【作者】 刘占林；

【导师】 洪德元； 王晓茹； 张大明；

【作者基本信息】 中国科学院研究生院（植物研究所） ， 植物学， 2002， 博士

【摘要】 被子植物的rRNA基因已经得到深入研究。二倍体被子植物一般拥有1-4对18S-5.8S-26S rDNA位点和1-2对5S rDNA位点。作为特殊的多基因家族成员，rDNA会受均一化力(homogenizing forces)的作用，通过基因转换、不等交换等机制，形成基因的致同进化(concerted evolution)。长期以来，我们一直认为动植物rDNA致同进化水平很高，各种拷贝的序列几乎完全一致，因此可以直接应用PCR测序的方法进行分子系统学研究。但是在裸子植物中由于研究资料的匮乏，使我们对裸子植物rDNA的变异模式了解甚少。松属植物作为裸子植物的最大类群，它的rDNA变异和进化有何特点、与被子植物是否相同，是这个重要类群的进化研究中目前尚未解决的问题。本文的研究内容从三个方面进行： (1)rDNA的染色体定位 目前，松属的18S-5．8S-26S rDNA的染色体定位研究只包括5种植物，其中的3种同时涉及到5S rDNA定位。这些研究结果表明，不同种存在相异的rDNA位点数目，甚至不同的个体的rDNA位点均有变化。其共同点是，18S-5．8S-26S rDNA位点数平均较被子植物多，5S rDNA除Pinus radiata外，在其它种里则与被子植物相似。这种现象是松属或裸子植物的共同特征，亦或是特例呢?有限的研究限制了对裸子植物rDNA的了解。本研究的目的之一就是研究松属植物rDNA的染色体空间分布特征，希望借此了解松属植物间的关系，比较裸子植物和被子植物rDNA在染色体组水平的差异。 (2)5S rDNA的分子进化 5S rDNA的序列水平的进化研究在松属中尚属空白。5S rDNA在染色体数目上没有显示裸子植物与被子植物的差异，是否意味着松属乃至裸子植物的5S rDNA也同被子植物一样——致同进化完全，序列高度一致呢?利用克隆测序方法对松属植物5S rDNA的研究无疑是有开创性的工作，可以探讨裸子植物的5S rDNA的进化机制和种间关系。 (3)杂种基因组研究 杂交物种的起源演化是当前生物学研究的热点，通过杂种基因组的研究，可以了解杂种的的基因组构成，组织方式和进化历史，探讨杂交事件对成种过程的影响及意义。这项研究涉及到高山松、云南松和油松。之所以采用这三种植物，因为等位酶、cpDNA和mtDNA证据证明高山松为油松和云南松的自然杂交种。但这些证据不足以反映杂种核基因组的重组特征和构成及其进化规律。我们利用rDNA-FISH、5S rDNA和基因组原位杂交分析三种松树间的基因组关系，为揭示高山松的进化机制和历史提供新的依据。 本项研究得到以下结果： 一．rDNA荧光原位杂交(FISH) 通过对华山松和白皮松两种单维管束亚属植物及油松、云南松、高山松、马尾松和南亚松等五种双维管束亚属植物的18S rDNA与5S rDNA的荧光原位杂交，结果表明： (1)裸子植物的18S rDNA位点数目明显多于二倍体被子植物。其中主要位点数目，油松有7对，高山松5对，云南松8对，马尾松10对，南亚松6对， 白皮松3对，华山松10对，平均在7对；另外，部分松树还存在弱位点。无论 强弱位点都有部分存在于染色体的着丝粒区，除了赤松 （Pinu dens伽raX在其 它松科植物中并没有发现这种现象。究竟是基因转移的结果或该位点是 18s riZNTA的原始起源位置还有待确证。 （2）SS fi3：NA位点相对变异较小，与被子植物相当。除了华山松 SS d3NA 有旱对位点，马尾松只有1对位点外，其它松树的SS riZNA位点数目均为2对，、并且在双维管柬亚属植物中有一对属于弱位点。 （3）两种 fi3NA存在不同连锁模式。双维管束亚属植物中，SS与 18s rDNA 连锁在同一染色体的同一臂或两条臂上。在同一染色体臂时，18s fi3NA在臂的 远端。单维管束亚属植物的SS与185 d7NA或连锁于同一染色体的同一臂上， 或分别处于不同染色体。前一情况，SS n3NA位于臂的远端。据此可以说明两个 亚属的rDNA结构在染色体组水平的很大分化。 （4）松属植物的关系及高山松核型特征。由于SS与18s n3NA连锁关系的 不同，可以将单维管束亚属和双维管束亚属分开。各亚属的不同物种可以依据杂 交位点的多少、位置、信号强弱构成的核型图加以区分，并且构成一定的系统关 系。杂交起源的高山松在染色体组上，表现出对油松和云南松两亲本不同染色体 特征的分别继承与重组，并产生独有的特征。其＊同源染色体之一 18s rDNA位 点的缺失，可能是染色体重组的痕迹。 二．SS if7：NA的序列变异与分子进化 利用分子克隆和DNA测序分析了油松、云南松、马尾松、白皮松和不同遗 传背景的高山松居群的SS iLNA基因序列变异及基因进化规律，得到以下主要 结果： （1）SS rDNA的结构特征。双维管束亚属植物长度在 658刁28 hp，白皮松则 为 49952 hp。长度差异体现在基因间隔区，而基因区极端保守，“基本为更多还原

【Abstract】 Ribosomal RNA genes (rDNA),a group of tandem repetitive sequence,are well-studied in angiosperms. Usually,1 - 4 pairs of 18S-5.8S-26S rDNA sites and 1-2 pairs of 5S rDNA sites are found in diploid genome of angiosperms. As a special type of multigene family,rDNA undergo concerted evolution by homogenizing forces through processes such as gene conversion,unequal crossing-over. For a long time we assumed strong concerted evolution in rDNAs of various animal and plant species and direct PCR-sequence approach has been widely used in molecular systematic research. However,the variation patterns of rDNA in gymnosperms are little known because of the limited sequence data. In Finns,18S-5.8S-26S rDNA localization were reported for five species,and 5S rDNA sequences were obtained for only three species. Till today there is no report on molecular evolution of 5S rDNA in Pinus. Thus,the rDNA variation pattern and organization in gymnosperm genomes are the primary focus of the present project. The evolutionary implication of our results for genome structure,gene evolution,and speciation inferences are discussed.In this study,fluorescence in situ hybridization (FISH) was used to localize rDNAs on chromosomes in seven Pinus species in order to reveal the rDNA organization. 5S rDNA cloning and sequencing were carried out to analyze the 5S rDNA variation pattern. Genomic in situ hybridization (GISH) was performed for better understanding of genomic relationship among closely relative pines. Among the species selected,P. densata is a putative hybrid between P. tabulaeformis and P. yunnanensis. The analyses of these three types of data in the hybrid and its parents could provide new information on genomic composition and evolutionary mechanisms of the hybrid. The main results are the followings:1. rDNA FISHChromosomal localization of 18S and 5S rDNA was carried out for two soft pines (Subgenus Strobus) and five hard pines (Subgenus Pinus) using FISH. The number of major 18S rDNA sites is generally much more than that in angiosperms and varies markedly among pines. There are seven pairs of 18S sites in Pinus tabulaeformis,five in P. desata,eight in P. yunnanensis,10 in P. massoniana,six in P. latteri,three in P. bungeana,and ten for P. armandi. Furthermore,weak signals are found on the centromeres of many chromosomes. Unlike 18S,pines have 1-2 pairs of 5S rDNA sites,except for P. armandi that has four pairs. Each pine could be discriminated by its rDNA FISH karyotype,although most of these pines cannot be distinguished by traditional karyotype analysis.The two types of rDNAs has different linkage pattern. In diploxylon pines,18S and 5S rDNA usually locate on the same or opposite arm of the same chromosome. 5S is more adjacent to the centromere if both rDNAs are on the same arm. In haploxylon pines,18S and 5S locate either on the same arm of the same chromosome or on different chromosomes. In the former situation,5S is near the telomere. This pattern shows the clear divergence of the two subgenera. At the same time,thedifferentiation of rDNA sites on chromosomes among pines correlates well to their phylogenic positions in Pimis reconstructed with other molecular data. P. densata resembles its parents by combining patterns characteristic of each parent as well as shows new features resulting possibly from recombination and genome reorganization.2 Heterogeneity and evolution of 5S rDNAPattern of intragenomic and interspecific variation of 5S rDNA hi five pines (including subgenus Strobus and Pinus) were studied through cloning and sequencing multiple 5S rDNA copies The length of 5S rDNA unit is 658-728 bp hi diploxylon pines while 499-521 bp hi the haploxylon P. bungeana. The conversed 120 bp gene region contains an intragenic control box related to the gene tanscription. The loops,identified in the secondary structure,are generally more conserved than stems. However,marked high transition/transversion was found on loop E. This could be caused by the presence of pseudogenes. In the spacer region,elements rel更多还原