The Investigation On Microbial Diversity Of Arctic Deep Sea Sediments

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.