Microbial Diversity In The Sediments Collected From The Northwest Pacific Ocean

The quality of DNA is very important in the research on microbial ecology by means of molecular biology. This includes three sides: quantity, integrality and whether the DNA extracted from samples can assess the microbial community. 1) In this research, crude DNA is extracted from sediments of NW Pacific Ocean by Zhou, Bath, Microwave and PVP method. The results indicate it is unfit to extract DNA from sediments using the Bath method and highest yield is got by Zhou and Microwave methods. Besides that Zhou method produces high molecular mass genomic DNA from sediments with faint shearing. 2) Agarose gel electrophoresis and DNA minicolumn methods are used for DNA purification. The reclaim efficiency of both purification methods is below 25% and it is higher of minicolumn than that of agarose gel electrophoresis. 3) The number of DGGE lanes show the microbial community abundance. The DDGE patterns based on two pairs of primers and DNA extracted by three methods and purified by two methods are compared each other. The results indicate more lanes can be received on the DGGE gel based on DNA extracted by Zhou method and purified by minicolumn. Meanwhile, the primer for 610 bp fragment is better than that for 210 bp fragment in DGGE examination.The PCR conditions affect the abundance of microbe when PCR is treated as a semiquantitative detection method. The bias of PCR tells us: 1) the template with high G+C contents is difficult to be amplified, the mismatch between template and primer is the vital factor of bias. 2) the bias produced by the G+C content and mismatch will be enhanced along with the annealing temperature rise; on the contrary, the bias can be reduced when the annealing temperature is below 47℃. 3) The bias can't be avoided effectively by changing cycle number of PCR. Besides that, the lanes in PCR products represent the predominant microbial community of samples.16S rDNA fragments of bacteria and archaea are amplified and libraries are constructed based on the purified DNA extracted from the sediments of NW Pacific Ocean. Through the ARDRA for libraries, OTU diversity tells us: 1) to bacteria, 1-3 fragments can be received when the insert fragments are cut by EcoRl and all of the clones of libraries are divided into 11 phylotypes. The results indicate the coverage C of the libraries is 59-76%, and the SH5 has the smallest C value. The similarity index between sample F120401 and SH5 is largest, between Fl20401 and A120614 is smallest. 2) to archaea, libraries of sample SH4 and SH5 are constructed. Most of (>80%) the inserted fragments of both libraries can't be cut by EcoRl. But the pattern of Rsal is much more complex which tells us archaeal diversity is much more complex in the sediments from deep sea. The coverage C is 47-60%, lower than that of bacterial 16S rDNA libraries.Parts of the OTU are sequenced based on the results of ARDRA. Phylogenetic trees are constructed using the sequenced and similar sequences in the GenBank. The results show: 1) to bacteria, four phylotypes are included in five samples. They are Protecobacteria, Planctomycetes, Acidobacte and Chlorofiexi. Among them, the Protecobacteria, including alpha> belta> gamma and delta subgroup, is predominant. 2) to archaea, half of the sequenced OTUs belong to crenarchaeote and others is unidentified or uncultured. Besides that, homology analysis shows there are sulfate-oxidizing bacteria, sulfate-reducing bacteria, methylotrophic bacteria, methylorophic archaea, but no methane producing archaea in sediments. A model about C and S circulation in deep-sea sediments is constructed based on others.27 clones which can represent the bacterial phylotype in deep-sea sediments are elected to amplify two different 16S rDNA fragments for DGGE. The results of DGGE show that the 610 bp fragments can be separated better than 210 bp fragments in DGGE gel. At the same time, more 610 bp fragments can be got than 210 bp fragments when total DNA extracted and purified directly from sediments as PCR template. The longer fragments have more information than shorter one.610 bp fragments are used for DGGE to compare the microbial diversity of five sediments from deep-sea. There are the same bacteria which are predominant butchangeable in the five samples. The similarly index got from DGGE pattern is higher than that from ARDRA. Under certain degree, the microbial diversity will be overestimated using the method ARDRA, but DGGE is on the contrary.