【作者】 熊立仲；

【导师】 张启发；

【作者基本信息】 华中农业大学 ， 分子生物学， 1999， 博士

【摘要】 杂种优势是生物界普遍存在的十分重要的生物学现象，而且在多种动植物中得到成功地利用。对杂种优势机理的研究已将近一个世纪，然而在许多方面仍是一个谜。水稻是杂种优势利用最成功的作物之一，近几年对水稻杂种优势的遗传学基础也有很深入地认识。本研究以杂种优势遗传基础研究较深入的水稻杂交组合为材料，通过检测亲本和杂种间基因表达差异以及DNA甲基化的差异，并结合所用材料的一些农艺性状杂种优势数据，从基因表达水平上探讨了基因差异表达或差异甲基化与杂种优势的关系。获得的主要结果如下： 1．首先以强优势组合油优63的剑叶为材料，用差异显示方法检测了亲本和F₁之间基因表达差异。表达差异主要分为：在亲本之一和杂种中表达（DMP₁或DMP₂），仅在两个亲本中表达（ABF₁）和仅在一个亲本或F₁中表达（UNP₁、UNP₂或UNF₁）。 2．以一套双列杂交组合（28个F₁，8个亲本）的剑叶为材料，用差异显示方法进一步分析了有表达差异的135条带在不同组合中上述6种差异带的数量。以差异带的数量为变量，与6个农艺性状（抽穗期、株高、生物学产量、穗粒数、千粒重和产量）的杂种表现、杂种优势和杂合性（数据为本课题组以前的研究结果）进行相关分析的结果表明：类似显性的差异表达的数量（DMP₁和DMP₂）与6个性状的所有数据都无关：亲本中特异表达（UNP₁和UNP₂）的数量与杂种优势或杂合性呈显著的正相关，而杂种中特异表达（UNF₁）的数量与杂种优势或杂合性呈显著的负相关。 3．分别以每条差异带对双列杂交组合中杂种的6个性状的杂种表现、杂种优势和杂合性进行显著性检验。有一关以上的差异带检测到对至少一个性状的一种或多种数据显著。其中在杂种优势和杂合性数据中显著的差异带占绝大部分，而且这些差异带绝大部分（70％）以带的缺失（表达受到抑制）一组为正效应。 4．进一步用65对引物，以油优63组合的剑叶为材料进行大量差异显示，共检测到1000多条差异表达的cDNA带，并回收了其中的326个（包括了各种差异表达类型）。回收这些cDNA带并克隆，一共获得384个克隆。 5．用高密度cDNA点杂交对上述方法获得的384个cDNA克隆进行了亲本和杂种间差异表达的鉴定。结果表明：40％的基因在亲本和杂种间存在明显的量上的表达差异。在每个时期，杂种相对于亲本既有表达增强的基因，也有减弱的基因。34％的基因在苗期叶片和剑叶之间存在明显表达差异，苗期具有表达优势的基因数目要多于抽穗期。 6．选用三个不同优势的杂交组合，对48个在油优63组合有显著表达差异cDNA克隆进行基因表达量的分析，结果表明，不同优势组合中一些基因的表达差异非常显著。 7.进一步选取存在显著的表达差异的。DNA克隆进行测序和染色体定位，己测序的34个克隆在数据库中既有高度同源的序列，但也有相当一部分没有找到同源序列;同源序列中，既有发育过程中十分重要的功能基因，也有一些调节基因。其中12个克隆被定位在染色体上。 8.普遍认为DNA甲基化与基因表达调控有关。为探讨DNA甲基化与杂种优势的关系，本研究使用了一种新的方法（甲基化敏感性扩增多态性，MSAP）去检测胞嗜陡甲基化（DNA甲基化的主要类型）。结果表明这种方法不仅效率很高，而且甲基化差异可以通过分子杂交加以证实。 9.基于这种方法检测到水稻基因组至少16.5%的CCGG位点存在胞嗜咙甲基化。甲基化位点具有明显的发育时期特异性。相对于亲本，杂种（汕优63）在某些位点上甲基化程度增强，而在某些位点上甲些化程度减弱。 10.用1司样的一套双列杂交组合的剑叶为材料，分析了杂种中甲荃化程度的变化和杂种优势的关系，结果表明:总体上甲荃化程度与杂种优势无关，而特异位点上甲荃化的改变对杂种优势有援河效应。而且还发现，不同位点处甲J否化减弱时，有的对杂种优势有正效应，而有的为负效应;在剑叶中，绝人部分有效应的位点上甲毯化增强（通常与丛因抑制有关）具有正效应。 11.部分搜异甲毯化片段的序列分析表明:这些序列可能来白墓因调控区域。 12.在同一套材料中，分别用差异表达和差异甲琏化去分析与杂种优势的关系所获得的结果能非常好地相互印证。 最后，基于控制特定农艺性状的功能基因在杂种和亲本之间不存在功能差异的认识，并结合本研究的一些结果，提出了一种调节因子互作假说，从表达调控水平上对杂种优势的产生机理进行了推理，并对深入开展杂种优势的生物学基础研究进行了讨论。更多还原

【Abstract】 Heterosis, a widely existing biological phenomenon, has been successfully exploited on breeding of many plants and animals. The efforts for exploring the mechanism of heterosis have lasted for nearly one century. However, many aspects remain unknown. Rice, one of the most important crops and showed greatest success on heterosis application, has deeply studied for the genetic basis of heterosis in recent years. Based on the rice materials with advanced studies on genetic basis of heterosis, this work was carried out for further dissecting the molecular basis of heterosis on gene differential expression level. The main results are as follows:1. By using differential display technique, the differential gene expression was compared between parents and hybrid in elite hybrid rice, Shanyou 63. Three types of differential expression patterns were detected: 1) expressed only in one parent and the hybrid (DMP1 or DMP2), 2) expressed only in two parents (ABF1), and 3) expressed only in one parents or only in the hybrid (UNP1, UNP2 or UNF1).2. In a diallel cross including 8 elite rice parental lines and 28 hybrids, totally 135 differentially displayed cDNA.bands were detected in flag leaf based on 6 primer combinations. The number of each differential banding patterns, which is variable in different crosses, was subjected to correlation analysis with hybrid performance, heterosis and heterozygosity of 6 important traits (heading date, plant height, biomass, seeds per panicle, 1000-grain weight and yield). No significant correlation was found between dominant-like patterns (DMP, and DMP2) and all the trait data. Significant positive correlations were detected between parent-specific expressions (UNP, and UNP2) and heterosis or heterozygosity. For hybrid-specific expressions (UNF,), however, significant negative correlations were found with the relationships to heterosis or heterozygosity for almost all the traits.3. Based on the presence or absence of specific cDNA band in different hybrids, T-test was performed for all the trait data. More than 60 cDNA bands were detected with significance on various trait data. For most significant bands, the heterosis or heterozygosity values of band-absent group were higher than that of band-present group.4. Using large-scale differential display analysis with 65 primer combinations in Shanyou 63 cross, more than 1000 differentially displayed cDNA bands were detected in flag leaves. Among them, 326 bands were recovered and cloned. Totally 384 cDNA clones were subjected for further confirmation.5. Expression analysis by high-density cDNA dot hybridization among the parents and hybrid of Shanyou 63 cross suggested that more than 40 per cent genes were differentially expressed. Compared to parents both increased expression and decreased expressions were found. A considerable number of genes were differentially expressed between flag leaves and seedlings.6. In three crosses of hybrid rice showing significant difference for heterosis, the expression analysis in seedlings also suggested significant difference of expression and some genes were differentially expressed with the same trend as the difference of heterosis.7. Homology search of 34 cDNA sequences showing significant differential expression suggested more than half of the sequences have no match in the database. For the homologous sequences, some are functional genes that are critical for plant development and some are regulatory genes. Out of the 34 cDNAs. 12 have been located on rice chromosomes.8. It is widely accepted that DNA methylation has involved in gene regulations. In this study, a new efficient method (Methylation-sensitive amplification polymorphism. MSAP) was developed and utilized to detect cytosine methylation in rice and this technique was further confirmed by southern analysis.9. Based on the MSAP technique, at least 16.5 per cent of the CCGG sites were cytosine methylated. Tissue-specific methylation was detected at many sites. A lot of sites with hypermethylation更多还原