Map-based Cloning And Function Analysis Of Leaf-Architecture Related Genes In Rice

Further improvement in rice plant architecture is one of the most effective ways to enhance field photosynthetic efficiency and crop yield.Leaf rolling is beneficial to shape plant architecture.Though several genes involved in leaf rolling have been cloned,the related molecular genetic mechanisms is still unclear.The plant materials of this research are the wild type rice Nipponbare?Oryza sativa L.?and Indica 9.To investigate the molecular mechanisms of rice leaf blade type,two new rice mutant populations produced by mutagenesis were screened.The method of map-based cloning and sequencing was used,and gene related to leaf blade phenotype in the mutant was identified and analyzed.1.Cloning and expression analysis of HD-ZIP IV family gene URL1Mutant plants had adaxially-rolled leaves,and leaf rolling index values?LRIs?in the url1 mutant were 0.59 to 0.67.Both the number and size of bulliform cellfrom mature leaves of url1 mutants was reduced.Results of genetic analysis showed that the abnormal character of url1 was a qualitative trait controlled by a semi-dominant gene.A large F₂ mapping population was then generated,allowing the fine-mapping of URL1 to a 53 kb region,and further amplification of the relevant DNA fragments and sequence comparison revealed that the 679 th base after termination codon of URL1 in the mutant carried a single base substitution?C to T?.qRT-PCR analysis indicated that the expression level of URL1 in mutant and F₁ was higher than the wild type.The fluorescence signals of the fusion protein revealed that URL1-GFP localized predominantly in nuclei.2.Function analysis of URL1 geneThe full-length URL1 genome DNA driven by the 35S promoter?pURL1::gURL1?was introduced into wildtype Nipponbare rice.Phenotypic survey presented that mature leaves of transgenic lines were adaxially rolled.The construct of RNAi was introduced into url1 and silencing of URL1 in the url1 mutant resulted in flat leaves which looks like wildtype.A typical EAR motif?LDLDL?was located at the C-terminal of URL1 protein,URL1functions as a transcriptional repressor and the EAR motif in URL1 was probably responsible for the repression activity of URL1.The research also suggested that URL1 3'UTR played certain important roles in regulating URL1 gene expression level.3.Interacting protein and downstream genes of URL1Yeast two-hybrid?Y2H?system and Co-IP was used to identify the fact that URL1interacted with ROC5 physically in vivo.Compared to wild type,the transcriptional level of ACL1 and SRL1 was reduced in url1 mutants.In the 3-kb regions upstream of the initiation codons of the ACL1 and SRL1,a L1 box-like site in the promoter of SRL1 and two in ACL1were identified.EMSA and CHIP were carried out to prove that URL1 interacted with L1box in the promoter of ACL1 and SRL1.4.Cloning and function analysis of OsCHR4 geneWe reported the rice Oschr4-5 mutant characterized by pleiotropic phenotypes,including narrow and rolled leaves,as well as enhanced cuticular wax deposition.The reduced leaf width was caused by a reduced number of longitudinal veins and increased auxin content.The cuticular wax content was significantly higher in the Oschr4-5 mutant,resulting inreduced water loss rate and enhanced drought tolerance.Molecular characterization revealed that a single-base deletion results in a frame-shift mutation from the second chromodomain of OsCHR4,a CHD3?chromodomain helicase DNA-binding?family chromatin remodeler,in the Oschr4-5 mutant.Expressions of seven wax biosynthesis genes?GL1-4,WSL4,OsCER7,LACS2,LACS7,ROC4 and BDG?and four auxin biosynthesis genes?YUC2,YUC3,YUC5 and YUC6?were up-regulated in the Oschr4-5mutant.