【作者】 Yuenyong Vanichpakorn；

【导师】 何光华；

【作者基本信息】 西南大学 ， 作物遗传育种， 2010， 博士

【摘要】 水稻是世界上最重要的粮食作物之一,约有一半以上的人口以其为主食,因此,提高水稻单产对维护世界粮食安全至关重要。水稻基因组较小,约430 Mb,是单子叶模式植物。实践证明,稳定的突变体是研究水稻基因功能和水稻分子育种的重要材料。其中,矮化突变体的发现与应用就是其中最典型的案例之一。育种技术的革新以及分子生物学发展的日新月异必然促进常规育种的改良,为实现水稻育种的更高目标创造条件。色素是植物从太阳光吸收光电子进行光合作用的靶物质,随着分子领域研究的发展,其在高等植物研究中引起了越来越广泛的关注。近来,通过叶绿体缺乏突变体取得了一系列研究成果,叶绿体缺乏突变体是植物体内某一种或几种光合色素缺失或缺乏,该类突变体在叶绿体发育、叶绿素代谢、光合作用等研究中具有重要的价值。在水稻育种中,该类突变体可以利用其叶色标记检测杂交组合和繁殖不育系的纯度,也具有重要的应用价值。尽管利用叶色突变体取得了一定的成果,与数量众多的叶色突变性状相比,许多研究尚需加强。我们实验室利用EMS处理自育优良恢复系缙恢10号,获得了2个遗传稳定的叶色突变体。本论文针对这两个突变体,系统研究了其遗传特性、色素含量、细胞学结构、分子定位等,为从分子水平进一步深入了解水稻色素合成机制创造了条件。主要研究结果如下：1.新型叶色突变体wyv1的鉴定与基因定位wyv1苗期叶片表现为黄白化,分蘖期转为黄绿,抽穗期至成熟期表现为淡绿,因此命名为白黄绿转换突变体（white-yellow-virescent leaf mutant, wyv1）。突变体的叶绿素含量显著下降,下降趋势与叶色表现成正相关。在苗期,表型最为典型,叶绿素含量最低,此时,测量叶绿素荧光动力学参数,结果发现：在叶色变化严重部位,光化学猝灭系数（qP）、PSII的实际量子效率（ΦPSⅡ）、电子传递速率（ETR）、初始荧光（Fo）、精光合速率（Pn）和PSII最大光化学量子产量（Fv/Fm）等均显著降低；然而,令人感兴趣的是,在新抽出叶,叶色淡绿部位,Pn和Fv/Fm与野生型相比无显著差异。遗传分析表明,该性状受核单隐性基因控制,为分子定位WYV1基因,利用籼型不育系西农1A与wyv1杂交,总共获得了1698株F2隐性定位群体。利用SSR等标记,最终把该基因定位在第3染色体上,位于SSR标记Y7和Y6之间,遗传距离分别为0.06 cM和0.03 cM。利用9311序列进行比对分析,发现物理距离仅为84 kb,这为WYV1基因的克隆和功能研究奠定了基础,也为该基因在育种的应用创造了条件。2.新型叶色突变体ygl5的鉴定与基因定位ygl5整个生育期均表现稳定的黄绿叶表型,总叶绿素含量值为1.09 mgg^-1到3.11mgg^-1不等,其中,叶绿素α和叶绿素b仅为野生型的66.1%和61.2%，为典型的叶绿素缺乏突变体。抽穗期进行细胞结构观察,结果发现,除了叶绿体基粒片层略有减少外,其它结构无明显变化。与野生型相比,有效穗、株高、千粒重等农艺性状均显著下降。遗传分析表明,该突变表型受一对单隐性核基因控制。配制西农1A和ygl5杂交组合,利用F2群体进行定位,初步把YGL5基因定位在第3染色体短臂,位于SSR标记RM12338和RM1332/RM3766之间,遗传距离分别为0.9 cM和5.5 cM/30 cM,最终把YGL5基因定位在SSR标记SWU3-1和RM14367之间,遗传距离分别为0.2 cM和1.9 cM。这为该基因的图位克隆和功能分析奠定了基础。更多还原

【Abstract】 As the key food crop for more than half of humanity, it is significant to increase paddy rice yield per unit area. Rice genome is small and only about 430 Mb, and this provides insight as model for improving monocots. Inherited mutations have proven to be a valuable system both for gene functional studies and for plant breeder. Of which, "Dwarf breeding" is one of the most famous examples. Innovative breeding methods and the emerging tools of molecular and genome biology as the "Biotechnological tools" must modify the conventional breeding methods and enhance the achievement of rice breeding goals. Pigments are special target in obtaining the electronic energy from the sun and photosynthesis is then conducted. Pigment synthesis is so important that it was paid more and more attention with the development of molecular biotechnology in higher plants. Recently, much interesting results have been obtained by studying chlorophyll-deficient mutants, which lacks one or over certain photosynthetic pigments. Besides its significance in basic studies in chloroplast development, chlorophyll metabolism and photosynthesis, such mutants are also important in breeding, for example, they can be used as a simple marker for genetic purity testing both in hybrid seed production and in CMS line multiplication. In despite of its essentiality and partially productions, compared with abundant mutations in leaf-color characters, much of work was still needed to gain further useful conclusion. Two leaf color mutant were obtained from the progeny of EMS treated seeds of Jinhui10, an excellent restorer line bred in rice institute, southwest university. This thesis reviewed their genetic characters, pigments contents, cell structure, molecular mapping, and etc. The results were as following:1. Identification and molecular mapping of novel leaf color mutant wyv1The wyv1 colored white-yellow leaves at seedling stage and then showed yellow-green at tillering stage, following virescent until to maturity. This mutant decreased chlorophyll contents significantly and the trendline was consistent with chlorotic level. At the seedling stage of showing typical phenotype, chlorophyll fluorescence kinetic parameters were measured and the results showed that coefficient of photochemical quenching （qP）, actual efficiency of PSⅡ（ΦPSⅡ）, electron transport rate （ETR） and initial chlorophyll fluorescence level （Fo）, net photosynthetic rate （Pn） and maximum photochemical efficiency of photosystemⅡ（Fv/Fm） were significantly lower in the severe chlorotic leaf. However, no significant differences were observed for Pn and Fv/Fm between mutational virescent leaf and normal green leaf. To molecular mapping WYV1 gene, XinonglA was crossed with wyv1, the F1/F2 populations were used for genetic analysis and the F2 population was used for mapping. Finally, the wyv1 phenotype was confirmed to be controlled by one single nuclear-encoded recessive gene, and the WYV1 gene was finally mapped between SSR marker Y7 and Y6 on rice chromosome 3. The genetic distance was 0.06 cM and 0.03 cM, respectively, and the physical distance was 84 kb according to indica rice 9311. These results must fascinate the gene cloning as well as its application in breeding.2. Identification and molecular mapping of novel leaf color mutant ygl5The ygl5 displayed stable yellow green leaf during the development, and its total chlorophyll ranged from 1.09 mg g^-1 to 3.11 mg g^-1. As a chlorophyll-deficient mutant, chlorophyll a occupied 66.1% and chlorophyll b occupied 61.2% of that of wild type, respectively. Compared with the wild type, chloroplast structure changed less except of granal stacks. The poor agronomic characters were identified, including effective panicle, plant height, 1,000-grain weight and seed setting rates. Genetic analysis showed that the mutational character was controlled by one major recessive nuclear gene according to F₁ and F₂ population of Xinong1A/ygl5. Four hundreds SSR markers were used for gene mapping and the YGL5 gene was primarily mapped between RM12338 and RM1332, RM3766 on the short arm of the chromosome 3, the genetic distance were 0.9 cM and 5.5 cM,30 cM respectively from the locus. The YGL5 gene was finally mapped between SSR marker SWU3-1 and RM14367 with genetic distances 0.2 cM and 1.9 cM respectively. These provide the basic information for gene cloning and function analysis of YGL5.更多还原