Gene Cloning And Functional Analysis Of YL1 Conferring The Leaf Color Through Characterizing The Rice Yl1 Mutant

Rice is one of the most important food crops in the world,and most of rice yield are from photosynthesis of leaves.Photosynthesis takes place in the chloroplast which was developed from proplastid,so the normal development of chloroplast is important to photosynthesis in rice leaves.Generally,the impaired chloroplast causes abnormal leaf color phenotype and negative effects on the photosynthesis and rice yield.Therefore,the leaf color rice mutants are usually used to identify the genes associated with the development of chloroplast.It is significant in theoretical research and production application to increase rice yield and photosynthesis by genetic engineering.In the present study,a rice yellow leaf mutant yll isolated from Shuhui 498,was characterized in leaf color and the chloroplast structure,and the YL1 gene was cloned by the map-based cloning strategy.The main results are as follows:1.Leaves of the yll mutant always appeared abnormal yellow in the whole growth duration.The pigment contents of chlorophyll in yll mutant were also lower than those in the parental Shuhui498 plants at various growth stages.TEM(transmission electron microscopy)analysis showed that the granal stacks and granal membranes of the chloroplast in mesophyll cells of yll mutants were less than those in the wild type both at the seedling stage and at the tillering stage.2.The genetic analysis showed that the yellow leaf phenotype of the yll mutant is controlled by a single recessive gene.Through map-based cloning,the YL1 gene was finally narrowed down to an approximately 29-kb region on the long arm of chromosome 1.The genomic DNA sequences of the 6 ORFs between the yll mutants and wild type revealed that the only gene(LOC_Os01g73450)was different,and yll mutant carries a single-nucleotide transition(C?T)at the fifth exon,resulting in an amino acid change from Arg to Cys.Furthermore,LOC_Os01g73450 was confirmed to be the candidate of YL1 by transgenic complementation and gene knockout through CRSIPR-Cas9 methods.3.qRT-PCR analysis showed that YL1 expressed in leaves,leaf shoots,and panicles,with the highest level in leaves.The distinct changes observed during 48-h conditions revealed that the transcripts of YL1 were much higher under light conditions than under dark conditions and YL1 was induced by light.The expression of other genes associated with chlorophyll biosynthesis,chloroplast development,and photosynthesis were significantly decreased in yl1 mutant compared with wild type at the seedling stage.4.Subcellular localization analysis indicated that YL1 protein was localized in the chloroplast.And the molecular weight of the YL1 protein was about 37 KD when expressed in prokaryotic expression system.Bioinformatics analysis showed that the YL1 is an uridylate kinase in rice,which is conserved between plant and bacterial.Allelic variation analysis showed that DNA polymorphism was mainly in the noncoding region in 43 rice germplasm,suggesting that YL1 was relatively conservative.5.To observe if primary carbon metabolites were affected in plants,the levels of sucrose,soluble sugars,and starch in source leaves at the seeding stage were determined before a light cycle and after a light cycle.The contents of sucrose,soluble sugars and starch in mutants were significantly less than those in wild type.In addition,expression of most genes associated with carbon metabolism and transport was decreased in mutants when compared with wild type.