分子伴侣在古菌多样性和进化研究中的应用

Limitations in culturing efficiencies have lead microbiologists to rely on molecular techniques to investigate the composition of microbial communities. Despite the utility of the 16S rRNA gene, there are well-documented shortcomings in assessing microbial diversity and for phylogenetic analysis. For example, the slow rate of 16S rRNA evolution results in nearly identical sequences for closely related organisms, reducing the resolution of phylogenetic trees and making differentiation of these taxa difficult. An alternative to the 16S rRNA gene has been the utilization of conserved protein-coding genes. Class II chaperonins in Archaea, also denoted as TF55 or thermosomes has been revealed in all archaeal species. The thermosome gene is an appealing target as its bacterial homologue, cpn60, has been demonstrated to be an excellent target for species detection, identification and quantification of individual bacterial species and strains as well as the metagenomic characterization of complex microbial communities. Thermosome gene sequences were more diverse than either of the other targets providing a higher resolution description of the archaeal community. So this research focused on archaeal chaperonin gene used as DNA barcode to anylyze extremophiles diversity and ecological distribution. In addition,in Archaea, organisms possess one to five parologous thermosome genes, mainly three,giving rise to alpha,beta and gama subunits. The multiple subunit nature of the thermosome genes resulted in distinct clustering of alpha, beta and gama subunits within the phylogenetic tree. This phenomenon, illustrating the recurrent paralogy of the thermosome, which are the result of the prespeciation and postspeciation duplications that the chaperonin undergoes within archaeal genomes.Analysis of archaeal chaperonin phylogenetic relationship would provide clues of how multiple subunits thermosome evolved.

分子伴侣作为生物细胞蛋白质折叠和组装的关键系统广泛存在于所有生物细胞中,该分子具有高度保守性；古菌广泛栖息于各种极端环境中，在生命起源和地球物理化学循环过程中扮演着极其重要的角色。而目前对古菌，尤其是极端环境古菌的群落结构、多样性、生态分布及分类地位研究还存在技术性问题，传统的16S rRNA分析方法不足以反映古菌群落组成的全貌，以分子伴侣cpn60基因的部分序列作为DNA条形码进行古菌多样性分析将提高近缘物种的分辨率，发现极端环境中的隐存物种。此外，古菌分子伴侣是由一到五种亚基组成的大分子复合体，通过古菌分子伴侣基因系统发育树的构建和分析可以解析分子伴侣多亚基形成的原因和先后顺序，为阐明真核生物分子伴侣多亚基的进化及功能上的协同作用奠定基础。本项目将开辟环境微生物种群多样性和分布研究的新的研究方法，并且本项目的实施将为多亚基生物大分子复合体的进化研究提供必要的资源和技术储备。

分子伴侣作为生物细胞蛋白质折叠和组装的关键系统广泛存在于所有生物细胞中，该分子具有高度保守性；古菌广泛栖息于各种极端环境中，在生命起源和地球物理化学循环过程中扮演着极其重要的角色。本研究采集了火山热泉水样、西南印度洋深海沉积物样品、沤麻池碱性废水水样等特殊极端环境样品，利用古菌分子伴侣特异引物对其中的古菌伴侣素基因序列进行了扩增，并对扩增得到的序列进行了系统发育学分析。研究发现火山热泉中的优势古菌为泉古菌门古菌，其伴侣素含有alpha、beta、gamma三种亚基，且alpha亚基与gamma亚基聚为一支；深海样品中的优势古菌为奇古菌门，其伴侣素也包含三种亚基，且各自聚为一支；同一物种来源的伴侣素不同种亚基间的基因差异大于种间的同种亚基间的基因差异，进一步阐明了古菌伴侣素作为二型分子伴侣的多亚基特性和进化特点。与此同时，积累了极端环境来源的伴侣素基因的几百条新序列，极大丰富了环境未培养微生物伴侣素基因资源库。此外，通过伴侣素特异抗体和免疫杂交的实验证实了泉古菌门古菌Acidianus tenchongensis的伴侣素部分分布在细胞表面，提示可以通过伴侣素蛋白特异性抗体识别并分选极端环境特定古菌亚群，为极端环境来源的古菌的基因资源挖掘奠定了基础。本项目建立了环境微生物种群多样性和分布研究的新的研究方法，本项目的实施将多亚基生物大分子复合体的进化和功能研究提供了必要的基因资源和技术储备。