【作者】 韩永华；

【导师】 宋运淳；

【作者基本信息】 武汉大学 ， 遗传学， 2003， 博士

【摘要】 本研究分析了玉米(Zea mays L.)及其近缘种基因组间重复DNA序列的同源程度和在染色体上的分布特征。对它们的单拷贝基因mirl和dad1进行了比较物理定位。鉴定了一种新的六倍体细胞类型的水生薏苡，并分析研究了它的起源。主要结果如下： 1．玉米在分类学上属于禾本科、玉蜀黍族的玉蜀黍属(Zea)。在玉蜀黍族中，摩擦禾属(Tripsacum)和薏苡属(COix)与玉蜀黍属的关系最近。因此玉米的近缘种主要指这三个属中的物种。在玉米和它的近缘种的基因组中重复序列占有很大比例。物种间重复序列的同源程度可以有效地反映它们亲缘关系的远近。一种新的通过基因组原位杂交进行的分子显带方法，即利用一个物种的基因组DNA作探针，对该物种或其它物种的染色体进行原位杂交，其杂交信号的检出程序是专门用于检出重复DNA顺序的。因此它是直观地显示不同物种间共享的重复序列在染色体上分布特征的一种简便方法。 本研究中，我们应用这一方法对摩擦禾属、薏苡属和玉蜀黍属的几个种进行了分析。结果表明，尽管三个属的供试物种之间保守的重复序列的杂交信号散在分布于每条染色体大部分区域，但杂交信号的强度比自杂交时要弱得多。这说明它们间重复序列的变异是明显的。我们分子显带所得到的杂交信号大体上表现为带状，可分为中间带(interstitial bands)、端粒带(telomeric bands)、着丝粒带(centromeric bands)和异染色质纽(knobs)。着丝粒和着丝粒两侧这一重复顺序密集区自杂交时被强而连续的信号所涂布，而属间杂交中仅有类似中间带的点状信号，表明不同属间这一区域的重复顺序是较不保守的。自杂交与属间杂交比较，端粒带和中间带信号的分布和强度相对一致，表明它们是比较保守的。异染色质纽重复顺序的保守性具有另外的特点。摩擦禾(TriPsacum dac州oides，Zn=72)异染色质纽的成分与供试的玉蜀黍属内几个种的异染色质纽的成分高度同源，而与慧该(Coixlacryma了obi，2n=20)异染色质纽的成分没有明显的同源性。这在一定的程度上反映，供试的三个属中，玉蜀黍属和摩擦禾属亲缘关系更为相近，而与慧该属的关系相对地远些。 2.染色体定量作图是近年来新发展的一项分子细胞遗传学技术，即通过计算机软件对染色体上着色深浅不同的区域或带纹进行定量分析，实现常规方法所难以达到的精确定量目标。通过这样的定量分析，可以大大提高染色体识别的准确度。例如，慧该中期的染色体长度差异不大，因而相邻染色体难以区分。而慧该前中期的染色体相对较长，经DAPI染料染色后染色体上显示明显不同的深染区和浅染区，深染区和浅染区分别相当于染色体上染色质纤维较浓缩和较伸展的区域。前中期染色体上深浅不一的染色区可以作为识别慧改特定染色体的重要标志。我们用MetaMorPh软件定量分析了惹该每条前中期染色体上(从短臂到长臂)DAPI荧光强度的变化，结合染色体的长度和臂比作为辅助参数，构建了慧该前中期染色体的定量染色体图。该定量染色体图不仅描述了每条染色体不同区域浓缩程度的不同，而且也反应出不同浓缩区域所占染色体长度的比例。因此该定量染色体图是实际的前中期染色体的一种直观模式，可作为识别慧该基因组中每条染色体的有力依据。 3.通过荧光原位杂交的方法，我们确定了55 rDNA和455 rDNA序列在玉蜀黍属内几个种和慧芭染色体上的物理位置。55 rDNA的定位结果表明，在玉蜀黍属中的栽培玉米(Ze am娜ssP.m弓心，Zn=20)、墨西哥玉米(Ze a mays ssP.mexicana，Zn=20)、二倍体多年生类玉米(Z ea diPloPerennis，2下20)和多年生类玉米(Z ea perennis，Zn二40)染色体的基因组中都只有一个位点，但在染色体上的物理位置有所不同。在栽培玉米和墨西哥玉米中，55 rDNA位置相同，均分布于第2号染色体长臂的近末端。而在二倍体多年生类玉米中55 rDNA则位于第五染色体短臂。多年生类玉米的55 rDNA位于在一对同源染色体的短臂上，其染色体序号尚难确定。455 rDNA的定位结果表明，在栽培玉米、墨西哥玉米和二倍体多年生类玉米的基因组中仅有一个455 rDNA位点，位于第六染色体短臂上的次级绕痕区。而在多年生类玉米的两对同源染色体短臂上的近末端区域都检测到455 rDNA的杂交信号，而且在一对染色体上的信号点总是比另一对染色体上的大。我们的实验结果为墨西哥玉米属于玉米种的一个亚种提供了进一步依据。 455 rDNA和55 rDNA序列在慧该染色体上的荧光原位杂交结果表明，尽管具有20条染色体的慧该是四倍体植物，但它的基因组中只有一个455rDNA和55 rDNA位点。455 rDNA序列位于惹该第2号染色体短臂上的次级缘痕区和随体上，55 rDNA序列位于第7号染色体长臂靠近着丝粒处，55 rDNA位点到着丝粒的百分距离是29.13士1.76。 4.玉米基因~了编码一种抗秋季粘虫(fal 1 armyworm)的半胧氨酸蛋白酶。利用RFLP作图mirj基因被定位在玉米第6染色体短臂上，但它在第6染色体短臂上的物理位置还不知道。在本实验中，我们以耐r1和455 rDNA为探针，通过双色荧光原位杂交技术确定了二z’r了基因在玉米中期和粗线期第6染色体上的物理位置。Sou更多还原

【Abstract】 In this study, homologous degree and distribution characteristics of repetitive DNA sequences were analyzed on the chromosomes of maize and its close relatives. The single copy genes mir1 and dad1 were also physically mapped onto the chromosomes of maize and its close relatives. A new hexaploid cyto-type of Coix aquatica was identified and its origin was analyzed. The main results are as follows:1. Maize belongs to genus Zea of tribe Maydeae in Gramineae. Genera Tripsacum and Coix are the close relatives of the genus Zea in the tribe Maydeae. Genomes of maize and its close relatives share their repetitive DNA sequences with a large proportion. Homologous degree of repetitive DNA sequences among different species reveals their evolutionary relationship in a certain degree. A new molecular banding technique has been developed through genomic in situ hybridization (GISH). In the GISH, a given genomic DNA was hybridized to chromosomes of their own or other species and signals of repetitive DNA sequences were exclusively detected. It is a simple approach for identifying distribution of the repeated sequences on chromosomes.Comparative studies were performed by molecular banding among several species of the genera Tripsacum, Coix and Zea. The results showed most of the corresponding chromosomal regions among the tested species share hybridization signals of the repetitive sequences. When a given genomic DNA was hybridized to their own chromosomes, the signals were the strongest. Whileit was hybridized onto the chromosomes of the other tested species, the signals were much weaker. It showed variation of repetitive sequences was obvious in different species. In the molecular banding, hybridization signals were banding-like in shape on the whole and could be divided into interstitial, telomeric, centromeric bands and knobs. For centromeric and pericentromeric regions, the signals were much stronger in the hybridization with their own genomic DNA than in that with other genomic DNA, while for interstitial and telomeric regions, the signals showed no much more differences in strength for the different genomic DNA probes. The results showed repetitive sequences on centromeric and pericentromeric regions were less conservative than those on interstitial and terminal regions in the tested genera. The knob signals were very strong and clear only for the hybridization of Tripsacum dactyloides (2n=72) with genus Zea instead of Coix lacryma-jobi (2n=20) with them. This provides evidence that the genus Zea is closer to the genus Tripsacum than to the genus Coix evolutionally.2. Quantitative chromosome mapping is a new developed molecular cytogenetic technique in recent years. It is undertaken by quantitatively analyzing the intensely and faintly stained regions or bands on chromosomes using computer software. Accuracy of chromosomal identification could be notably improved by the quantitative analysis. Mitotic metaphase chromosomes of C. lacryma-jobi studied cytologically have been proved to be hardly distinguishable due to their morphological similarity and the lack of distinct chromosomal landmarks. In contrast to the metaphase chromosomes, prometaphase chromosomes of C. lacryma-jobi were longer and showed obvious differences among intense and faint regions stained by DAPI. The intensely and faintly stained regions corresponded to more condensed and more dispersed chromatin fibers along chromosomes, respectively. The uneven stained regions can be used as key markers to characterize chromosomes of C. lacryma-jobi and facilitate identification of homologous chromosomes. Distributional DAPI stained patterns along each chromosome were quantified by MetaMorph software. Based on the data besides chromosomal relative length and arm ratio, the quantitative chromosome map was constructed. The chromosome map could illustrate not only the more distinctive chromosome features, but also fractional length of differential condensation regions. The quantitative chromosome map could provide essential information更多还原