Map-based Cloning And Functional Analysis Of A Narrow And Rolling Gene NRL3 In Rice (Oryza Sativa L.)

Rice is an important crop that provides nutrition and energy to the global population.In recent years,the global demand for rice has increased rapidly,but output has grown by less than 1%.Therefore,increasing rice yield is the ultimate goal of rice breeding.Rice leaves are the main photosynthetic organs.Leaf shape affects the efficiency of light capture and energy conversion.Appropriate leaf shape can increase plant photosynthesis efficiency and increase plant yield.However,the molecular regulation mechanisms that affecting rice leaf morphogenesis are unclear and need further study.In this study,the mutant nrl3 was identified from a library produced by using ethyl methanesulfonate?EMS?treatment with an indica rice variety Zhongjiazao 17?YK17?.The main results were as follows:?1?Compared to the wild type,the mutants exhibited a narrower leaf,a roller leaf,an increase in grain length,a decrease in grain width and panicle length,and a decrease in grain weight;Histological analysis showed that the mutant had large veins.The number of microvessels and the number of microvessels decreased,and there was a defect in the development of thick-walled cells on the leaf surface of the leaves.Scanning electron microscopy?SEM?further confirmed the defects in the development of the distal leaf surface of the mutant leaf;the chlorophyll content assay showed that the chlorophyll a and chlorophyll b content in the mutants is higher than that of the wild type.?2?The F₂ population was obtained by crossing the mutant nrl3 with the tropical japonica rice variety D50,and the F₂ population was separated and examined.The results showed that the ratio of normal leaf morphology and narrow leaf was about 3:1,which proved that the narrow leaf trait was controlled by single invisible gene.To fine-map NRL3,936 F₂ individuals with the nrl3 phenotype were chosen for genotyping,which further narrowed the mapping interval down to a 221-kb region between InDel marker Indel 3 and SSR marker RM2322.The analysis of the ORFs in the interval revealed the 8^thh ORF,LOC_Os03g19520,a single substitution of guanine?G?to adenine?A?was found on the 8^thh exon,which led to premature termination of protein coding.Further genetic experiments demonstrated that LOC_Os03g19520 was the target gene.?3?The results of qRT-PCR showed that NRL3 was constitutively expressed and its expression was highest in leaf sheath;subcellular localization experiments showed that NRL3 localized in cytoplasm,nucleus and membrane;The expression levels of genes associated with PCD,chlorophyll synthesis,chlorophyll degradation,and leaf development were examined in the wild-type,nrl3 and overexpression lines,respectively.The results showed that the expression levels of PCD and chlorophyll degradation-related genes were significantly down-regulated in the mutants,but the expression levels in the over-expressed lines either recovered or exceeded the levels of wild-type;the expression level of leaf development-related genes was found in both mutants.Some degree of change occurred;these results indicate that there was an abnormality in the degradation of PCD and chlorophyll in the mutant.?4?Using NRL3 as bait,we screened and sequenced many independent fragments interacting with NRL3,and the selected monoclonal was sequenced.The results showed that NAL9/VYL might interact with NRL3.Yeast duplex?Y2H?and bimolecular fluorescence complementation?BIFC?showed that NRL3 interacts with NAL9/VYL protein on the membrane;and NRL3 forms a complex with NAL9/VYL and its subunits Clp3,Clp4,Clp5and ClpT.Regulates leaf morphology and chlorophyll accumulation.