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底栖有孔虫的不同壳质类型DNA提取技术比较及青岛湾潮间带分子多样性研究
吕曼
Subtype硕士
Thesis Advisor类彦立
2015-05-19
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline生物工程
Keyword底栖有孔虫 Dna提取 Dna保存 Rdna 高通量测序
Abstract底栖有孔虫有钙质玻璃壳、钙质瓷质壳、砂质壳 (胶结壳) 和无壳 (有机质膜) 等多种类型,其中前三种类型有孔虫在古海洋学、地质学、生物指示、生态系统等众多研究领域具有重要研究价值。对有孔虫进行准确分类鉴定是其开展应用的前提,然而有孔虫种类之多,形态变异之大使得许多种类有孔虫的分类很困难。经典分子生物学技术在有孔虫的分类鉴定及系统发育等方面有巨大应用价值,然而国内有关有孔虫分子生物学的研究几乎为空白。本论文首先对现生底栖有孔虫的基因提取和保存方法学进行了比较研究,摸索出了分别适用于三种壳质类型的方法;另外利用优化的分子方法,结合高通量测序技术对青岛湾潮间带底栖有孔虫的分子多样性进行了探索研究,并与环境关系进行了分析。
方法学部分,对现生有孔虫的DNA进行有效保存、提取及扩增是开展分子生物学研究工作的前提。为了探索出分别适合钙质玻璃壳类、钙质瓷质壳类底栖有孔虫的DNA提取、扩增及保存方法,及适合砂质壳类底栖有孔虫的DNA提取和扩增方法,本研究通过对青岛湾潮间带沉积物进行样品采集,进行如下系列实验:1) 对于钙质玻璃壳类有孔虫,以卷转虫属Ammonia spp.为材料,以SSU rDNA 3’端为标记基因,研究了五种DNA提取方法——DOC (Na deoxycholate, 脱氧胆酸钠) 裂解液法、Guanidine裂解液法、Qiagen Dneasy Plant Mini Kit、Dneasy Blood & Tissue Kit以及REDExtract-N-AmpTM Tissue PCR Kit对Ammonia spp. DNA提取效能的差异;并以LSU rDNA 5’端为标记基因,比较了三种DNA保存方法——不同温度条件的烘干 (20℃~120℃)、冷冻 (-20℃、-80℃,有水、无水) 以及不同裂解液处理 (含和不含EDTA) 对Ammonia spp. DNA保存效果的差异。2) 对于钙质瓷质壳类有孔虫,以五玦虫属Quinqueloculina spp.为材料,以SSU rDNA 3’端为标记基因,研究了五种DNA提取方法对Quinqueloculina spp. DNA提取效能的差异;以及三种方法对其DNA保存效果的差异。3) 对于胶结质壳类底栖有孔虫,以砂轮虫属Trochammina spp.为材料,以SSU rDNA 3’端为标记基因,研究了五种DNA提取方法对Trochammina spp. DNA提取效能的差异。
     方法学部分研究结果表明,1) 对于玻璃质壳类有孔虫,DOC裂解液法、Guanidine裂解液法和REDExtract-N-AmpTM Tissue PCR Kit对其 DNA的提取效能较好,但DOC法更廉价且操作简便;DNA保存方法中,烘干保存法的20℃组和30℃组显著优于40℃组;冷冻保存法中无水组显著优于有水组;DOC裂解液保存法各组间无显著差异;三种保存方法综合效果表现为冷冻组显著优于烘干组和裂解液组。2) 对于瓷质壳类有孔虫,Guanidine裂解液法对其DNA的提取效能最好;DNA保存方法中,烘干保存法的110℃组效果最好,冷冻保存法中-20℃无水冷冻效果最好,但统计学上均不显著;Guanidine裂解液保存法中-20℃含EDTA组效果显著优于其他组。3) 对于砂质壳类有孔虫,Guanidine裂解液法对其DNA的提取效能最好。
       在分子多样性研究部分,将经过优化的有孔虫经典分子生物学方法运用到高通量测序的前期实验研究中,以有孔虫SSU rDNA 3’端的一段高变区为标记基因,对5月份至10月份采自青岛湾潮间带的沉积物样品进行高通量测序,研究有孔虫分子多样性的月变动情况及与环境因子 (温度、盐度、pH) 之间的关系。
分子多样性研究部分, 通过高通量测序,共得到506 251条Tags,聚合为1527个OUT (Operational Taxonomic Units)。初期分析结果表明,这些OTUs主要为目水平的Rotaliida和Miliolida,青岛湾潮间带沉积物中有孔虫群落的分子多样性在七月 (夏季) 最高,九月、十月 (秋季) 次之,五月 (在春季) 最低;pH是影响底栖有孔虫物种丰富度的主要环境因素,相对丰度最高的五个属中,钙质玻璃质壳类有孔虫Ammonia, Epistominella, Operculina, Bulimina四个属的相对丰度变化趋势均与pH呈正相关,而另一砂质壳类有孔虫Textularia属的相对丰度与pH呈负相关。
       本论文给出了分别适用于玻璃质壳类、瓷质壳类和砂质壳类底栖有孔虫的DNA提取、扩增和保存的优化方案,将为有孔虫分子鉴定工作的开展提供实用
的技术方案。此外,本研究摸索出了一套运用高通量测序技术调查青岛湾潮间带底栖有孔虫的分子多样性的方法,并与环境关系进行了分析和讨论。
Other AbstractBenthic foraminifera consist of many types, including hyaline, porcelain, agglutinated or organic tests, and the first three types play significant role in study of paleoceanography, geology, biomonitoring, ecological system and so on. For better application of foraminifera in these fields, we must indentify the species accurately. However, the vast diversity and wide morphological variability of foraminifera make it very difficult to clarity their taxonomic status. The application of molecular biology techniques will help solve some misidentification problems that derived from morphological taxonomy. However, there was limited study on molecular biological research of domestic foraminifera. In this study, we compared the efficiency of different methods for DNA-extracting and DNA-preserving from benthic foraminifera. Furthermore, we utilized the improved molecular methods to investigate the molecular diversity of benthic foraminifera in intertidal zone of Qingdao Bay using high throughput sequencing.
In order to establish an efficient method for preserve, extraction and amplification of DNA from hyaline and porcelain benthic foraminifera, and to extract and amplify agglutinated benthic foraminiferal DNA efficiently, some experimental series were set up: 1) We used the partial SSU rDNA as DNA barcode to test the different efficiency of five DNA extraction methods for the representative hyaline foraminifera Ammonia spp. The DNA extracting methods include DOC lysis buffer method, Guanidine lysis buffer method, Qiagen Dneasy Plant Mini Kit, Dneasy Blood & Tissue Kit and REDExtract-N-AmpTM Tissue PCR Kit. In order to establish efficient methods for preserving DNA from hyaline benthic foraminifera Ammonia spp., the partial LSU rDNA was used as DNA barcode and three experimental series of DNA preservation were set up including air-dried (from 20℃ to 120℃); frozen (-20℃、-80℃, with or without seawater); DOC lysis buffer with vs. without EDTA. 2) We used the partial SSU rDNA as DNA barcode to test the different efficiency of five DNA extraction methods for Quinqueloculina spp. which represent porcelain foraminifera, and three methods for DNA preservation. 3) We also used the partial SSU rDNA as DNA barcode to test the different efficiency of five DNA extraction methods for Trochammina spp. which represent agglutinated foraminifera.
And the result showed that 1) DOC method, Guanidine method and REDExtract-N-AmpTM Tissue PCR Kit were all effective for hyaline foraminifera, but the DOC method was more economic and convenient; DNA was better preserved under the condition of air-dried under 20℃and 30℃ than 40℃ (P<0.05); Frozen without seawater group was better than with seawater group (P<0.05); There was no significant difference between the DOC lysis buffer with or without EDTA treatment (P>0.05); And the best results were obtained in the treatment with frozen samples. 2) For porcelain foraminifera, Guanidine lysis buffer was the best method for DNA extracting; And DNA was better preserved under the condition of air-dried under 110℃and 30℃ but the difference was not significant (P>0.05); There was no significant difference among frozen groups (P>0.05); Samples in Guanidine lysis buffer with EDTA and stored in -20℃ were better preserved (P<0.05); And the best results were obtained in the treatment with samples stored in Guanidine lysis buffer. 3) For agglutinated foraminifera, Guanidine lysis buffer was the best method for DNA extracting.
Benthic foraminifera is an important component of Marine micro-food-cycle, occuping an important position in the Marine ecosystem. The traditional method to investigate the diversity of foraminifera is time-consuming and occasionally omits some species. High-throughput sequencing technology is helpful to solve this problem. Targeting a short hypervariable region of foraminiferal SSU rRNA gene, we used the next-generation sequencing method to examine foraminiferal molecular diversity in Qingdao Bay intertidal sediments sampled from May to October, and the relationship between foraminiferal diversity and environmental factors (temperature, salinity, pH) was also assessed.
We got 506 251 tags which were assembled from reads and 1527 OTUs (Operational Taxonomic Units) for 6 samples. Preliminary analysis demonstrated that 1) the OTUs mainly belonged to Rotaliida and Miliolida; 2) The diversity of foraminiferal communities in intertidal zone of Qingdao Bay is highest in July (summer), moderate in September and October (autumn), and lowest in May (spring); 3) pH was the main environmental factor affecting benthic foraminiferal richness; Among the top five genus, the relative abundance of Ammonia, Epistominella, Operculina, Bulimina (hyaline foraminifera) were positively associated with pH; On the contrary, the relative abundance of Textularia (agglutinated foraminifera) was negatively associated with pH.
In summary, an optimal DNA-preservation and -extraction protocols were tested respectively for hyaline, porcelain and agglutinated foraminifera. Moreover, we optimized the method to apply HTS to investigate the molecular diversity of benthic foraminifera in intertidal zone of Qingdao Bay and analyzed the relationship between the molecular diversity and the environment.
Subject Area海洋生物分类学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/22736
Collection海洋生物标本馆
Affiliation中国科学院海洋研究所
Recommended Citation
GB/T 7714
吕曼. 底栖有孔虫的不同壳质类型DNA提取技术比较及青岛湾潮间带分子多样性研究[D]. 北京. 中国科学院大学,2015.
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