Identification And Functional Analysis Of Osarf19 Gene For Floral Organ Development And Dss Gene For Grain Size In Rice(Oryza Sativa L.)

This thesis includes two parts:the first is "identification and functional analysis of OsARF19 for floral development in rice",and the other part is "map-based cloning and functional analysis of DSS gene for grain size in rice".Part one:Rice,an important crop,provides a great deal of energy for people around the world.Abnormal floral organ development can damage seed quality and yield in rice.Many genes classified to groups based on ABCDE model were reported to regulate floral organ development.In addition,the phytohormone auxin can also affect floret formation,but the underlying mechanism is largely unknown.In this study we isolated a T-DNA insertion mutant that displayed abnormal florets as well as altered plant architecture.Identification and functional analysis of OsARF19 gave us a better understanding of auxin regulating floret development,and also provided a new material for plant architecture improvement.1.We identified a mutant with abnormal florets from a T-DNA insertion pool.PCR and qRT-PCR analysis confirmed that the gene OsARF19 was disrupted.Thus osarf19 was a knock-out mutant.2.The mutant osarfl9 displayed three types of floral abnormalities,including florets with an additional lemma,an enlarged palea and variably degenerated paleas.Each type of abnormal florets only possessed a relatively low proportion,and resulted in low floret infertility.In addition,osarf19 displayed altered plant architecture with increased plant height.The increased plant height was caused by elongated bottom internodes.3.We generated OsARF19 knock-down lines by RNAi,which showed three types of abnormal florets and increased plant height.These mutant phenotypes simulated the effects of reduced OsARF19 transcripts and confirmed that the various phenotypic alterations in osarfl9 mutant were caused by diminished OsARF19 expression.4.Cellular morphology analyses by light and scanning electron microscopy showed that cell length of osarfl9 was longer than that of WT.Meanwhile,cell wall synthesis genes were up regulated and cell cycle related genes were down-regulated.These results further confirmed that cell elongation rather than cell proliferation was the cause of the enlarged plant architecture.5.We studied the expression pattern of OsARF19 by qRT-PCR and GUS activity analysis.OsARF19 transcripts were present in various tissues,but preferentially in leaves,basal internodes,and young panicles or florets.The abundant transcripts of OsARF19 in specific tissues were consistent with influencing roles in stem,leaf and floral organ development.Subcellular localization analysis showed OsARF19 functioned in the nucleus.6.By qRT-PCR analysis,we found that disruption of OsARF19 increases expression levels of OsYUCCA,OsPIN,OsARF family members,while reducing OsGHs transcription activity.The high auxin performance greatly up regulated two floral organ regulators,OsMADS29 and OsMADS22,possibly responsible for palea abnormalities in osarf19.Part two:Grain size,a complex agronomic trait,plays an important role in determining yield and quality in rice.In this study,a mutant named decreased seed size(dss)was identified by screening a regenerated plant population derived from the 60Co y-ray-irradiated japonica cultivar Nanjing 35.The mutant displayed obviously small seed,dwarfism,as well as more tillers.Map-based cloning and functional analysis of DSS provided new evidence to elucidate molecular mechanism of grain size regulation,and should be useful for increasing grain yield in rice.Results are summarized as follows:1.The dss mutant has a small seed size,including decreased grain length,width,and weight.Besides,the mutant displayed dwarfism which was due to reduced internode length,as well as more tillers.We analyzed the cell length and number of parenchymal and epidermal cells from lemmas,and found decreased cell number was the cause of small seed size in dss.2.The dss locus was finally narrowed to a region of 140-kb on the long arm of chromosome 3 by map-based cloning.After sequencing,we found a nearly 18-kb genomic segment deletion which blocked transcriptions of 3 candidate genes.Transgenic analysis of three candidate genes by RNAi and over expression(OE)demonstrated that ORF13 was the gene DSS,involved in seed size regulation.3.DSS encodes a protein of 473 amino acids.The N terminus of DSS contains 7 transmembrane domains,and the C terminus contains a typical C3HC4 type RING domain,which was proved to have E3 ligase activity by in vitro ubiquitination assay.4.We studied the expression pattern of DSS by qRT-PCR and GUS activity analyses.OsARF19 transcripts were present in various tissues,but preferentially in young panicles.DSS protein functioned in endoplasmic reticulum(ER)and the N terminus 7 transmembranes conferred such ER-localization.5.QRT-PCR analysis of well-known seed size regulators demonstrated that both BG2 and GS5 were down regulated in DSS RNAi lines while up-regulated in DSS OE lines.These results indicated an uncertain relation between DSS and GS5 or BG2.