Map-Based Cloning And Functional Analysis Of Two Genes Related To Brassinosteroid In Rice(Oryza Sativa L.)

Plant architecture and grain shape are very important for increasing rice yield,and it has always been a concern for breeders.A group of plant steroid hormones,Brassinosteroids(BRs),play an important role in regulating rice plant architecture and grain shape.BRs regulate many processes in plant growth and development,including promoting cell elongation and division,vascular differentiation,response to light,improving the resistance to disease.The study shows that BRs are involved in regulating plant growth and development,such as plant senescence,stem elongation and seed germination.At present,many studies on BR-related mutants and transgenic plants show that BRs have great potential to improve rice yield.Although many factors regulating BR synthesis and signal transduction pathways have been identified and studied,the understanding of BR synthesis and signal pathway regulation is limited.Therefore,it has an important biological and practical significance to carry out the research on BRs in rice.This thesis mainly focused on two BR-related mutants.The first part is "Map-based cloning and functional analysis of a brassinosteroid insensitive gene DS1 in rice";the second is "Map-based cloning and functional analysis of a rice brassinosteroid biosynthesis gene LHDD10".Part 1:In this study,we isolated a rice mutant from Nanjing 35 radiation mutagenesis population,which showed dwarfism,small grain size and erect leaf phenotype.We named the mutant as dwarf and small grain 1(ds1).Through genetic mapping and functional analysis,we elucidated the genetic and intrinsic molecular regulation mechanism of the phenotype of dsl.We also systematically studied the dsl in cytology,physiology and biochemistry.It not only deepened the understanding of the regulation mechanism of rice plant architecture,but also provided an important theoretical support for breeders to cultivate high yield rice varieties with“ideal plant architecture".The main results are as follows:1.Compared with the wild type,dsl had dwarfism,small grain size and erect leaf phenotypes.These mutant phenotypes were similar to those of BR biosynthesis and signal deficient mutants,such as brdl and d61.Through the BR sensitivity test,we found that the dsl mutant was insensitive to BR treatment,which indicated that the mutation of DS1 affected the BR signaling pathway.Scanning electron microscope observation of stem and glume showed that the alteration in cell size was responsible for the mutant phenotypes of dsl,and the expression of genes related to cell expansion and cell wall synthesis was significantly down-regulated in the dsl mutant.We crossed dsl with wild type to construct an F2 genetic analysis population.Genetic analysis showed that the phenotypes of dsl were controlled by a single recessive gene.2.We crossed dsl with Dianjingyou to construct an F2 genetic separation popUlation.The dsl locus was mapped to a 80-kb genomic region on the long arm of chromosome 1 by map-based cloning.Based on the rice gene base and genome sequencing,we found that the deletion of bases in the first and second exons of LOC_Os01g12890(OsEMFl).This mutation caused a frame shift in OsEMFl,which encoded a protein that lost its normal function.Through the complementation experiment,we further proved that the mutation of LOC_Os01g12890 led to the phenotypes of dsl.The total length of the DS1 genome is 4655 bp consisting of 4 exons and 3 introns,and the total length of CDS is 3174 bp,and DS1 encodes a putative EMF1-like protein consisting of 1057 amino acids,which is homologous to Arabidopsis AtEMFl.DS1 contains a nuclear location signal(NLS)in the N-terminus,binding to the GTP-ATP in the middle region,and a LXXLL domain in the C-terminus.3.Quantitative RT-PCR showed that the expression of BR signal-related genes decreased significantly in the mutant,and OsBRIl was the most down-regulated.The mutation of DS1 led to the altered expression of BR signal-related genes,which ultimately affected rice growth and development.Tissue expression analysis showed the transcript level of DS1 is strong in young leaves,panicles and seeds,relatively high in stem apex,stem and root.Interestingly,the DS1 expression pattern in the mutant was consistent with the wild type,and there was no difference in the expression of DS1 between the wild type and dsl.We found that the expression of DS1 increased after BL treatment.This result indicates that BR can induce the expression of DS1.4.Yeast two hybrid showed that DS1 could interact with auxin response factor OsARF11,and also showed that the C-terminus of DS1 and the middle region of OSARF11 were necessary for interaction.Further,the interaction between DS1 and OsARF11 was confirmed by BiFC in vivo and pull-down in vitro.We found that the knock-out positive plants showed dwarf and erect leaf phenotypes by Crispr-Cas9 system.These phenotypes were very similar to those of dsl.In the mutant osarf11,OsBRI1 expression was significantly reduced.These results indicated that DS1 regulates rice growth and development by interacting with OsARF11.5.Previous studies showed that OsARFs can directly regulate the expression of OsBRI1 through binding to the AuxRE(TGTCTC)motif on the promoter.We found that DS1 regulated OsBRI1 expression by interacting with OsARF11 by yeast one hybrid and transient expression experiments.Part 2:In this study,we isolated and characterized a rice BR deficient mutant late heading date and dwarf(lhdd10),which showed the phenotypes of dwarfism,small grain size and delayed heading date from a collection of EMS mutation progeny of indica variety 93-11.The genetic and regulatory mechanism of LHDD10 that regulated plant height,grain shape and heading stage were revealed via mapping and functional analysis.This research provided an important theoretical support for the creation and cultivation of excellent high yield rice varieties by genome editing.The main results are as follows:1.Compared with 93-11,lhdd10 showed short grain,decreased grain weight,dwarfism,delayed flowering time,and dark green leaves.Through the observation of the longitudinal paraffin section of stem cells,we found that the intemode cells of lhdd10 were significantly smaller than wild type,which may be the main reason for the smaller grains and dwarfism.Genetic analysis indicated that the phenotype of lhdd10 was controlled by single recessive gene.2.Exogenous BR treatment showed that lhdd10 was a BR deficient mutant.QRT-PCR showed that most of the BR synthesis and signal related genes were down-regulated in the mutant.The variation of LHDD10 leads to the change of BR synthesis and signal gene expression,and finally affects the growth and development of rice.3.lhdd10 and KetanNanga hybridization were used to construct F2 location population,and lhdd10 was mapped to a 130 kb genomic region on the long arm of chromosome 10.The flanked region contained 11 candidate genes.Sequencing showed that there was a single base substitution in the coding region of LOC_OslOg25780.QRT-PCR showed that LHDD10 was strongly expressed in young panicle and stem apex.LHDD10 transcripts also showed higher expression in leaf sheaths,young roots and leaves.LHDD10 encodes a putative FAD oxidoreductase.4.lhdd10 showed delayed flowering time under LDs and SDs.The expression of genes related to heading date under LDs and SDs was analyzed.DTH8,OsMADS51,OsGI and Ghd7 were not significantly different between wild type and mutant,while Ehd1,OsMADS50 and RFT1 were significantly down-regulated under LDs and SDs.5.Ihdd10 had smaller grain size and shorter grain length.The expression of the known grain size regulatory factors GL3.2,GL7,PGL2,GW5,GS3,SGI and OsSPL13 in the mutant were significantly down-regulated,suggesting that LHDD10 might control grain size through regulating the expression of grain size-related genes.