| 摘要: |
| 采用间歇产气试验方法对红树林淤泥中的混合菌群进行产氢菌群富集, 并对混合产氢菌群发酵产氢过程中发酵液的pH 值和氧化还原电位(ORP)的变化进行分析。此外, 在产氢速率最高时段,发酵液中菌群经过常规的基因组提取, 分别采用通用的梭菌属Fe-氢酶基因和16S rRNA 基因引物进行PCR 扩增, 扩增产物经变性梯度凝胶电泳(DGGE)分析。结果发现产氢菌群中至少有6 种, 而Fe-氢酶基因只含有一种。将Fe-氢酶基因的扩增片段切胶纯化之后, 经PCR 重新扩增、纯化, 克隆测序。NCBI 序列比对结果表明, 该片段序列与产气荚膜梭菌Fe-氢酶基因的序列相似性达99%。根据已知产气荚膜梭菌Fe-氢酶基因序列设计引物, 经两轮PCR 扩增获得Fe-氢酶基因全序列。混合菌群中Fe-氢酶基因GenBank 数据库的登录号为EU590683。此外, 采用Bioedit 和Mega2 软件构建了Fe-氢酶的NJ 系统进化树, 结果表明该Fe-氢酶基因与产气荚膜梭菌(Clostridium perfringens)聚为一类。推测的氨基酸序列与产气荚膜梭菌的相应序列相似性达97%—99%。PfamHMM 结构域查找结果发现, 此氢酶含有五个结构域, 分别为1 个2Fe-2S 铁硫簇结合区域、2 个4Fe-4S 结合区域、Fe-氢酶大亚基和Fe-氢酶小亚基。
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| 关键词: 红树林, 产氢菌群, 变性梯度凝胶电泳, Fe-氢酶基因, 序列分析 |
| DOI：10.11693/hyhz200904020020 |
| 分类号: |
| 基金项目:国家高技术研究发展计划(863 计划)资助项目, 2006AA05Z112 号; 天津市科技支撑计划重点项目, 07ZCGYSH03400 号; 青岛市科技计划项目, 07-2-3-7-jch 号; 973 计划前期研究专项项目, 2007CB216410 号。 |
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| CLONING AND SEQUENCE OF Fe-HYDROGENASE GENES OF CLOSTRIDIUM SP. FROM MANGROVE OOZE |
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ZHU Da-Ling1, WANG Guang-Ce2, PAN Guang-Hua1, QIAO Hong-Jin2, CAI Jin-Ling1
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1.Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and Technology;2.Institute of Oceanology, Chinese Academy of Sciences
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| Abstract: |
| Hydrogen-producing bacteria groups were studied for seeking a bioenergy source. The bacteria were sampled in mangrove ooze from Beihai intertidal zone Guangxi, South China, and were enriched in batch fermentation culture. The pH and ORP (oxidation reduction potential) values were measured. During the peak hydrogen production by the bacteria, Fe-hydrogenase gene and 16S rRNA gene of the bacteria was PCR amplified and analyzed in DGGE (denaturing gradient gel electrophoresis). Six operational taxonomical units (OUT) were obtained on the 16S rRNA gene but only one on the Fe-hydrogenase gene of Clostridium. The segment of Fe-hydrogenase gene was cloned and sequenced. NCBI blast result indicates that the segment of Fe-hydrogenase gene was 99% identical to that of C. perfringens. Thus, forward and reverse primers were designed according to the reported Fe-hydrogenase gene of C. perfringens. After two PCR amplifications, the full-length of Fe-hydrogenase gene was obtained. The accession number of this gene is EU590683. The sequence of this gene was arranged and analyzed with Mega software package having a phylogenetic tree built up in DNADist and Neighbor. The deduced amino acid sequence shared 97%—99% homology with the Fe-hydrogenase of C. perfringens. Five domains were found in the Fe-hydrogenase by PfamHMM domain search, namely, a 2Fe-2S binding domain, two 4Fe-4S binding domains, an Fe-hydrogenase large subunit, and an Fe-hydrogenase small subunit.
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| Key words: Mangrove, Hydrogen-producing bacteria group, Denatured gradient gel electrophoresis, Fe-hydrogenase gene, Sequence analysis |
