| Rice (Oryza sativa L.) is not only one of the most important food crops in the world, but also the model plant for molecular biology study of monocotyledon. The formation and development of rice panicle is the important determinate on grain yield and quality, so it is necessary to elucidate the mechanism on its’ reproductive growth, especially the formation and development of panicle. In the last twenty years, studies on floral tissue structure and molecular mechanisms had became a hotspot to researchers, and the knowledges went more and more thoroughly. Based on the studies of Arabidopsis thaliana and Antirrhinum majus, the insights of floral development of dicotyledonous were well understood at present, but the study on monocot is dramatically less than that of dicotyledonous. More recently, several genes associated with rice panicle development had been identified, and most of them were investigated via mutants. In the present study, three mutants associated with rice panicle development were identified, and their phenotype was characterized and the mutated genes were fine mapped or cloned, the main results were as flowerings.1. Identification and gene cloning of the leafy head mutant plal-SA rice leafy head mutant plal-5was derived from progenies of japonica Taipei309treated with60Co-y ray irradiation. Comparing with its’ wild type, the pla1-5mutant tended to be dwarf and had smaller and more leaves, with a reduced tiller number, tillering on upper nodes, plal-5failed to produce a normal panicle at maturity stage. Because the panicle development of homozygous mutant was blocked and no seed harvested, we then preserved it with heterozygous mutant. The mutation traits could inherited stably with successive years planting, an F2population was generated between heterozygous plal-5and indica cultiva Nanjingll, genetic analysis implied that the pla1-5phenotype was controlled by a single recessive nuclear gene. Two DNA pools derived from the wild type and mutant plants were assembled respectively, the SSR marker named CHR1003on chromosome10was found to be polymorphism between the two DNA pools, Subsequently,45individual mutants from F2population were genotyped, the results confirmed that the marker CHR1003was linkage to PLA1-5, By using map-based cloning strategy, PLA1-5was finally narrowed down to a58kb region between SSR markers CHR1027and CHR1030on the long arm of chromosome10and co-segregates with the molecular marker CHR1028and CHR1029. Based on Rice Genome Annotation Project, five intact candidate genes were predicted within this region, they were LOC_Os10g26300, LOC_Os10g26320, LOC_Os10g26330, LOC_Os10g26340and LOC_Os10g26360. Among them, LOC_Os10g26340encoded a cytochrome P450CYP78A11and all the other four genes encoded putative transposon protein. Specific primers were designed based on the gene annotation sequence of Nipponbare, and the nucleotide sequences of the candidate gene were compared among the wild type and the mutant alleles. The results showed that no mutation site could be found in the other predicted genes as LOC_Os10g26300, LOC_Os10g26320, LOC_Os10g26330or LOC_Os10g26360, the plal-5mutant harboured1bp deletion of nucleotide T in exon1of LOC_Os10g26340encoding cytochrome P450CYP78A11, which might result in a downstream frame shift and a premature termination. These results implied that the cytochrome P450CYP78A11gene might be the candidate gene of PLA1-5. We tentatively designated the gene as PLA1-5for it was the same candidate gene of PLA1.2. Identification and gene mapping of the panicle development failure mutant pdflA panicle development failure mutant pdfl was derived from the progenies of japonica cultivar Wuxiangjing9after tissue culture, it has no obviously difference with its wild type during vegetative stage; but at reproductive stage, the axillary buds of the mutant were non-dormant and continue to grow, and upper tillers were formed ultimately. The panicle rachis cells of mutant pdfl was necrotic after a period of panicle development. During plant growth, the mutant was treated with gibberellin (GA), photoperiod and temperature, respectively, the results showed that the mutated trait was regulated not by GA or photoperiod, but by high temperature. The panicles of mutant pdfl could develop normally under low temperature during growth stage, but they showed enclosed panicle at different degree. A segregation population was generated with the cross between the mutant pdfl and the indica cultivar Nanjing11. Genetic analysis showed that the mutation character was controlled by a single recessive nuclear gene. By using bulked segregation analysis and map-based cloning strategy, the PDF1gene was ultimately mapped between the SSR markers CHR539and CHR516on the short arm of chromosome5. The results laid a foundation for fine mapping and gene cloning of PLA1-5and further investigation on molecular regulation mechanism of rice panicle development.3. Genetic analysis and gene cloning of a triangular hull mutant trilA rice triangular hull mutant tril was obtained from the progeny of a japonica rice variety Taipei309treated with60Co-y ray. Compared with the wild type, the tril mutant presents triangular hull, exhibits increase of grain thickness and protein content, but a slight decrease of plant height and grain weight. Genetic analysis indicated the mutation characters were controlled by a recessive nuclear gene which can be steadily inherited. By using map-base cloning strategy, we fine mapped tril to a47kb region between the molecular markers CHR0122and CHR0127on the long arm of chromosome1, and co-segregates with the molecular marker CHR0119. According to the rice genome sequence annotation there are six predicated genes in the mapped region, these are LOC_Os01g52680, LOC_Os01g52690, LOC_Os01g52700, LOC_Os01g52710, LOC_Os01g52720and LOC_Os01g52730. Because the partical sequences of the predicted gene LOC_Os01g52730were not included in the region, so only the other five intact annotated genes were analyzed, the five candidated genes encode OsMADS32-MADS-box family gene with MIKCc type-box, putative retrotransposon protein, hypothetical protein, putative glycosyl transferase8domain containing protein and putative Complex I intermediate-associated protein30domain containing protein respectively. Among these, OsMADS32has been reported to be a transcription factor related to flower development. Sequencing analysis between the mutant and wild type indicated that there was a nucleotide A deletion in exon3of OsMADS32of the mutant, which might result in a downstream frameshift and a premature termination, there were no nucleotide difference of the other four annotated genes. RNA of different organs at different phases in Japonica cultiva Nipponare was extracted, both semi-quantitative and real-time RT-PCR analysis showed that the gene OsMADS32was highly expressed in young inflorescence and very low in other tissues. These results implied that the OsMADS32gene might be a candidate of TRI1.By identification, genetic analysis, gene mapping and cloning of the panicle associated mutants, The study provide import materials and gene resources for further investigation on molecular regulation mechanism of rice panicle and caryopsis development. |
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