【作者】 陆青；

【导师】 张启发；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 光敏感雄性核不育水稻农垦58S具有长日高温不育、短日低温可育的特点，是发展水稻“两系”法育种的重要种质资源。此前的研究发现，根据杂交组合中材料遗传背景的不同，农垦58S的不育性受到1个或2个位点的控制，位于第12染色体上的pms3位点是导致正常水稻品种农垦58突变为光敏雄性不育水稻农垦58S的根源。利用只在pms3基因位点发生育性分离的农垦58S／DH80 F₂群体，pms3基因已被精细定位到两个RFLP分子标记C751和M36之间，距离分别为1.6 cM。 本研究的最终目的是遵循图位克隆法的策略，分离克隆pms3基因。我们通过对农垦58S／DH80 F₂群体进行扩大，从大约7000株的F₂群体中获得了892株极端雄性不育单株，利用C751和M36进行RFLP分析，分别获得了极端不育重组单株43株和17株用于对pms3基因的精细定位。根据美国Clemson大学基因组研究所释放的日本晴全基因组物理图谱信息，找到了可能覆盖pms3基因区域的日本晴BAC重叠群，结合DNA指纹技术和分子标记对60株重组单株的分析，确定了包含13个BAC克隆在内的pms3区段物理图谱。 通过利用157个BAC克隆的亚克隆作为探针对亲本农垦58S和DH80进行多态性分析，发现了4个来自不同BAC克隆的与pms3基因紧密连锁的分子标记P9、135、C11和M2，利用这4个标记对C751和M36筛选出的重组单株进行基因型分析，发现M2和P9将目标基因限定到两个相互重叠的BAC克隆73M2和71J16上。采用“鸟枪”法对可能携带pms3基因的BAC克隆73M2进行测序，获得了约160kb的基因组序列，通过搜索Monsanto公司的数据库，得到了另外108 kb的序列，将该区域的序列延伸至268 kb，同时结合对亚克隆P9和M2的序列测定，将目标基因限定到230 kb的范围内。在230 kb范围每隔5 kb左右挑选1个亚克隆作为探针，共49个探针对亲本进行分析，找到了11个在亲本间有多态性的亚克隆，利用它们作为探针继续分析该区间重组事件发生的情况，将pms3基因范围缩小至两个分子标记LJ25和LK40之间，二者相距28.4 kb，与pms3基因之间分别还有4个和2个重组单株。该28.4 kb DNA片段上包含了1在水稻花和成熟叶片中表达的基因、1个在愈伤组织和早期的幼穗中表达的基因以及5个预测的ORFs。 为了进行pms3基因的功能互补测验以及比较农垦58、DH80、农垦58S之间序列上的差异，以农垦58叶片为材料，构建了农垦58基因组BAC文库，该文库更多还原

【Abstract】 Photoperiod-sensitive genie male sterility rice （PSGMS） Nongken 58S, which under long-day condition is sterile while under short-day condition is fertile, is a very important germplasm to develop the "two-line" hybrid in rice breeding program. Previous studies have established that the sterility of Nongken 58S is controlled by one or two loci, depending on the genetic backgrounds of the material used in the crosses, and the locus located on rice chromosome 12 designated pms3 is the one at which the original mutation occurred in the cultivar Nongken 58 to give rise to Nongken 58S. Using an F₂ population from a cross between Nongken 58S and DH80, in which the fertility trait showed a typical single-locus segregation at the pms3 locus, the pms3 gene was mapped to two molecular markers C751 and M36, with the genetic distance of about 1.6 cM each.The overall goal of this study was to clone the pms3 gene following a map-based cloning method. Firstly, we got 892 highly sterile individuals from Nongken 58S/DH80 F₂ population and then genotyped them by using C751 and M36. The two markers identified 17 and 43 recombinants respectively. Based on the whole genomic physical map information released, we anchored the pms3 gene to one possible Nipponbare BAC contig. Combining the DNA fingerprinting with the 60 recombination individuals analysis by molecuar markers, the physical map of the pms3 gene region containing 13 BACs was identified.157 BAC subclones were then used as probes to reveal the polymorphisms between Nongken 58S and DH80 and hence analyze the recombination plants. 4 markers were found to be closely linked to pms3 with M2 and P9 limiting the pms3 to two overlapping BACs 73M2 and 71J16. By shotgun sequencing method, 73M2 was sequenced and assembled into 3 contigs with a total length of 162 kb. By searching the sequence data provided by Monsanto Company, we got additional 108 kb DNA sequence and extended the contiguous sequence to 268 kb. Sequence analysis of the two RFLP markers P9 and M2 showed that they were located on 230 kb apart in the 268 kb range. Additional 11 polymorphic probes from the 49 ones spaced at distance of one clone per ～5 kb in the更多还原