三亚湾壮实鹿角珊瑚组织内共附生固氮菌多样性分析  

Molecular diversity and phylogenetic analysis of nitrogen-fixing (nifH) genes in diazotrophic bacteria associated with corals in Sanya Bay

作　　者：陈蕾[1,2,3] 张燕英[1,2] 董俊德[1,2] 凌娟[1,2,3] 潘虎[1,4] 张偲[1] 龙丽娟[1] 

机构地区：[1]中国科学院南海海洋研究所海洋生物资源可持续利用重点实验室,广州510301 [2]中国科学院南海海洋研究所海南热带海洋生物实验站,三亚572000 [3]中国科学院研究生院,北京100049 [4]湖南农业大学生物科学技术学院,长沙410128

出　　处：《科学通报》2013年第17期1596-1603,共8页Chinese Science Bulletin

基　　金：国家科技支撑计划(2009BAB44B03);国家高技术研究发展计划(2012AA092104);国家自然科学基金(41006069;40676091);中国科学院知识创新工程重要方向性项目(KSCX2-EW-G-12)

摘　　要：以海南省三亚鹿回头珊瑚礁自然保护区中一种常见的壮实鹿角珊瑚(Acropora robusta)为研究对象,用PCR-RFLP方法研究其组织内共附生固氮微生物固氮酶nifH基因的多样性;用TaqⅠ和HinfⅠ这两种限制性内切酶对所获得的196个阳性克隆子进行RFLP分析,得到24个不同的OTUs(operational taxonomicunits),文库覆盖率为87.65%.利用Clustal W与Mega软件对nifH基因构建的系统进化树聚成了3大类:变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和绿菌门(Choloria),大部分的文库序列(70.83%)与变形菌门的α亚簇和β亚簇具有较高的相似性,其中α-变形菌簇占66.67%,是本文库中的优势种;次优势类群为厚壁菌门,占整个文库OTUs的16.67%;绿菌门仅占12.5%.此外,在这株珊瑚样本中获得的24个克隆子中有13条nifH基因序列与它们最相近序列的相似性小于97%.综上结果表明该壮实鹿角珊瑚组织内的共生固氮菌的具有较高的多样性,而且还可能含有大量未被培养的新的固氮基因.This study aimed to investigate the molecular diversity and pertbrm phylogenetic analysis of nitrogen-fixing bacteria associated with the coral tissues of Acropora robusta in the Luhuitou fringing reef of Sanya Bay, Hainan Province, China. To measure the nitrogen-fixing gene （nifH） fragment, diverse PCR products were analyzed by cloning, restriction fragment length polymorphism （RFLP） analysis and sequencing. The results showed that 196 clones and 24 different RFLP patterns were obtained from the sample, which were digested by the restriction enzymes TaqI and HinfI. The clone library coverage was 87.65%. The phylogenetic tree was constructed using Clustal W and Mega software. A total of 24 sequences could be subdivided into 3 clusters in the phylogenetic tree, and the majority （70.83%） had high sequence similarity to a-proteobacteria and fl-proteobacteria, a-proteobacteria （66.67%） were the significant dominant group. The second largest group of sequences found was most closely related to the bacterial phylum Firmicutes （16.67%） with four sequences being representative of this division. Additionally, three sequences （12.5%） were obtained from the phylum Chlorobia. Thirteen nifH gene sequences had a similarity of less than 97% to the closest relatives known in the National Center for Biotechnology Information （NCBI） database. This result demonstrated that there is a high diversity among the N2-fixing bacteria and that they contain a large number of unstudied nitrogen-fixing genes associated with the reef building coral Acropora robusta, in Sanya Bay, South China Sea.

关 键 词：壮实鹿角珊瑚 固氮菌 固氮酶基因nifH PCR-RFLP 多样性 

分 类 号：Q938.8[生物学—微生物学]