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题名	滇金丝猴细菌人工染色体文库构建及Neurotrypsin基因的分子进化研究
作者	徐怀亮
学位类别	博士
答辩日期	2004-07
授予单位	中国科学院研究生院
授予地点	北京
导师	宿兵
关键词	细菌人工染色体 染色体定位 荧光原位杂交 滇金丝猴 分子进化 纯化选择 功能限制 灵长类
其他题名	Construction of Bacterial Artificial Chromosome Library of Yunnan Golden Monkey and Molecular Evolutionary Study of Neurotrypsin Gene
中文摘要	本文包括两方面有关灵长类的研究工作。一是构建了一个非人灵长类（滇金丝猴）的人工染色体文库（BAC文库），为进一步研究灵长类的基因组进化奠定了一定的基础，二是对一个认知相关基因neuotrypsin在灵长类中的分子进化进行了详尽的研究，以期从分子进化角度认识该基因在灵长类高级认知功能发展中所起的作用。滇金丝猴（Rhinopithecusbieti），一个处于严重濒危状态的旧大陆猴，中国特产灵长类之一。在系统发育上它占据着旧大陆猴与小猿的中间地位，是研究灵长类进化的一个重要物种。，在本研究中，我们构建了一个滇金丝猴的细菌人工染色体文库（BAC文库）。该文库含有136320个BAC克隆，平均插入片段大小为148kb，小片段（50-100kb）所占的比例为2.74％，非重组克隆仅占2.67％。假定金丝猴与亲缘关系较近的灵长类有着相似的基因组大小，该文库至少有6倍的基因组覆盖率。对随机选取的BAC克隆进行末端测序，我们获得了201个序列标签（STS）。通过荧光原位杂交（FISH）技术，139个经末端测序的BAC克隆被精确定位到滇金丝猴的染色体上。荧光杂交实验还表明了人和金丝猴染色体产存在着高度的同线保守性。在人类基因组数据库中的Blast搜寻的结果显示染色体上的克隆数目与染色体大小呈很好的相关性，表明该文库克隆比较均匀地覆盖了滇金丝猴的基因组。该金丝猴文库及所定位的克隆将会成为非人灵长类的比较基因组研究和大规模基因组测序的一个宝贵的资源。Neurotrypsin是与个主要在脑中表达的，同神经发育和认知功能相关的胞外丝氨酸蛋白酶。它在人体中发生的突变与常染色体上隐性的非综合性精神发育迟缓（MR）紧密相关。我们通过对n个非人灵长类，包括大猿、小猿、旧大陆猴和新大陆猴neurotrypsin的编码区的测序，研究了neurotrypsin在灵长类中的分子进化。结果显示出，在灵长类进化过程中，neurotrypsin保持着由纯化性选择（负沟选择）所致的强烈的功能限制，这暗示着neurotrypsin在灵长类的认知发展中起着关键的功能性作用。进一步的分析表明，纯化性选择实际上只作用于neurotrypsin的介导其结合到其它细胞表面或胞外蛋白质的SRCR功能结构域上。另外，通过灵长类和其它哺乳动物目的比较，我们还发现，在鼠neuotrypsin中，一个人SRCR结构域（外显子2和3）的缺失是由于在鼠科动物中所发生的片段丢失事件所致。
英文摘要	In this paper, two studies on primates were conducted. Firstly, a bacterial artificial chromosome (BAC) library was constructed, which provided a valuable resource for primate genomic study. Secondly, the molecular evolution of neurotrypsin gene was dissected, which provided insight into the development of high-order cognition function in primates at molecular level. he Yunnan snub-nosed monkey (Rhinopithecus bieti), one of the rarest and most endangered primates in the world, is indigenous to china. Based on morphological study, it occupies an intermediate position between the Old World monkeys and the lesser apes, therefore, a keystone species in understanding primate evolution. We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136,320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100- kb) is 2. 74%, and only 2. 67% non-recombinant clones were observed. Assuming-a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times genome-coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAG clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescent in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAG library. This library and the mapped BAG clones will serve as a valuable resource in comparative genoinics study and large-scale genome sequencing of onhuman primates. eurotrypsin is one of the extra-cellular serine proteases that are predominantly expressed in the brain and involved in neuronal development and function. Its mutations in humans are associated with autosomal recessive non-syndromic mental retardation (MR). We studied the molecular evolution of neurotrypsin by sequencing the coding region of neurotrypsin in 11 representative non-human primate species covering great apes, lesser apes, Old World monkeys and New World monkeys. Our results demonstrated a strong functional constraint of neurotrypsin which was caused by strong purifying selection during primate evolution, an implication of an essential functional role of neurotrypsin in primate cognition. Further analysis indicated that the purifying selection was in fact acting on the SRCR domains of neurotrypsin, which mediate the binding activity of neurotrypsin to cell surface or extra-cellular proteins. In addition, by comparing primates with three other mammalian orders, we demonstrated that the absence of the first copy of the SRCR domain (exon 2 and 3) in mouse and rat was due to the deletion of this segment in the murine lineage.
语种	中文
公开日期	2010-10-15
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6198]
专题	昆明动物研究所_比较基因组学
推荐引用方式 GB/T 7714	徐怀亮. 滇金丝猴细菌人工染色体文库构建及Neurotrypsin基因的分子进化研究[D]. 北京. 中国科学院研究生院. 2004.

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