【作者】 陈丽蓉；

【导师】 杨季芳；

【作者基本信息】 浙江理工大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 深海沉积物由于数以万年的沉积作用，所含成分繁复，是最复杂的微生物栖息地。北极地区蕴含着丰富的微生物资源，由于其特殊的地理位置和气候条件，处于其中的微生物大多有其特殊的生理生化特性。因此，对于北极地区深海沉积物中微生物的研究具有重要的意义。依靠传统的分离培养的方法只能获得其中0.1%-1%的菌种，大量的未知微生物未被发现，需要依靠现代分子生物学的方法进行分析研究。本研究利用PCR-DGGE结合克隆测序的方法对北极地区沉积物中微生物的多样性进行了初步的研究，为进一步深入开展极地深海微生物资源与环境研究打下基础。本文研究材料是取自白令海和楚科奇海海域的沉积物样品。实验采用了包含试剂盒法在内的九种DNA提取方法，以期获得最适的提取北极沉积物中微生物总DNA的方法。选用细菌通用引物，优化PCR反应退火温度。对PCR扩增出的16S rDNA的V3-V5区产物进行变性梯度凝胶电泳检测，进而进行16S rDNA的多态性分析。选取其中较为清晰的特征条带克隆测序，最终获得沉积物中微生物的多样性分析结果。九种DNA提取方法结果显示：这些方法都能获得大小在23kb左右的DNA样品。将样品经过初步处理后，颜色较深的直接使用OMEGA公司试剂盒提取总DNA。而对于颜色较淡的则通过方法Ⅰ（SDS-蛋白酶k法）提取总DNA后进行纯化。通过巢式PCR的方法可获得大小在570bp左右16S rDNA的V3-V5区片段。两次PCR优化后的退火温度分别为55℃和53℃。采用变性梯度范围为40%-60%，丙烯酰胺浓度为6%的变性胶进行DGGE检测。不同地点不同深度的沉积物样品，其DGGE图谱均有所差异，条带数目，位置及亮度均有不同。数据统计显示，最终获得的DNA序列大多为未可培养微生物序列。克隆获得的类群包括变形细菌门(Proteobacteria)、放线菌门(Actinobacteria)，酸杆菌门(Aciodobacteria)，拟杆菌门(Bacteroidetes)，硝化螺旋菌门(Nitrospirae)，厚壁菌门(Firmicutes)，异球菌属(Deinococcus)，其中变形细菌（包括α，β，γ，δ，ε-Proteobacteria）为明显的优势类群，与文献报道基本相同。各表层样品间优势菌群基本相同,尤其是相近海域的样品。而相距较远的沉积物样品间DGGE指纹图谱差异相对较大，只有优势条带相同，其余条带在位置和数量方面均有所差异。柱式样品中大多为中间偏上层数的DGGE指纹图谱较为特殊，出现一些特有条带。经测序后发现大多为一些沉积物中较为少见的菌种。整体而言，柱式样品各层间DGGE指纹图谱基本相同，随深度增加条带数目有所减少，亮度减弱。本研究对北极深海沉积物中微生物群落的多样性进行了初步的研究，克服了传统方法的局限性，获得了较为丰富的微生物遗传信息。其中包括一些未培养或鉴定的潜在新菌，针对它们特殊的生理生化性质可以进行深入的开发利用。更多还原

【Abstract】 Due to tens of thousands of years of sedimentation, the deep sea sediments’ compositionsare complex. It is one of the most complex microbial habitats. The Arctic region contains awealth of microbial resources, because of its special geographical position and climateconditions, in which microorganisms have the special physiological and biochemicalcharacteristics. Therefore, The investigation on microbial diversity of arctic deep sea sedimentsis very important. Rely on traditional culture methods, we can only get the0.1%-1%strain, alarge number of unknown microorganisms are undetected, we need to use the method ofmodern molecular biology. The study combine the PCR-DGGE with cloning and sequencingmethod to research microbial diversity of arctic deep sea sediments, and will provide basis forfurther study and reference.The studies’ samples are the sediments，which are from the Bering Sea and the ChukchiSea. In this experiment we use nine methods of DNA extraction, which are including kitmethods. We want to get the best way to extract microorganisms’ total DNA in the Arcticsediments. Selection of bacteria universal primers, optimized PCR reaction annealingtemperature. The16S rDNA V3-V5products separated by denaturing gradient gelelectrophoresis, and then analysis of16S rDNA polymorphism. Select the characteristic strip toclone, finally get the results of microbial diversity of sediments.Nine methods of DNAextraction results show: these methods can obtain the DNA, which are all about23kb in size.After initial treatment, if the color is dark, use the OMEGA kit for extracting the total DNA. Asfor the lighter use methodⅠ(SDS-protease K) to extract total DNA,then purified it. By nestPCR method we can obtain the size of570bp16S rDNA V3-V5fragment. Two PCR annealingtemperatures were55℃and53℃. PCR products were separated in6%polyacrylamide gelswith a denaturant gradient from40%to60%. Ultimately determine the optimal time for9-10h,and select the EB staining method for subsequent cloning experiments. The sediments ofdifferent locations, different depth the DGGE profiles are different, the band number, locationand intensity are different.The results showed the DNA sequences mostly are uncultured microorganismsequence.IncludingProteobacteria,Actinobacteria,Aciodobacteria,Bacteroidetes,Nitrospirae,Fir micutes, Deinococcus. Proteobacteria (including α，β，γ，δ，ε-Proteobacteria)is the dominantgroup.That is same to the report. Surface sediments have the same dominantMicrobes.,especially samples from the near sea. The middle upper layers’ fingerprint is mostlyspecial in Column sample,and have some unique strip, After sequencing, we found that speciesare mostly rarely in sediment. As a whole,the column sediments’ each layer are similar, withthe depth increasing, the strip of column station’s DGGE map is gradually reduced.更多还原