【作者】 彭海；

【导师】 吴先军； 汪旭东； 周开达；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 近年来，植物多倍体进化与基因表达研究已成为多倍体研究的前沿，取得的主要成果是多倍化后迅速发生了基因表达多态性分化，包括多倍体群体内不同单株间多态性表达与器官特异性表达变化。然而研究主要集中于小麦(Triticum)、棉花(Gossypium)、芸苔(Brassica)等偶数倍的人工异源多倍体，同源多倍体甲基化与基因表达变异研究仅有少数几个例子：如酵母(Saccharomyces cerevisiae)和拟南芥(Arabidopsis)等；自然多倍体中也仅少数几个最近起源的多倍体如大米草(Spartina)、婆罗门参(Tragopogon)和千里光(Senecio)，知道并能获得其起源亲本而对其进化与表达有所研究；自然发生的同源三倍体水稻DNA甲基化与基因表达变化研究几乎没有，然而同源多倍体与水稻多倍体都与进化相关，因而同源多倍体与水稻多倍体基因表达与进化的研究也不应该被忽视。 本研究中采用双胚苗水稻SARⅡ-628中自然发生的同源三倍体为研究材料，利用同裂酶MspⅠ和HpaⅡ分析了三倍化后甲基化变异；利用基因芯片、cDNA-AFLP与RT-PCR三种方法相结合分析了三倍化后基因表达变化(基因芯片分析中兼顾了单倍化后基因表达变化)。结果表明SARⅡ-628自然同源三倍化后当代甲基化与基因表达都迅速发生了不同程度的变异，取得主要结果如下： 一、甲基化变异分析：1)三倍化后甲基化程度变化不大，由17.24％变化为三倍化后的17.20％；2)29个甲基化变异位点中甲基化程度上升、下降与不定的比例分别为29.51％、47.54％和22.95％，甲基化程度下降多于上升；3)甲基化变异遍布于整个基因组且存在位点特异性。 二、芯片分析结果：芯片分析从两个角度进行，一是单倍体、二倍体和三倍体联合分析；二是对三倍化过程单独分析。联合分析主要结果如下：1)大部分染色体对倍性敏感程度不一样，即有些染色体上的位点在倍性变化时更容易发生表达变化；2)倍性敏感位点(倍性变化时表达发生变化的位点)存在成丛分布；3)倍性敏感位点在功能上分布不均衡；4)单倍化中表达变异频率高于三倍化但表达量与功能都差异不大；5)单倍化与三倍化中都有相当比例沉默与激活的极端表达调节类型且激活多于沉默，类似地，表达量上升也多于下降；6)单倍化与三倍化对同一基因有类似的影响，与基因组剂量效应不一致。三倍化过程中基更多还原

【Abstract】 In recent years, the researches on evolution and gene expression of polyploid plants are going front of polyploid research fields. The main result from those studies was the rapid gene expression differentiation after polyploidization, including polymorphic expression-change patterns among individuals and among organs. Those researches focused on synthetic allopolyploid with even ploidy such as wheat, corn, brassica et al. Only a few case were made on autopolyploid such as in yeast and Arabidopsis; things were the same when it came to natural polyploid except such several recent allopolyploids as Spartina, Tragopogon and Senecio; Few researches were conducted on DNA methylation mutation and gene expression change of rice natural-genesic triploid. However, both of autopolyploid and rice polyploid are related to evolution, which should not be ignored in the research of polyploid evolution and gene expression.Autotriploids, occurring naturally from SARⅡ-628 twin-seedling rice, was employed as research material in this study. Isoschizomers HpaⅡ and MspⅠ　 were employed to analyze methylation mutation during triploidization; Gene chip, AFLP-cDNA and RT-PCR were combined together to analyze gene expression change during triploidization （haploid expression was also included in chip analysis）. The results showed that rapid DNA methylation mutaion and rapid gene expression change emerged in M₀ generation after SARⅡ-628 natural autotriploidization. The main results are as follows:Methylation mutation analysis: 1) The variation of methylation level, from 17.24% to 17.20%, was trivial after triploidization; 2) Among the 29 methylation mutated sites, the ratios of increase, decrease and undecision for methylation levels were 29.51%, 47.54% and 22.95%, respectively, which suggested that increased sites of methylation level were more than deceased ones; 3) Methylation mutaion sites distributed on the whole genome and were site-specific.Chip analysis: The chip data were checked from two aspects. That is, associated analysis of haploid, diploid and triploid, and analysis only with triploid. The results of associated analysis were as follows: 1) Most of the chromosomes had different sensitivities to ploidy change. That is, sites on some chromosomes had more tendencies for expression change; 2) Some ploidy-sensitive sites clustered together on chromosome; 3) Ploidy-sensitive sites had an uneven function distribution; 4) Change frequency of haploid was higher than that of triploid but with similar expression change amounts and function classes; 5) There were quite a number of silent and activated sites during both haploidization and triploidization. Activated and increased sites were more that silent and decreased ones; 6) Against genome dose effect, there was similar effect on the same gene during both haploidization and triploidization.更多还原