【作者】 圣忠华；

【导师】 陈立云；

【作者基本信息】 湖南农业大学 ， 种子科学与工程， 2013， 博士

【摘要】 株1S是当前我国广泛用于两系法杂交早稻组合配制的低不育起点温度高配合力的温敏核不育系。本研究以株1S为材料,对其花粉败育的细胞学、不育性状的经典遗传学、不育基因的精细定位进行了研究。水稻中1W温敏白条纹叶突变体来源于日本晴经EMS诱变。该突变体苗期低于28℃条件下表现白条纹叶,随着发育的进行以及温度的升高,其白条纹叶逐渐转变为与野生型日本晴几乎无差异的绿叶。本试验对中1W温敏白条纹叶的农艺性状、不同发育时期叶绿素含量、叶色不同部位叶绿体的亚显微结构、白条纹叶基因的遗传规律、精细定位等进行了研究,主要结果如下：1花粉败育细胞学观察表明,株1S花粉败育起始于小孢子早期,一直持续至花粉成熟期,由于绒毡层细胞解体迟缓,不能提供其发育所需的营养物质而导致花粉败育。株1S花粉败育花药壁亚显微结构观察进一步证实了这一研究结果。2育性经典遗传学分析表明,株1S不育基因与安农S-1,福龙S的不育基因部分等位；与C815S,培矮64S的温敏核不育基因完全不等位。株1S温敏不育性状受1对隐性核基因控制。3不育基因精细定位结果表明,株1S不育基因位于第2染色体短臂上,定位于In-Del标记In101与In91之间的30.2Kb的区域内。该基因暂命名为tms9。4生物信息学分析表明,在该30.2Kb的区域内存在7个候选基因(ORF),根据各候选基因在株1S可育和不育条件下的表达以及各候选基因测序,最终第6个候选基因(LOC_Os02g12290)被认为最有可能是株1S不育目标基因,进一步的转基因功能互补验证实验正在进行。5中1W温敏白条纹叶除株高显著低于野生型外,其他农艺性状与野生型无显著差异。6中1W温敏白条纹叶田间条件下四叶期、分蘖期、孕穗期的叶绿素含量显著低于野生型,抽穗期及以后则与野生型差异不显著。7中1W温敏白条纹叶白色部分叶绿体数量明显少于野生型；叶绿体结构异常,具体表现为内囊体数目较少,基粒片层排列疏松不连续,嗜锇小体较多,淀粉体较少。叶片转绿后,叶绿体结构与野生型无显著差异。8遗传分析表明：中1W温敏白条纹叶性状由1对隐性核基因控制。该基因定位在水稻第9染色体端粒附近SSR标记RM23742和RM23759之间约486.5Kb的区域内,该区域内含有5个BAC克隆子,该基因暂命名为wsl1。更多还原

【Abstract】 ZhulS is a thermo-sensitive genic male sterile line of rice with low critical temperature of fertility alteration and excellent combining ability, which has been widely exploited as a female parent in Chinese two-line hybrid rice breeding. In this study, the cytological observations of pollen sterility, the classical genetic of male sterility trait were analysised and the thermo-sensitive genie male sterile gene of ZhulS was fine mapped, respectively. The rice thermo-sensitive white striped leaf mutant Zhong1W was mutated from cv. Nipponbare by EMS. Zhong1W showed white striped leaves at the seedling stage and tillering stage when the temperature was below28℃, then the white striped phenotype restored gradually to green until mature along with the temperature rising. In this study, the agronomic traits, chlorophyll content during different development stages, chloroplast ultrastructure within different leaf color parts, the classical genetic analysis and molecular mapping of Zhong1W leaf color mutant gene have been investigated. The all major study results are as follows:1The cytological observations of ZhulS pollen sterility indicated that the pollen abortion was dated from early microspore and which continued to pollen maturation, it can not provide the nutrients for microspore development because of the tapetum cell delayed disintegration which lead to pollen abortion. The anther wall cells ultrastructure observations during Zhu1S pollen abortion stage further confirmed the results of cytological observations, that due to the not timely disintegration of tapetum cells then the microspores can not get enough nutrients during the development stage which lead to pollen abortion.2Genetic analysis showed that Zhu1S thermo-sensitive genic male sterility was controlled by a single recessive nuclear gene. There was partly allelic sterile gene between Zhu1S and AnnongS-1and FulongS. However, there was no allelic sterile gene between Zhu1S and C815S and Peiai64S.3The thermo-sensitive genic male sterile gene of Zhu1S, which was named tms9temporarily, was finally mapped on the short arm of the chromosome2between the Indel markers Indel101and Indel91with the physic distance of30.2Kb.4There are seven candidate genes in the30.2Kb positioning interval according to the bioinformatics analysis. Finally, according to the results of candidate genes expression under the fertile and sterile conditions of Zhul S and the candidate genes sequencing, the sixth candidate gene (LOC_Os02g12290) was at last considered as the possible target gene of thermo-sensitive genic male sterile gene for Zhu1S, and futher genetically modified function complementary verification tests are underway.5At maturity, the plant height of the thermo-sensitive white striped leaf mutant Zhong1W was significantly shorter than that of the wild type. While the other agronomic characters between Zhong1W and its wild type showed no significant difference.6During the fourth leaf stage, tillering stage and booting stage, the Chlorophyll a content, Chlorophyll b content and the total Chlorophyll content of Zhong1W were all lower than that of its wild type, while during the heading stage, the Chlorophyll a content, Chlorophyll b content and the total Chlorophyll content between Zhong1W and its wild type have no significant difference.7The number of chloroplast in the white part of Zhong1W leaves was much less than that of its wild type, the thylakoids were vague and degraded, and the grana lamellar became more loosely-packed, discontinued and significantly reduced in number. In addition, the number of osmium corpuscle in the chloroplasts of Zhong1W was much more than that of its wild type, while the starch granule of which was significantly reduced. Within the green part of Zhong1W, the leaf color showed light green, and the number of chloroplast in the mesophyll cell was increased, and there were also some chloroplasts with well-developed grana lamellar structure similar to that of the wild-type in the mesophyll cells.8Genetic analysis showed that the rice thermo-sensitive white striped leaf mutant trait of ZhonglW was controlled by a single recessive gene. ZhonglW thermo-sensitive white striped leaf gene was finally mapped on the telomere region of chromosome9and positioned between SSR markers RM23742and RM23759, which are separated by approximately486.5kb. There are five BAC clones in the mapping interval. Zhong1W thermo-sensitive white striped leaf gene was temporarily named wsll.更多还原