贵州喀斯特地区碳酸盐岩表生古菌群落结构及多样性研究——以南江大峡谷为例

为了研究贵州喀斯特地区碳酸盐岩表生古菌群落结构的多样性，应用16S rDNA文库技术，对采集于贵州南江大峡谷的白云岩和石灰岩样品进行基因文库的构建和限制性片段长度多态性(RFLP)分析。用限制性核酸内切酶MspⅠ对两个文库中分别随机挑选的300个阳性克隆子进行酶切分型，白云岩和石灰岩16S rDNA基因文库各得到14和13个基因型，其覆盖率分别为95.4%和91.3%，香农指数分别为2.14和1.93。系统发育分析表明白云岩和石灰岩表生古菌克隆子全部归属于泉古菌门（Crenarchaeota），代表性克隆与GenBank数据库已有16S rRNA序列的相似性为96%～100%，且最高相似性序列均来源于土壤及岩石环境的未可培养古菌序列。 

Archaeal community structure and diversity of the carbonate rocks in karst regions,Guizhou:A case study of the Nanjiang canyon

Guizhou is one of the three largest karst regions in the world where vast expanses of karst develop well. The carbonate rock area is 13×104 km2, covering 73% of total land surface area of the province. Carbonate rock is very challenging for organisms to live in it owing to its features, such as arid environment, nutritional deficiency, and great temperature fluctuations. However, there are various microorganisms on its surface and in its cracks, which play a crucial role in biogeochemical cycle. The Nanjiang canyon is a typical karst canyon, which is praised as "Karst Ecosystem Museum". It is located in Kaiyang County of Guizhou Province, southwestern China. And there are a lot of exposed carbonate rocks in this area. In order to study the archaeal diversity of carbonate rocks in Guizhou karst areas, we investigated the archaeal community structure in dolomite and limestone rocks from Nanjiang canyon using 16S rDNA gene clone libraries combined with Restriction Fragment Length Polymorphism (RFLP) analysis. Genomic DNA of rock samples were collected using the UltraCleanTMSoil DNA Isolation Kit. PCR amplification of the archaeal 16S rRNA gene was performed using universal primers 27F and 958R. The PCR products were ligated into the pGEM-T Easy Vector SystemⅠand transformed into competent E.coli JM109 cells and then constructed the dolomite and limestone clone libraries. Randomly selected 300 positive clones from each clone library were identified by RFLP with restriction endonuclease Msp I. In total, 14 and 13 genotypes were obtained from dolomite and limestone clone libraries, respectively. The rarefaction curves indicate that the major part of the diversity in the clone libraries was covered. The coverage C values of dolomite and limestone clone libraries are 95.4% and 91.3%, and Shannon-Wiener indexes are 2.14 and 1.93, respectively. The diversity of indexes shows that the diversity of archaea is much lower than the bacterial and fungal diversity, and the archaeal diversity of dolomite is slightly higher than that of limestone. The phylogenetic analysis reveals that all clones from the two clone libraries are affiliated to the phylum Crenarchaeota. The similarities between the representative clone sequences from carbonate rocks and their closest sequences deposited in GenBank range from 96% to 100% and all of the matches are from uncultured archaeal clones. Meanwhile, most of the archaeal sequences are closely related to environmental clones from soil and rock environments.