-4 mutant.Overexpression assays showed that lax panicle was rescued in correspondence with the gradually increased expression level of the wild type LAX2-4 transcript.Thus,LAX2-4 could be a new allele of LAX2.CRISPR/Cas9 mediated knockout of the fourth exon verified that LAX2-4 participates the regulation of sterile lemma and lemma development in rice.LAX2-4 could interact with an E-class gene,Os MADS1,and the fourth exon was necessary for its function.2.Apical abortion appeared at“rapid elongation of rachis and branches”stage,when the floral organs differentiation had completed,but the degenerated spikelets development was stunted and gradually withered.Abortion spikelets were mainly appeared at the top of primary branches in the upper half of panicle.Cytological analysis and hydrogen peroxide content measurement revealed that the overaccumulation of hydrogen peroxide in the apical spikelets of paa7 may trigger programmed cell death.Histological analysis showed that the number of vascular bundles of the spikelet pedicles in paa7 mutant was decreased.The results of auxin content determination indicated that auxin content in the degraded spikelets was significantly lower than that in the wild type.Panicle apical abortion phenotype of paa7 mutant is controlled by a recessive nuclear gene.A 3 bp insertion and 4bp deletion were found in the 3’-UTR of FZP,and expression level of FZP was decreased in paa7.Complementary assay showed that panicle apical abortion was rescued by the wild type fragment,indicated that PAA7 could be a new allele of FZP.The auxin content in apical spikelets of panicle before degeneration was significantly lower than that in the WT.Exogenous IAA treatment experiments at young panicle development stage showed that10^-5 mol/L IAA solution could rescue panicle apical abortion phenotype best,following by10^-6 mol/L.These results suggested that auxin deficiency caused panicle apical abortion in paa7.FZP could combined with the promoter of Os ARF23,Os PID and Os YUCCA6,which contained CAAT-box and MYB binding elements,then regulating the auxin content in panicle.3.Besides lax panicle and apical abortion,frizzy panicle phenotype same with the severe mutant of FZP was also observed in F₂ of lax2-4/paa7,indicating that LAX2-4（LAX2）and PAA7（FZP）could interact with each other genetically.The cr-L2-paa7 transgenic plants also showed frizzy panicle phenotype.Interaction between LAX2-4（LAX2）and PAA7（FZP）was confirmed by Y2H and BiFC assays.These results indicated that LAX2and FZP could interact with each other and regulate panicle development together.In this study,we confirmed the function of LAX2-4（LAX2）and PAA7（FZP）and the interaction between them,enriched the network of young panicle development in rice,provided a reference for breeding new rice varieties with large panicles.