| 摘要: |
| 采用PCR扩增、文库构建、限制性片段长度多态性分析、序列分析和系统学分析等方法,初步研究了夏季胶州湾上层海水浮游桡足类核糖体小亚基RNA基因(18SrDNA)约1.5kb片段的序列变异。从浮游生物混合DNA中选择性扩增桡足类18SrDNA,建立桡足类18SrDNA变异类型文库,并从文库中随机挑选的30个克隆进行分析。结果表明,VspI限制性内切酶能将这些克隆分成频率分别为0.17、0.23和0.6的3种操作分类单元(OTUs),遗传多样性指数达到0.95。3条OTU代表克隆序列与甲壳纲桡足亚纲核苷酸差异数在75.4—97.8之间,而与其他亚纲的差异都高于100。3条OTU代表克隆序列均属于桡足亚纲,其中,AY437861和AY437862属于哲水蚤目。3条OTU代表克隆序列可分为2个高变异区和3个相对保守区,其GC%分别为47.37%、48.16%和48.57%。研究结果表明,混合DNA提取方法简单,设计的引物可选择性地扩增浮游桡足类18SrDNA,根据18SrDNA序列序列变异描述浮游桡足类多样性是可行的。研究结果也为在浮游桡足类分类中引入18SrDNA序列奠定了基础。 |
| 关键词: 18S核糖体RNA基因 桡足类 序列变异 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金资助项目,40176028号 |
附件 |
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| AMPLIFICATION AND VARIATION ANALYSIS OF JIAOZHOU BAY PELAGIC COPEPOD 18S RIBOSOMAL RNA GENE (18S rDNA) |
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MEN Rong-Xin, YANG Guan-Pin, LIU Yong-Jian, Guan Xiao-Jing
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College of Marine Life Sciences, Ocean University of China, Qingdao, 266003
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| Abstract: |
| Zooplankton plays a crucial role in material and energy circulat ions in marine ecosystem. Copepod are major members of zooplankton in both biomass and species number. DNA descriptions of marine copepod diversity, community structure and community dynamics are therefore very important.
Traditional approaches for copepod diversity evaluation are based on morphological taxonomy. These traditional methods are experience dependent and integration of data from different sources is difficult. Difference in DNA sequence, however, is the most fundamental difference between living beings. Descriptions of diversity based on DNA sequences is more objective and data integration is easier. In order to describe molecular genetic diversity and develop a community structure partitioning method for marine pelagic copepod. The variation within 1.5kb pelagic copepod 18S ribosomal RNA gene (18S rDNA) fragment was determined using molecular biological techniques, including polymerase chain reaction amplification, library construction, restriction fragment length polymorphism analysis, sequencing and systematic analysis.
Pelagic copepod 18S rDNA was amplified from DNA mixture extracted from plankton and ligated into plasmid vector. A library containing all possible 18S rDNA variants of copepod was constructed and from it 30 recombinants were selected randomly for further analyses. The inserts of these 30 recombinants were reamplified, purified and cut with Vsp I restriction endonuclease, yielding 3 restriction fragment length polymorphism (RFLP) types, or operational taxonomy units (OTU). The frequencies of clones covered by these three OTUs are 0.17, 0.23 and 0.6 respectively, giving a genetic diversity index of 0.95. The nucleotide differences between OTU representative clone and known sequences of copepod range from 75.4 to 97.8, while those between OTU representative clone and other subclasses of crastacea are higher than 100. Systematic analysis showed that the sequences of three OTU representative clones merge always into copepoda clade, and two of them, AY437861 and AY437862, into calanoida clade. The sequences of three OTU representative clones can be divided into 2 high variable regions and 3 relatively conservative regions according to the concentration of variable nucleotide sites. The GC contents of these three clones are 47.37% , 48.16% and 48.57% respectively.
Results show the DNA extraction method used in this study is both simple and efficient , and the primers designed can amplify pelagic copepod 18S ribosomal RNA gene fragments selectively from mixed plankton DNA. It is feasible that pelagic copepod diversity can be described according to 18S rDNA sequence variations. Our results also provided the basis for the utilization of 18S rDNA sequence in the taxonomy of pelagic copepod. |
| Key words: 18S ribosomal RNA gene, Copepod, Sequence variation |
