玛珥湖好氧不产氧光合细菌pufM基因DNA和mRNA的定量及多样性分析

Genetic diversity and quantification of aerobic anoxygenic phototrophic bacteria in Hugangyan Maar Lake based on pufM DNA and mRNA analysis

摘要:

【目的】湖光岩玛珥湖是一类特殊的火山口湖, 它完全封闭, 地质年代久远, 尚未受人类活动的剧烈影响, 孕育着丰富而特殊的微生物种群。好氧不产氧光合细菌(AAPB)是以其在有氧情况下能行使光合功能而定义的一类专性异养细菌, 其生理生态特征独特, 进化年代久远, 在水生生态系统的上层水体中广泛分布。目前, AAPB在玛珥湖水体中是否有分布仍是未知。【方法】构建和比较夏季湖水1 m、5 m、12 m三个水层的总DNA和总RNA的AAPB光合中心合成的关键基因pufM的6个克隆文库, 并结合定量PCR技术, 分析了不同水层AAPB的分布、系统发育多样性及其在总细菌中的比重。【结果】6个文库覆盖率和稀释曲线显示样本初步揭示了各水层优势AAPB类群的多样性。BLAST核苷酸同源性介于80%?93%; 多样性指数表明, 湖光岩表层和底层多样性相当, 中间层最低, 总RNA的多样性高于总DNA。系统发育分析结果表明, OTU21?24所含的序列(占总序列的49.43%)与β-变形细菌的进化距离最接近, 是湖光岩玛珥湖的优势AAPB菌群。定量PCR结果显示1 m水层中AAPB在总细菌中的比重最高, 可达38.06%; 而5 m水层中AAPB所占的比重最低, 仅为0.85%; 12 m为9.54%。【结论】湖光岩玛珥湖孕育着丰富而多样的AAPB类群。

关键词:湖光岩, 玛珥湖, 好氧不产氧光合细菌, pufM, 多样性, 荧光定量PCR, 系统发育分析

Abstract:

[Objective] Maar lake is a special type of Crater Lake. Huguangyan Maar Lake was formed about 140 k?160 k years ago, fully closed, and has not yet been affected by human activities, where abundant and novel microbial species might dwell as reported previously. Aerobic anoxygenic phototrophic bacteria (AAPB) is a functional bacterial group with long evolution history in Earth possessing unique physiological and ecological characteristics. To date, our knowledge about AAPB distribution in Maar Lake is still blank. [Methods] Here, by constructing and analyzing six clone libraries of the photosynthetic reaction center pufM gene from total DNA and RNA, respectively, with 1 m, 5 m, and 12 m water layers in Huguangyan Maar Lake, and combining quantitative Real-time PCR, we studied AAPB’s distribution, phylogenetic diversity and the proportion in the total bacteria in different water layers. The results of coverage value and rarefaction curves of six libraries showed that AAPB diversity was sampled well for the purpose of revealing the diversity of main AAPB groups in each water layer. [Results] BLAST analysis showed that pufM sequences in Maar Lake were 80%?93% similar to public sequences. Diversity index indicated that the AAPB diversity in surface and deep layers was similar, whereas diversity in the intermediate layer was lowest. In view of total RNA and DNA data, pufM RNA diversity was higher than that of DNA. Phylogenetic and statistical analysis revealed that 49.43% sequences are fell into the OTUs 21?24 which were closely related to β-proteobacteria and represent dominated AAPB groups. Quantitative PCR results showed that the percentage of AAPB in total bacteria in 1 m water layer reached a highest value of 38.06%, whereas only 0.85% and 9.54% in 5 m and 12 m, respectively. [Conclusion] Huguangyan Maar Lake is occupied by rich and diverse AAPB groups.

Key words:Huguangyan Maar Lake, Aerobic anoxygenic phototrophic bacteria, pufM diversity, Q-PCR, Phylogenetic analysis