【作者】 施雯；

【导师】 欧晓昆； 耿宇鹏；

【作者基本信息】 云南大学 ， 生态学， 2012， 硕士

【摘要】 DNA甲基化是植物基因组中广泛存在的一种表观遗传修饰,不仅在生物快速适应环境变化和表型进化过程中发挥着重要作用,而且在生物体发育和器官分化时参与调控基因表达。因此,DNA甲基化组不同于基因组,可能存在显著的器官特异性。然而,口前对DNA甲基化模式的个体间变异和个体内器官间变异的相对重要性却知之甚少,不利于在生态表观遗传研究中制定合理的采样策略。本研究以莲子草属的两种植物(喜旱莲子草Alternanthera philoxeroides和刺花莲子草Alternanthera pungens)为材料,采用扩增片段长度多态性(AFLP)和甲基化敏感扩增多态性(MSAP)技术,对硅胶干燥处理条件下的植物不同器官(叶VS茎)和不同发育时段(幼叶VS成熟叶)的基因组甲基化模式进行比较研究。实验所用的15个喜旱莲子草个体和5个刺花莲子草个体分别采自同一个自然种群。总共筛选了10对引物,分别扩增得到936个、772个位点。经GENALEX6.1软件分析数据,得到如下结果：(1)同一种群不同个体间DNA序列的遗传变异水平较低。AFLP分析显示,喜旱莲子草和刺花莲子草的遗传多态性位点分别为3.59%和4.80%,Nei遗传多样性指数(He)分别为0.012和0.024,Shannon多样性指数(I)分别为0.017和0.031。表明本研究所采集的喜旱莲子草和刺花莲子草的遗传背景比较一致,MSAP的条带变异主要源于DNA甲基化模式的差异。其中,刺花莲子草的变异水平略高于喜旱莲子草,可能是因为前者由种子繁殖而后者以无性繁殖为主。(2)同一个体不同器官间的DNA甲基化存在显著差异,高于个体间变异。MSAP分析显示,同一个体的不同器官(叶和茎)之间的DNA甲基化模式存在显著差异,并且器官间差异远高于个体间相同器官的差异。两个物种得到的结果基本一致。表明植物DNA甲基化模式存在不可忽视的器官特异性。(3)同一个体不同发育阶段叶片间的DNA甲基化也存在显著差异。MSAP分析显示,同一个体的幼叶和成熟叶之间的DNA甲基化模式存在显著差异。在喜旱莲子草中,不同叶片间的差异甚至高于个体间差异；在刺花莲子草中,不同叶片间的差异与个体间变异大致相当。(4)刺花莲子草的同一对叶片中的大叶和小叶DNA甲基化差异不明显。刺花莲子草具有特殊的异形叶：同一茎节的一对叶片形状相似,但一大一小。MASP分析表明,刺花莲子草的同一茎节上的大叶和小叶之间在DNA甲基化模式上没有明显差异。表明这种叶片大小的表型变异可能只受少数基因位点控制,并不伴随着系统性的表观遗传差异。(5)“硅胶干燥处理”对植物材料DNA甲基化的水平影响很小。将喜旱莲子草的同一个新鲜叶片沿中脉一分为二,一半用硅胶干燥后提取DNA,另一半直接提取DNA,进行平行检测。筛选7对引物进行选择性扩增后,发现两种处理的甲基化模式几乎完全相同。表明硅胶干燥不会对植物DNA甲基化产生显著影响。以上结果表明植物的不同器官、同一器官的不同发育阶段在DNA甲基化模式上均可能存在显著的差异。在今后的生态表观遗传学研究中,应该充分考虑植物DNA甲基化的器官和发育阶段特异性,尤其在采集野生植物种群时,其采样策略应该比常规的DNA序列分析更加有针对性,不能将不同器官的材料和同一器官不同发育阶段的材料混淆。同时,硅胶干燥是一种可接受的、安全的样品处理手段。更多还原

【Abstract】 DNA methylation, an epigenetic modification which exists broadly in plant genome, plays a significant role not only in coping with quick environmental changes and phenotypic evolution process, also in gene expression regulation during biological development and organ differentiation in organisms. Therefore, differed from genome, the mehylome is organ-specific. But by now, the importance of the DNA methylation variation among different individuals and organs is acquainted scarcely, which is disadvantageous for setting up reasonable sampling strategies during ecological epigenetic researches. In this study, we used Alligator weed(Alternanthera philoxeroides (Mart.) Griseb.) and Khaki weed (Alternanthera pungens) as the experimental materials, the technique of AFLP and MSAP were employed to detect DNA methylation patterns in different organs(leaves VS stems) and leaves of different development stages(young leaves VS mature leaves).15individuals of Alligator weed and5individuals of Alternanthera pungens were collected from the same natural population respectively. A total of936and772MSAP bands were scored from10primer combinations. The data analyzed by software GENALEX6.1, the research results were summarized as follows:(1) All samplings from the same population showed very little genetic variation.The results of AFLP showed that the percentages of polymorphic bands of Alligator weed and Khaki weed were3.59%and4.80%respectively. The Nei’s index of genetic diversity(He) and the Shannon diversity index(I) were close to zero at the species level that indicated very little genetic variation both in Alligator weed and Khaki weed, and the variation of MSAP bands was almost attributed to the difference of DNA methylation patterns. The genetic variation of Khaki weed was a little more than that of Alligator weed, and the reason might be that the former could propagate seeds while the latter propagate by cloning mainly.(2) There was variation of DNA methylation patterns between leaves and stems.MSAP results of different organs (leaves VS stems) from Alligator weed and Khaki weed showed notable difference of DNA methylation patterns between leaves and stems, that is, organ-specific. And furthermore, organic specificity of DNA methylation was much more significant than variation of the same organ between individuals in each population which indicated that the organic specificity of DNA methylation cannot be ignored in plants.(3) There was variation of DNA methylation patterns between young leaves and mature leaves.The DNA methylation patterns of young leaves were distinctly different from that of mature leaves in both Alligator weed and Khaki weed by comparison of MSAP results. The differences implied that dynamic changes really exist during biological development. Interestingly, the DNA methylation variation between two types of leaves was more than that inter individuals in Alligator weed, but the difference was almost the same no matter between leaves or individuals in Khaki weed.(4) The DNA methylation patterns were almost the same in small leaf samplings and big leaf samplings which from the same pairs of leaves.After two separate MSAP reactions on Khaki weed’s small leaf samplings and big leaf samplings from the same pairs of leaves, the results came out that despite the size of leaves the DNA methylation patterns were the same.between two leaves from one pair. Therefore this kind of phenotypic differences might not be caused by epigenetic variation.(5) Silica gel dyring treatment can impact plant DNA methylation patterns slightly.Each fresh leave sampling was separately hemisected firstly and MSAP was taken immediately on one half, the other was dried in silica gel antecedent to detecting. The results showed that DNA methylation patterns were approximately consistent between dry leaves and fresh leaves that suggested silica gel influence slightly on plant DNA methylation levels.In conclusion, the results obtained here provide new evidence of the DNA methylation specificity in development stages and organs of plant genome. Based on all the results, we suggest that the organic and developmental specificity of DNA methylation should be fully considered in ecological epigenetic researches, especially the sampling stratagies should be more elaborate than convertional analysis of DNA sequences when collecting plant materials in field. Meanwhile, silica gel drought is a kind of safe and acceptable treatment for DNA methylation.更多还原