【作者】 封功能；

【导师】 刘巧泉； 徐辰武；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2013， 博士

【摘要】 水稻(Oryza sativa L.)不仅是世界上最重要的粮食作物之一,还是禾本科作物分子生物学研究中重要的模式植物。水稻穗的形成与发育是决定产量和品质的重要因素,也是人类食物的主要来源,因此,有必要对其生殖发育特别是小穗发生与发育调控的分子机理进行相关研究。近二十年多来,科研工作者对植物生殖器官组织结构和分子遗传机理的研究越来越多,也越来越深入。基于拟南芥和金鱼草等植物的研究,目前对双子叶植物花发育机制有了比较清晰的认识,而水稻等单子叶植物花器官的研究明显落后于双子叶植物。突变体的获得对研究植物发育过程中基因作用有着重要的意义,目前在水稻中已报道的许多与穗发育相关的基因大多是通过突变体发现的。本研究以三个与穗发育相关的突变体为研究对象,通过形态鉴定、遗传分析和基因定位,最终将目的基因进行了精细定位或克隆,所获主要研究结果如下：1、水稻类树状突变体pla1-5的鉴定与基因克隆在粳稻品种台北309的60Co--γ辐射诱变后代中发现了一份水稻类树状突变体plal-5(plastochronl-5),表现为植株矮化、叶片小且数量增多、分蘖数降低、高位分蘖、穗分化受阻等特征。由于纯合突变体plal-5穗发育受阻,不产生种子,因此利用杂合突变体进行材料的保存。连续多代种植发现,该性状能稳定遗传。利用杂合突变体与籼稻南京11号构建F2群体,遗传分析表明,该性状受一对隐性核基因控制。分别构建野生型DNA池和突变型DNA池,发现第10染色体上的分子标记CHR1003在两池间呈多态,标记CHR1003对F2群体中的45个突变型单株进行基因型检测,证实CHR1003与PLA1-5连锁,利用图位克隆技术,将目的基因定位于第10染色体长臂上两个分子标记CHR1027和CHR1030之间,物理距离为58kb,并与SSR标记CHR1028和CHR1029共分离。根据水稻基因组序列的注释,该区域内存在5个完整的注释基因,分别为LOC_Os10g26300、 LOC_Os10g26320、LOC_Os10g26330、LOC_Os10g26340和LOC_Os10g26360,其中LOC_Os10g26340编码细胞色素P450CYP78A11,其它4个基因均编码假设的转座子蛋白(Transposon protein, putative)。分别设计引物扩增突变体及其野生型台北309中5个注解基因,并进行序列测定。测序分析表明,LOC_Os10g26300、LOC_Os10g26320、 LOC_Os10g26330和LOC_Os10g26360四个基因在突变体plal-5与野生型间无序列差异。但突变体的LOC_Os10g26340第1外显子内缺失了一个碱基T,导致移码突变和翻译提前终止。据此,初步推测细胞色素P450CYP78A11基因为PLA1-5的候选基因。由于本研究候选基因P450CYP78A11与PLA1为同一基因,故将本研究所定位基因暂定名为PLA1-5。2、水稻穗发育受阻突变体Pdfl的鉴定与基因定位在粳稻品种武香粳9号的组织培养后代中发现了一个穗发育受阻突变体pdfl (panicle development failurel)。该突变体在营养生长期与野生型比较无明显差异,但进入生殖生长后,突变体上部腋芽不休眠,而是持续发育,并形成高位分蘖,小穗发育一段时间后,穗基部细胞逐渐死亡,并导致小穗死亡。分别对突变体抽穗前进行赤霉素、光周期和温度处理,结果表明,该突变性状不受赤霉素和光周期的影响,但受温度的影响,在突变体生长期降低温度小穗能正常发育,但出现不同程度的包穗。利用该突变体与籼稻品种南京11号杂交构建分离群体,遗传分析表明,该突变性状由单隐性核基因控制。通过群体分离分析法和图位克隆法最终将目的基因定位于第5号染色体短臂的SSR标记CHR539与CHR516之间的417kb范围内,为该基因的精细定位、克隆及水稻颖花发育的分子调控机理奠定了基础。3、水稻三角颖突变体tril的遗传分析与基因克隆以在经60Co-γ射线辐射粳稻品种台北309的后代中分离获得一个三角颖突变体tril (triangular hull1)为研究对象,与野生型相比,tri1籽粒颖壳呈三角形,粒厚增加,蛋白质含量升高,株高和千粒重降低。遗传分析表明,该突变性状能稳定遗传,并受一对隐性核基因控制。采用图位克隆法将目的基因精细定位于水稻第1号染色体长臂上分子标记CHR0122与CH0127之间,物理距离约47kb,并与分子标记CHR0119共分离。在该区域内共有6个候选基因,分别为LOC_Os01g52680、LOC_Os01g52690、LOC_Os01g52700、 LOC_Os01g52710、LOC_Os01g52720、LOC_Os01g52730,其中LOC_Os01g52730部分序列位于47kb区域之外,对剩余5个注解基因进行分析,分别编码MIKC类的OsMADS32、假定的逆转录转座蛋白、假定的蛋白、糖基转移酶8结构域蛋白和复合物Ⅰ中间体相关蛋白。测序分析表明,tri1突变体中一个释义基因OsMADS32的第3外显子内缺失了一个碱基A,导致移码突变和翻译提前终止,其它基因在突变体和野生型间无碱基差异。分别提取粳稻日本晴不同时期不同组织的RNA,半定量RT-PCR和荧光定量RT-PCR分析表明OsMADS32主要在水稻幼穗中表达,在根、茎、叶和发育的种子等组织中表达量极低,说明OsMADS32基因与花的发育与关。据此,推钡OsMADS32基因可能为TRI1的候选基因。本研究通过对三个水稻穗发育相关突变体的鉴定、遗传分析、基因定位和克隆,为进一步研究水稻小穗和籽粒发育的分子调控基理提供了重要的材料与基因资源。更多还原

【Abstract】 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.更多还原