Microbial Diversity Of Different Altitude Wetlands In Changbai Mountain By PCR-DGGE

Changbai Mountain is the highest mountain in the northeast of China as it is calld "Northeast Ridge".It is located between Jilin Province in China and North Korea.The ecosystems is relatively completed,the natural environment is complex and diverse,the mountain climate is variable,the plant species is rich and colorful,which are the reasons that why Changbai Mountain is called "Museum of Natural History" and huge "biological gene library".In this study,we collected soil samples from different altitude wetlands of Changbai Mountain.Extracted total soil DNA were amplified in PCR and the PCR products were analysed with denaturing gradient gel electrophoresis(DGGE).1.Optimized the PCR reaction system and the reaction conditions of the variable region of the 16S rDNA V3.Explored the dosage of Taq enzyme,dNTP mixture and primers,and select primers.Last,the most appropriate system for PCR amplification:0.25 ?L Taq enzyme(5U/?L),2.5 ?L 10×PCR Buffer(Mg2+),2 ?L dNTP mixture,5 ?L DNA,primers(GC-338F,5.18R)1 ?L,add ddH20 to 25 ?L.PCR cycling program:94? denaturation step for 9 min,then 30 cycles of denaturing step of 94? for 30 s,an annealing step of 55? for 30 s and an extension step of 72 ? for 30 s.The last step in the PCR program is a final extension at 72 ? for 7 min.2.Optimized the PCR-DGGE conditions,the results:Electrophoresis 14 hours in 1×TAE buffer,the voltage is 100 V,the electrophoresis temperature is 60 ?,the range of the denaturing gradient is 40%?60%,and then stain in EB(ethidium bromide)staining solution.Finally,observed and photographed on the gel imaging system.3.Analyzed 11 different altitude wetlands soil samples in Changbai Mountain by PCR-DGGE.Finally recycled and sequenced nineteen clear bands,of which 16 bands are uncultured microorganisms.Band 2,Band 7,Band 8,Band 9,Band 19,they are Acidobacteria bacterium(similarity:91%?98%).