基于核酸DNA/RNA同位素示踪技术的水稻土甲烷氧化微生物研究
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

国家自然科学基金项目(41501276,31270147)和中国科学院战略性先导科技专项(B类)(XDB15040000)资助


The Application of Biomarker Genes for DNA/RNA-Stable Isotope Probing of Active Methanotrophs Responsible for Aerobic Methane Oxidation in Six Paddy Soils
Author:
Affiliation:

Fund Project:

Supported by the National Science Foundation of China (Nos. 41501276, 31270147) and the "Strategic Priority Research Program" of the Chinese Academy of Sciences (No. XDB15040000)

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    稳定性同位素示踪复杂土壤中微生物DNA/RNA的技术难点是13C-DNA/RNA的鉴定。本研究针对我国六种典型水稻土,利用稳定性同位素13CH4示踪活性的甲烷氧化菌,超高速密度梯度离心获得不同浮力密度DNA/RNA后,以甲烷氧化菌独有的pmoA功能基因和16S rRNA特异基因作为分子标靶,通过半定量凝胶电泳技术评价了特异基因作为分子标靶判定13C- DNA/RNA的可行性,进一步利用克隆文库技术研究水稻土中的活性甲烷氧化菌群落结构。结果表明:甲烷氧化菌功能基因pmoA作为分子标靶,能够准确鉴别13C-DNA,而甲烷氧化菌特异的16S rRNA基因则能较好地区分12C和13C标记的RNA,但13C-RNA中的非目标微生物污染高于13C-DNA示踪技术。进一步以13C-DNA和13C-RNA为模板,分别构建了pmoA和16S rRNA基因的克隆文库,系统发育分析表明I型菌主导了土壤甲烷氧化过程,其中江西鹰潭和黑龙江五常土壤中活性甲烷氧化菌全部属于Ia型,而四川资阳、浙江嘉兴、江苏常熟和江都土壤中Ia型和Ib型甲烷氧化菌均有发现,并且后者比例较低。这些结果表明分子标靶基因能够有效判定复杂土壤中的甲烷氧化菌13C-DNA/RNA,在DNA和RNA水平的结果基本一致,我国典型水稻土中活性甲烷氧化菌可能存在一定的地理分异规律。

    Abstract:

    Rice fields are major source of atmospheric methane (CH4). However, 30%~90% of CH4produced in paddy soils is oxidized by methanotrophs before it escapes to the atmosphere. China holds the largest rice production in the world, but it remains largely unknown about the active methane oxidizers in paddy soils. In this study, soil microcosms of six paddy soil incubated with 13CH4 were constructed to assess active methanotrophs by tracing the isotopically labeled 13C-DNA/RNA. Six paddy soils collected from Yingtan City of Jiangxi Province (YT), Ziyang City of Sichuan Province (ZY), Jiaxing City of Zhejiang Province (JX), Changshu City of Jiangsu Province (CS), Yangzhou City of Jiangsu Province (YZ), and Wuchang City of Heilongjiang Province (WC), were incubated with 400 µmol-1 L labeled 13CH4 or unlabeled 12CH4 to determine aerobic methane oxidation kinetics. The destructive sampling was conducted when 400 µmol-1 L CH4 was consumed. 13C-DNA and 13C-RNA were obtained through ultracentrifugation of total DNA and RNA, respectively. Clone library of pmoA genes from 13C-DNA and 16S rRNA genes from 13C-RNA were constructed to analyze composition of active methanotrophic community. After ultracentrifugation of total DNA and RNA, the agarose gel electrophoresis of pmoA gene amplicons and methanotrophic 16S rRNA reverse transcription amplicons from the fractionated DNA and rRNA, respectively, were performed, indicating the incorporation of 13C-substrate into methanotrophs during the aerobic methane oxidation. DNA-SIP and rRNA-SIP each have their advantages. In contrast to DNA, the incorporation of labeled substrate into rRNA is much faster, and a greater unspecific background of ‘heavy’ nucleic acid was observed in ‘heavy’ fractions in rRNA-SIP than DNA-SIP, indicating the more efficient separation for DNA. The separation of differentially labeled rRNA was effective, however, it was not as quantitative as for DNA. This resulted in a greater unspecific background of ‘heavy’ rRNA in ‘light’ fractions, which may be caused by their strong tendency to form secondary structure. Phylogenetic analysis of pmoA gene from total DNA of background paddy soils indicated that dominant methanotrophs in situ were type II in six paddy soils. It may be explained by the fact that type II methanotrohs can be better adapted to oligotrophic environments. Interestingly, consistent results were obtained from both clone libraries of pmoA genes from 13C-DNA and methanotrophic 16S rRNA transcripts from 13C-RNA, indicating that type I methanotrophs dominated active aerobic methane oxidation in the six paddy soils. All type I was composed of type Ia in YT and WC sample, whereas type I was composed of Ia and Ib in ZY, JX, CS and JD sample. The fast growth found for type I methanotrophs are in agree with a r-strategy lifestyle. Sufficient available nutrient e.g. CH4 may be prerequisite for the proliferation of type I methanotrophs. Phylogenetic analysis of pmoA gene from 13C-DNA and 16S rRNA transcript from 13C-rRNA revealed that the active methanotrophs responsible for aerobic methane oxidation in six paddy soils were type I. The results indicated that methanotroph-specific 16S rRNA and pmoA genes can be of great help for identification of 13C-DNA/RNA from methanotrophs grown on the labeled substrates.

    参考文献
    相似文献
    引证文献
引用本文

郑 燕,贾仲君.基于核酸DNA/RNA同位素示踪技术的水稻土甲烷氧化微生物研究[J].土壤学报,2016,53(2):490-501. DOI:10.11766/trxb201509020090 ZHENG Yan, JIA Zhongjun. The Application of Biomarker Genes for DNA/RNA-Stable Isotope Probing of Active Methanotrophs Responsible for Aerobic Methane Oxidation in Six Paddy Soils[J]. Acta Pedologica Sinica,2016,53(2):490-501.

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2014-03-03
  • 最后修改日期:2015-11-08
  • 录用日期:2015-12-17
  • 在线发布日期: 2015-12-17
  • 出版日期: