OsHAP家族基因的功能研究和粒形基因GL3.2的图位克隆

发布时间：2018-07-06 08:38

本文选题：扩增模式 + 关联分析　；参考：《华中农业大学》2016年博士论文

【摘要】：水稻是全球的主要粮食作物,地球上近一半的人以大米为主食。提高水稻产量是水稻生产和科学研究的目标。水稻开花是决定水稻生产的一个关键性状,它决定水稻品种的生态适应性,使水稻在合适条件下最大限度的利用温度和获得阳光。鉴定和利用开花相关基因是开展水稻设计育种的基本前提。目前,候选基因的关联分析在重要农艺性状的发掘方面有成功的应用。Ghd8是水稻HAP家族HAP3亚家族基因,它调控水稻的开花。为了鉴定水稻HAP基因在开花调控机制中的作用,我们对HAP家族基因在水稻核心种质和普通野生稻中的核酸多样性,以及HAP家族的扩张模式进行了分析;利用关联分析研究水稻HAP家族基因与水稻开花性状之间的关联性;通过转基因技术和突变体材料对水稻HAP家族中大部分基因进行功能验证。鉴于HAP复合体以三聚体形式存在的假设,进一步验证了HAP家族蛋白间的互作模式及其与Hd1,Ghd8的互作关系,研究结果如下:通过相关网站搜索到35个水稻HAP家族的基因,利用529份世界水稻种质资源和107份普通野生稻对其进行核酸多样性分析,发现HAP家族基因的平均核酸多样性比整个水稻基因组的核酸多样性高。在籼稻和粳稻中,Fst值存在较大差异(高于0.25),表明HAP家族基因有很强的种群分化。Os HAP2E/2G,Os HAP2F/2H,Os HAP3D/3E,Os HAP3F/3I,Os HAP5A/5B和Os HAP5D/5E为区段复制而得,Os HAP3G和Os HAP3J为串联复制而得。利用529份世界水稻种质资源对HAP基因和水稻开花性状进行关联分析发现,仅有4个基因与开花相关,其中HAP2亚家族无基因与开花关联。通过对23个HAP家族基因的转基因或突变体植株的分析,结果发现包含已克隆的Ghd8在内,有4个HAP基因调控开花,其中有3个基因被关联到。HAP5与HAP2,HAP3亚家族成员之间形成三聚体,酵母双杂交实验结果表明:Ghd8分别与HAP2,HAP5亚家族的部分蛋白互作,且Os HAP5A和Os HAP5B都与Hd1蛋白互作,为研究Os HAP5B如何调控开花提供了方向,也为HAP5,Ghd8互作调控水稻开花的分子机制的研究提供了可能。水稻粒形在自然界中存在广泛的变异,水稻粒形是人工选择的一个重要的农艺性状,它决定了水稻的外观品质和粒重。利用大粒品种“南洋占”和小粒品种“川7”为亲本的重组自交系群体定位到一个同时影响粒长与粒重的主效QTL。通过构建近等基因系,遗传分析及图位克隆,确定了候选基因GL3.2。将抑制川7基因型表达的RNAi-GL3.2转入川7品种中发现,RNAi转基因材料粒长为6.3 mm,比野生型川7材料的粒长(6.0mm)长0.3 mm。通过关联分析和测序发现,一个使转录提前终止的关键SNP导致了两种基因型粒长的差异。
[Abstract]:Rice is the main food crop in the world, and nearly half of the world's people feed on it. Increasing rice yield is the goal of rice production and scientific research. Rice flowering is a key trait that determines rice production. It determines the ecological adaptability of rice varieties and enables rice to maximize the use of temperature and sunlight under suitable conditions. Identification and utilization of flowering related genes is the basic premise of rice design and breeding. At present, candidate gene association analysis has been successfully applied in the discovery of important agronomic traits. Ghd8 is a HAP3 subfamily gene of rice HAP family, which regulates the flowering of rice. In order to identify the role of HAP gene in flowering regulation in rice, we analyzed the nucleic acid diversity of HAP family gene in rice core collection and wild rice, and the expansion pattern of HAP family. Correlation analysis was used to study the relationship between rice HAP family genes and flowering traits of rice, and the function of most genes in rice HAP family was verified by transgenic techniques and mutant materials. In view of the assumption that the HAP complex exists in the form of trimer, the interaction patterns of HAP family proteins and their interaction with Hd1 / Ghd8 are further verified. The results are as follows: 35 genes of rice HAP family were searched through relevant websites. The nucleic acid diversity of 529 rice germplasm resources and 107 common wild rice was analyzed. It was found that the average nucleic acid diversity of HAP gene was higher than that of the whole rice genome. The difference of Fst value between indica and japonica rice (> 0.25) indicates that the HAP family genes have strong population differentiation. OOs HAP2E / 2G / 2HAP2F- / 2HAP2F- / 2HAP3D- / 3EOHAP3D / 3EO-HAP3F3IHAP5A / 5B and Os HAP5D- 5E are duplicated and OS-HAP3G and OS-HAP3J are duplicated in series. Using 529 rice germplasm resources in the world, the correlation analysis of HAP gene and flowering traits of rice showed that only 4 genes were related to flowering, and HAP2 subfamily had no association with flowering. By analyzing the transgenic or mutant plants of 23 HAP family genes, it was found that 4 HAP genes regulated flowering, including the cloned Ghd8, and 3 of them were related to the formation of trimers between the HAP5 and HAP2HAP3 subfamily members. The results of yeast two-hybrid experiments showed that part of the proteins of the HAP2HAP5 subfamily were interacted by 1: Ghd8, and both Os HAP5A and Os HAP5B interacted with the HD1 protein, which provided a direction for the study of how Os HAP5B regulates flowering. It is also possible to study the molecular mechanism of the interaction between HAP5 and Ghd8 in regulating rice flowering. Rice grain shape has a wide variety in nature. Rice grain shape is an important agronomic character which determines the appearance quality and grain weight of rice. The main QTLs which affect grain length and grain weight at the same time were located by using the recombinant inbred line population of the large grain variety "Nanyang" and the small grain variety "Chuan7" as their parents. The candidate gene GL3.2 was identified by constructing near-isogenic lines, genetic analysis and map cloning. When the RNAi-GL3.2, which inhibited the expression of Chuan7 genotype, was transferred into Chuan7, it was found that the grain length of the transgenic material was 6.3mm, which was 0.3mm. longer than the 6.0mm of the wild-type Chuan7. By association analysis and sequencing, it was found that a key SNP leading to early termination of transcription resulted in differences in grain length between the two genotypes.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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