Microbial Diversity Analysis Of Different Altitude Wetlands In Changbai Mountain

Changbai Mountain Nature Reserve is one of the nature ecological systems that maintains relatively complete in China,which is a natural "Museum of Natural History" and huge "Biological Gene Library".There are many nature wetlands in the area,they are influenced by the unique topography,climate,hydrology and other factors,which determines its abundant microbial species.Therefore,this article mainly focuses on the microbial community structure and microbial diversity of different altitudes wetland soil in Changbai Mountain.In this study,we collected twelve kinds of soil samples from different altitude wetlands of Changbai Mountain North slope and Western slope,two different methods were used to analyze the microbial community structure and microbial diversity:Eight kinds of dominant strains that isolated and purified from different altitudes wetland soil were identified using MIDI microbial identification system,the results show that:strains R2A 1-6-2,NA 1-5-3 as Bacillus,Bacillus-licheniformis;strain R2A 2-6-2 as Bacillus.sp;strains R2A 1-6-4,NA 2-4-1 as Bacillus,Virgibacillus-pantothenticus;strain NA 1-4-2 as Bacillus,Bacillus-cereu;strain NA 1-5-1 as Micrococcus-luteus;strain NA 2-4-2 as Paenibacillus-alginolyticus.Different superior strains have lower similarity level.Using PCR-DGGE molecular biology technique and phylogenetic analysis method,analyzed microbial diversity of different altitudes wetland soil in Changbai Mountain.The V3 variable region of the bacterial 16S rDNA gene fragment of total DNA in the wetland soil were amplified by PCR,the amplification system and reaction conditions were optimized.The results show that the amplification volume was 25.0 ?L,containing 2.5 ?L of 10×PCR buffer(Mg2+),2.0 ?L of dNTPs mixture(0.25 mM),1.0 pL of each primer(10 ?M),0.3 ?L of TaKaRa Taq DNA polymerase(5 U/?L)and 5.0 ?L of template DNA(soil DNA),final reaction system was supplemented with 13.2 ?L ddH2O and the thermocycler program as follows:initial denaturation at 94? for 9 min,followed by 30 cycles of denaturation at 94? for 30 s,annealing at 55 ? for 30 s,and elongation at 72? for 30 s,and ending with a final elongation step at 72 ? for 7 min,230 bp length of target gene fragment was obtained.The PCR products were analyzed by DGGE,and the parameters were optimized.The most optimum system for DGGE as follows:the voltage is 80 V,gel concentration is 8%,the denaturing gradient ranges from 55%to 70%,about 13.5 hours in 1×TAE buffer.Twenty-six clear bands were cut from DGGE gel profiles,eight characteristic bands were choose for sequencing,the results show that the advantages strain of Changbai Mountain wetland soil are Uncultured bacterium,Uncultured actinobacterium,Uncultured Pseudonocardia sp.,Uncultured Methylobacterium sp.,Uncultured Rhizobiales bacterium.