【作者】 翁建峰；

【导师】 翟虎渠；

【作者基本信息】 南京农业大学 ， 遗传学， 2009， 博士

【摘要】 水稻(Oryza sativa L.)作为全球最重要的粮食作物之一,随着耕地的减少和人口的增长,提高其产量对于解决未来粮食安全问题具有十分重要的战略意义。水稻粒形(包括粒长、粒宽、长宽比与粒厚)是稻米最重要的外观品质指标之一,影响稻米的产量和加工品质。粒形是受多基因控制的复杂数量性状,目前大部分对于粒形的研究仅局限于利用初级定位群体进行QTL分析。本研究利用具有粳稻(宽粒)品种Asominori遗传背景、籼稻(窄粒)品种IR24供体片段的染色体片段置换系材料CSSL28(窄粒)与背景亲本Asominori回交构建F2次级分离群体,并以水稻谷粒和糙米的粒宽、粒长和长宽比为研究对象,进行遗传分析、初步定位和单基因分离。最终我们精细定位与克隆了控制水稻粒宽、长宽比和千粒重的QTL GW5,并进行了GW5的初步功能分析,同时还比较了目标置换系CSSL28与背景亲本Aosminori在产量性状及其水稻颖壳细胞形态学等相关性状的差异。主要结果如下：Asominori与CSSL28的粒形差异显著(P<0.01),其中CSSL28稻谷的粒宽与长宽比分别为Asominori的83.6%与126.6%,糙米分别为91.6%与123.7%,而其他农艺性状(如株高、株形和穗形等)无显著性差异；在产量相关性状,置换系CSSL28与背景亲本Asominori的分蘖数、结实率无显著性差异,而CSSL28的单株粒重、单穗粒重和千粒重显著低于Asominori (P<0.01)。颖壳细胞形态学分析表明,CSSL28与Asominori之间粒宽的差异是由其颖壳外围薄壁细胞数目的差别引起,并且CSSL28籽粒果柄横切面的维管束面积明显低于Asominori; Asominori开花后5-20天的灌浆速率高于CSSL28,同时前者垩白性状指标在籽粒成熟后偏高；另外,加工品质分析表明,Asominori的糙米率(P<0.01)与精米率(P<0.05)显著高于CSSL28。利用CSSL28与背景亲本Asominori回交构建F2次级群体,将控制水稻粒宽和长宽比的QTL分解为单基因GW5,宽粒表型受单隐性核基因控制；水稻籽粒表型和分子标记分析表明,由于F2群体粒形性状受籼粳杂种部分不育位点S31(t)影响,窄粒单株／宽粒单株比例偏离3：1分离；我们利用包含2,180个宽粒单株的F2次级群体和自行设计的SSR、CAPS与InDel标记,将GW5最终定位在第5染色体短臂位于标记Cw5与Cw6之间的一个交换重组热点区域,距两标记的物理距离分别为10kb和11kb左右,且标记Indel1、Indel2与GW5共分离。21kb范围序列与转录本分析表明,ORFl与ORF3在两亲本中核苷酸和转录水平未发生明显的变化,而宽粒材料Asomincri相对于窄粒材料CSSL28和IR24主要存在1.2kb基因组片段的缺失,通过软件预测发现该缺失区域内存在开放阅读框ORF2,我们把该ORF定为GW5的候选基因。同时,对46个栽培品种和12个野生稻资源的该区段序列分析发现,宽粒材料1.2kb基因组片段均缺失,而野生稻和窄粒品种则含有这一区段,据此推测该缺失区域内的ORF2是GW5的候选基因,且GW5可能与水稻的驯化相关。RT-PCR分析表明,GW5基因仅在窄粒的材料中表达；亚细胞定位与酵母双杂交结果显示,GW5定位于细胞核中并可能参与蛋白的泛素降解途径；然而,利用2.5kb包含ORF2的基因组和过量表达ORF2并未使转基因的Asomincri籽粒变窄,该结果表明通过预测得到的ORF2并不完整；我们通过RACE并结合Southern blot技术获得了部分GW5的cDNA。更多还原

【Abstract】 Rice is the most important staple food crop and a primary food source for about half of the world’s population. As farmland is decreasing and the global population increasing, there is an urgent need to secure grain production. The grain shape, including grain length, grain width, length/width ratio and grain thickness, is one of important apperence qualities in rice, which may have an effect on both grain yield and milling quality. The grain shape is a complex quantative trait controlled by multiple genes, and most of the grain-based researches are limited to the use of the primary populations targeted QTL at present. In this study, we have gotten a chromosome segement substitution line with narrow grain, CSSL28, derived from Asominori (wide cultivar)/IR24 (narrow cultivar) with Asominori as the recurrent parent. The mapping population was constructed from the cross between CSSL28 and parental line, Asominori. Traits of grain length, grain width and length/width ratio were used for characterization, genetic anaylsis, primary mapping and high resolution mapping QTL anaylsis. We identied and preliminarily characterized the QTL GW5 underlying grain width, length/width ratio and grain wight, and also investigated the differences of yield components and cell morphological between CSSL28 and Asominori. The main conclusions are as follows:The significant differences existed in grain shape between Asominori and CSSL28 (P<0.01). CSSL28 shows 83.6%and 126.6%in width and length/width ratio of paddy rice, 91.6%and 123.7%in brown rice grain, which were compared with these of Asominori respectively. But, none of other agronomic traits show significant difference, such as plant height, plant and panical shape, etc. For factors influencing rice yeild, there are also no significant differences between the substitution lines and background parent in tiller number, seed setting rate up, but in grain weight per plant, grain weight per panicle and 1,000-grain weight(P<0.01). Cytological analysis showed that the number of external parenchyma cells in rice hull caused the grain width differences between Asominori and CSSL28, and the vascular cross-sectional area in carpopodium of CSSL28 was significantly lower than that of Asominori. Meanwhile, the grain filling rate of Asominori was higher than the CSSL28 in 5-20 days after flowering, and the former has a higher percentage of grains with chalkiness. Moreover, the brown rice rate (P<0.01) and milled rice rate (P<0.05) in Asominori were significantly higher than that in CSSL28.Based on secondary F2 populations derived from the backcross of CSSL28 and Asominori, the QTL GW5 controlling both the grain width and length/width ratio was dissected into a single gene despite the nontraditional Mendelian segregation ratio between the plant with slender grains and wide ones in the F2 population. Meanwhile, the ratios of genotypes of SSR marker and frequency distribution of grain width in BC4F2 indicated that the indica-japonica hybrid partial sterility gene S31(t), being linked with GW5, was the reason of the observed distortion of segregation in the BC4F2 population. For genetic anaylsis, the phenotype of narrow grain in CSSL28 was controlled by a dominant nuclear gene. Then, the GW5 was further mapped between CAPS makers Cw5 and Cw6 on chromosome 5, within a 21kb recombination hotspot region of BAC clone (OJ1097_A12), using the 2,180 segregants showing the wide grain phenotype in BC4F2 population and markers developed in our laboratory. The result of sequencing indicated that the genome of Asominori habored a 1.2kb deletion compared with that in CSSL28. Within targeted region, two opening reading frams(ORFI and ORF3) was predicted in Asominori, but three ones (ORF1, ORF2 and ORF3) in CSSL28. In another words, ORF2 was located in the deleted region. Also, there is no difference exsited in the coding region of ORFI and ORF3. Thus, we teneatively designated the ORF2 as the GW5 gene. Genotyping analyses in 46 rice cultivars and 12 wild rices showed that the deletion prevailed in wide grain rice cultivars and defined a domestication related rice gene.The expression of GW5 in narrow cultivar was confirmed using the RT-PCR analysis. The protein was predicted to contain a nuclear localization signal (NLS) and an arginine rich domain. Transient expression in onion epidermal cells showed the GW5-GFP fusion protein is exclusively localized to nuclear. The yeast two-hybrid showed that the GW5 may physically interact with polyubiquitin and played an important role in the ubiquitin proteasome pathway. However, the 2.5kb fragement covering the ORF2 and the over-expression of ORF2 could not restore the narrow phenotype in Asominori. These results suggensted the ORF2 was incompleted for GW5, and the partial cDNA was isolated using RACE, which was detected with Southern blot.更多还原