【作者】 陈磊；

【导师】 张洪海； 马建章；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2010， 博士

【摘要】 作为现存食肉目动物中分布最为广泛的类群,犬科动物与人类生活有着千丝万缕的联系。在对犬科动物的研究过程中,消化生理学领域的研究具有举足轻重的地位,其中有关消化道菌群生态的研究近年成为学术界的热点话题。尽管目前对家犬肠道菌群的研究已经比较深入,但是狼的消化道菌群目前仍是个未知数。为了解狼的肠道菌群组成和多样性特征,本文第一部分通过构建菌群16S rRNA基因文库和进行系统发育分析,对采集自中国内蒙古自治区达赉湖自然保护区附近的3只健康成年狼的粪便菌群结构进行了分析。我们将3只狼的粪便样品分别构建16S rRNA基因文库,从中随机挑取550个克隆进行检测,共得到307个不同的非嵌合体的细菌16S rRNA基因序列。根据97%的序列相似性,这些序列可划分为65个临时分类单元(OTU),其中有17个OTU(代表着98个克隆的16S rRNA基因序列,占克隆总数的31.9%)与GenBank和RDPⅡ数据库中现有序列的相似度小于98%,为未分类、未鉴定的细菌。系统发育分析表明,狼粪便菌群共有5个细菌门组成,按照多样性高低依次是：Firmicutes (60% of total OTUs), Bacteroidetes (16.9%), Proteobacteria (9.2%), Fusobacteria (9.2%)和Actinobacteria (4.6%)。Clostridiales是狼粪便菌群中最大的细菌目,占据了OTU总数的53.8%。这一细菌目共由5个科组成,其中Lachnospiraceae是丰富度最高的一个科,其余四个科依次是：Ruminococcaceae, Clostridiaceae, Peptococcaceae和Peptostreptococcaceae。通过将本文研究结果与人类和家犬等其它哺乳动物肠道菌群结构进行比较发现,狼粪便菌群结构与其它哺乳动物基本相同,狼和家犬在Actinobacteria门和Fusobacteriales门的细菌组成和数量上存在较大差别。线粒体基因组是比较基因组学研究的理想工具。家犬(Canis lupus familiaris)的线粒体全基因组测序于1997年完成,至今已测序的犬科动物有四种：家犬、狼(Canis lupus)、郊狼(Canis latrans)和赤狐(Vulpes Vulpes)。本课题组于2008年对分布于青海省的西藏狼(Canis lupus laniger)的线粒体基因组进行了测序。然而,中国分布的其他种群的狼和其他犬科动物的线粒体基因组数据未见报道。因此,本文第二部分内容是应用Long-PCR和引物步移法,结合克隆测序法,对中国分布的豺、貉和内蒙古地区分布的狼的线粒体基因组进行测序,同时通过与已报道的犬科动物线粒体基因组进行比对分析,探讨犬科动物线粒体基因组组成和结构的异同,从而总结出犬科动物线粒体基因组的结构特点。经测序,蒙古狼、豺和貉线粒体基因组全长分别是16709 bp、16672 bp和16713 bp,三种犬科动物线粒体基因组均包含13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和1个非编码区。序列碱基的组成存在明显的A-T偏好性。tRNA基因中除tRNA-Ser (AGY)缺少双氢尿嘧啶(DHU)臂以外,其余均能折叠成典型的三叶草二级结构。在编码蛋白质基因的所有密码子中,以编码亮氨酸(Leu)的其中一个密码子(CTA)和编码异亮氨酸(Ile)的三个密码子(ATT, ATC, ATA)使用频率最高。大多数密码子的使用犬科各种动物高度一致,蒙古狼和西藏狼的某些起始和终止密码子与其他犬科动物存在差别。通过对4个属8个种(其中包括狼的三个亚种)犬科动物的线粒体基因组的碱基组成和基因结构进行比较发现,犬科动物线粒体基因组序列大部分区域高度一致,序列差异主要存在于非编码区的两翼区域(ETAS区和CSB区)。序列长度的差异主要由非编码区重复序列(RS-3)重复次数的差异所致。通过对犬科动物之间碱基替代情况进行分析发现,不同种犬科动物的同一功能部位碱基替代速率较为一致,同种犬科动物的不同功能基因的碱基替代率差异较大。13种蛋白质编码基因的碱基替代率也有明显不同,其中以L链编码的ND6基因的碱基替代率与其他蛋白质编码基因的差异最为显著。犬科动物线粒体基因组各功能部位中以tRNA基因的碱基替代率最低,说明tRNA基因在进化过程中高度保守。犬科动物系统发育地位的探讨在食肉目进化生物学研究中占有重要的地位。已有学者基于形态学和某些分子生物学数据进行过相关的分析,但由于测序方法的限制和数据库中基因序列的缺乏,未见基于线粒体基因组全序列的犬科动物分子进化的研究。作为本文研究的第三部分内容,我们在对蒙古狼、豺、貉线粒体基因组测序以后,应用所测得的蒙古狼和早期测得的西藏狼的线粒体基因组序列结合数据库中现有狼的亚种的数据,对这两个地区狼的系统发育地位进行定位。同时利用测得数据结合GenBnak中已有线粒体基因组全序列的数据,进行了犬科和犬型亚目的系统发育重建。基于12SrRNA+16S rRNA+H链上的12个蛋白质编码基因的联合数据的系统发育分析表明,在已报道的狼亚种数据中,西藏狼的分化时间最早,其次为阿拉伯狼(Canis lupus arabs),蒙古狼与欧亚狼(Canis lupus lupus)的系统发育地位最为接近。对犬型亚目系统发育分析发现,犬型亚目可明显划为Cynoidea和Arctoidea两大类群。犬科的4个属(犬属、豺属、狐属、貉属)可分为3个进化地位平等的分支,进化时间估算貉属最先从犬科中分化出来。最大简约法、最大似然法和贝叶斯法的推断结果均验证了除郊狼以外的其他犬属动物的单源性。拓扑结构所显示的狼和家犬的分化地位暗示着应将家犬作为狼的亚种进行划分。通过碱基替代率估算的家犬与狼的分化时间大约为1.56～1.92百万年以前。更多还原

【Abstract】 Canidae, as the most widely distributed taxa in Carnivora, is inextricably linked to our life. Among the studies on canine animals, the researches of digestive physiology play a very important role, and the study on the digestive tract microbial communities is becoming a much hotter topic in recent years. Although the microbial communities in the different segments of the intestinal tract of domestic dogs (Canis lupus familiaris) have been good characterized, to our knowledge, no molecular study has assessed the composition of the microbial communities in the intestinal tracts of wild wolves(Canis lupus). Therefore, the first purpose of the present study is to characterize the microbial diversity in the distal gut of a group of healthy wolves by using the approach of 16S rRNA gene sequences analysis. Fecal samples were collected from 3 healthy adult wolves and we created clone libraries for the samples from each wolf separately. A total of 550 clones were randomly picked from each library aND307 non-chimeric near-full-length sequences were finally used for subsequent phylogenetic analysis. With 97% sequence similarity, a total of 65 OTUs were defined. Seventeen OTUs (31.9% of total clones) showed less than 98% sequence similarity to existing 16S rRNA gene sequences in the GenBank and RDP databases, and may represent uncharacterized bacterial species. Five major phylogenetic lineages were identified:Firmicutes (60%), Bacteroidetes (16.9%), Proteobacteria (9.2%), Fusobacteria (9.2%) and Actinobacteria (4.6%). The majority of clones fell within the order Clostridiales (53.8% of total OTUs). It was predominantly affiliated with five families: Lachnospiraceae was the most diverse bacterial family in this order, followed by Ruminococcaceae, Clostridiaceae, Peptococcaceae and Peptostreptococcaceae. We compared our results with human beings and other mammals and got a similar composition among the microbial communities in the intestinal tracts of them. The differences of the intestinal flora between wolves and domestic dogs were mainly in the component and diversity of bacteria in the phyla Actinobacteria and Fusobacteriales.Mitochondrial genome as an extra-chromosomal genome is extensively used for testing hypotheses of microevolution, studying population structure and phylogenetic relationships at various taxonomic levels. Since the domestic dog’s mitochondrial genome was sequenced in 1997, there are four Canidae species whose mitochondrial genomes have been reported, they are Canis lupus, Canis familiaris, Canis latrans and Vulpes Vulpes. In 2008, we sequenced the mitochondrial genome of Tibetan wolves (Canis lupus laniger) captured from the Qinghai province of China. But the mitochondrial genomes of wolves in other regions of China have not been reported till now. So in the second part of this article, we sequenced the mitochondrial genomes of Mongolian wolf and other two species of Canidae in China, dhole and raccoon dog, by using the long and accurate polymerase chain reaction. We compared our results with the mitochondrial genomes of other canine animals for the purpose of detecting the common characteristic of the mitochondrial genomes of Canidae. Our results were as follows:The lengths of the mitochondrial genomes of Mongolian wolf, dhole and raccoon dog were 16709 bp,16672 bp aND16713 bp, respectively. The entire mitochondrial genome sequence contained 2 ribosomal RNA genes,13 protein-coding genes,22 transfer RNA genes aND1 non-coding region. A strong A-T bias was found in the mitochondrial genomes of the three canine animals. All the transfer RNA genes could be folded into the typical cloverleaf-shaped structure except tRNA-Ser (AGY), which lacked the dihydrouridine arm. The overall codon usages in the mitochondrial genome protein-coding genes were unification among canine animals. The frequencies of CTA (Leu), ATT (Ile), ATC (Ile), ATA (Ile) were higher than those of other codons. Some of the start or stop codons were different among wolves, dhole and raccoon dog. The comparison of the mitochondrial genome sequences of 4 genera (representing 8 canine species or subspecies) of Canidae exhibited a very high consistency among different canine animals. The differences of the Canidae animals were mainly found in the two peripheral domains (the ETAS domain and the CSB domain) of the control region of the mitochondrial genome. The difference in the length of the mitochondrial genomic sequences was mainly cause by the different number of the repeat units in the repeat sequence (RS-3). From the comparison of the substitution rate of the mitochondrial genomes we found that same gene in different animals had a similar substitution rate while different genes in the mitochondrial genome of the same animal had different rates. Different protein-coding genes also had different substitution rates and the ND6 gene expressed the most prominent difference to other protein-coding genes. Among all the mitochondrial genes, tRNA genes had the lowest substitution rate and implicated a slower evolution rate.The discussion about the relationships and the evolution mechanisms of the Canidae animals is another hot topic which has been considerable studied based on a lot of morphological data and various molecular data. But because of the limits of the sequence method and the spare of complete mitochondrial genome sequence in the database, there is no report about the phylogenetic relationship of Canidae based on the complete mitochondrial genome sequence. Thus, as the third composition of this article, we analyzed the phylogenetic relationship of different subspecies of wolves by using the concatenated data set of 12S rRNA +16S rRNA+12 heavy-strand encoded protein-coding genes and reconstructed the phylogenetic relationship of the suborder Caniformia by using 23 representative species, and pay a special attention on the evolutionary relationships of Canidae animals. Our result indicates that, within the subspecies datas of Canis lupus, Canis lupus laniger was polarized first, then was Canis lupus arabs. Canis lupus chanco and Canis lupus lupus were sister species, which had a similar phylogenetic position. Within the suborder Caniformia, there was a basal caniform divergence between Cynoidea and Arctoidea. The four genera in Canidae were divided into three clades with an equal phylogenetic position. From the estimates of divergence time of canine animals we found that the genus Nyctereutes splited first. The result of the maximum parsimony, maximum likelihood and Bayesian inferences all indicated that the genus Canis is a unilateral unit besides the Canis latrans. All the topology of the three inference methods indicated that Canis lupus familiaris should be defined as a subspecies of Canis lupus. The estimated divergence time of dog and wolf was 1.56～1.92 million years before present.更多还原