| Floral organ development in rice,which signals the transformation of vegetative stage to reproductive stage,is a key process involved in rice reproduction and yield formation.Studies on the regulators and molecular mechanism of the rice reproductive development by using the rice floral organ development and male sterile mutant has been always a focal point for rice bioresearch.In this study,we analysis the phenotype and gene mapping on two sterile mutants(ohms1 and difo1)with deformed floral organs.In addition,phenotype analysis and map-based cloning on a genic male-sterility mutant,strs1,were performed.The expression pattern and preliminary function of OsSTRS1,a regulator involved in rice anther and pollen development,were analyzed.The main results are as follows:1.Research on ohms1 mutant: The open hull and male sterile 1(ohms1)mutant was characterized as open hull and lemma and palea like structure between anthers and stigma that made the spikelet of the mutant showed three “triodia-like ” flower glumes.The mutant was self sterile but 60%-70% pollen grains were fertile.Genetic analysis and gene mapping showed ohms1 was controlled by a single ressessive gene and the mutant gene was fine mapped to a region of 42 kb on the short arm of chromosome 3 between markers KY2 and KY29.Sequence analysis of the four open reading frames(ORFs)in this region revealed that LOC_Os03g11614 was probably corresponding to ohms1,which encoded a MADS box gene allelic with OsMADS1,and had a single nucleotide transformation(A to G)at the bottom of the fifth intron.Enzyme digestion and cDNA sequencing furtherly indicated that the variable splicing was responsible for the deletion of the sixth exon of ohms1,but no structure changes in MADS domain or amino acid frameshift occurred.Additionally,real time fluorescence quantitative PCR analysis showed that the expression level of OHMS1 decreased significantly and the expression level of rice flowering factors and floral glume development related genes changed significantly.All those results demonstrated that OHMS1 may play an important role in rice floral organ development,particularly in floral glume development and floret primordium differentiation.2.Research on difo1 mutant: Outer whorls of floral organs in deformed interior floral organ 1(difo1)mutant were normal compared with the Zhonghui8015(Zh8015)spikelet,but the interior floral organs of the difo1 mutant present various numbers of stamens and stigmas,with no typical filament and no mature pollen grains.Most difo1 flowers exhibited an increased number of stigmas that were attached to the stamens and an intumescent ovule-like cell mass in addition to the ovary.Transverse sections of spikelets and scanning electron microscopy analysis revealed an indeterminate number of interior floral organs and abnormal early spikelet development for the difo1 mutant.Instead of the linear-shaped surface of wild-type stamens,difo1 displayed a glossy stamen surface resulting in immature stamens and complete sterility.In addition,the difo1 mutant exhibited delayed anthesis,rapid anthesis and non-extended stamens compared with wild type.Genetic analysis and gene mapping revealed that difo1 was controlled by a single recessive gene,which was fine-mapped to a 54-kb interval on the short arm of chromosome 4 between markers S22 and RM16439 harboring nine ORFs.Sequence analysis revealed that the mutant carried a single nucleotide deletion in its promoter region,which was likely corresponded to the phenotype,in a C2H2-type zinc finger protein gene(LOC_Os04g08600).Moreover,qRT-PCR analysis showed a significantly down-regulated expression pattern for DIFO1 and many floral organ identity genes in the interior floral organs of difo1.DIFO1 is therefore an important floral organ development gene in rice,particularly with regard to interior organ meristem identity and floret primordium differentiation.3.Research on OsSTRS1:The vegetative growth,panicle and spikelet structure of the strs1 mutant were almost the same with wild type Zh8015,but the mutant exhibited thin and milky white anthers with completely aborted pollen.Compared with the wild type,the strs1 demonstrated delayed tapetum degeneration,shrunken microspores at mitosis stage,and atrophy contents with olny single nucleus pollen in anther chamber.It was confirmed by the electron microscopy observation that the strs1 mutant exhibits a dense and chaotic anther wall,residual sporopollenin and ubischbodies,shrunken pollen exine,and no starch grains in pollen.These observations suggested that the disequilibrium of nutrition supply in the later stages caused the abnormal development of the anther and tapetum,resulting in complete abortion of pollen in strs1.The STRS1 male sterile gene was mapped to a 20-kb region between markers JS6 and JS7 on the short arm of chromosome 3.Sequencing results revealed that a two nucleotides deletion was detected in the fourth exon of a strictosidine synthase gene(Os03g0263600)which resulted in an mRNA frameshift and a premature stop codon.RNA interference and functional complementation assay further confirmed that loss of function of OsSTRS1 is responsible for pollen abortion phenotype in the strs1 mutant.OsSTRS1 was homologous with the male sterile gene MS45 in Maize.Expression pattern analysis showed that OsSTRS1 mainly expressed at mitosis stage and highly focused in anther wall and pollen exine,expression level of rice anther development related genes changed significantly.Chemical composition analysis indicated that cutin and wax components of anther in strs1 mutant were significantly decreased compared with the wild type.Subcellular localization of the protein showed that the OsSTRS1 protein localized to the plasma membrane in rice protoplast,and all the functional domains of OsSTRS1 localized to nucleus after removal of the transmembrane region of the protein.This provide a basis for further study and understanding on the regulation mechanism and biological function of strictosidine synthase genes in rice anther development and pollen exine formation. |
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