Analysis In Marine Oil-Degrading Microbial Community Structure Based On PCR-DGGE Method

Traditional techniques about screening bacterias has not attained the modern experiment goal.Based on 16 SrDNA gene amplification and sequencing analysis,Denaturing gradient gel electrophoresis(DGGE)is becoming one of the important technologies on studying microbial community structure.The molecular biology method has been successfully used in achieving dynamic changes of microorganisml in nature.And it is more effective compared to traditional way.Considering bioremediation of oil spill in Tianjin Bohai bay,PCR and DGGE are applied to make the preliminary study on petroleum-degrading communitues.The aim is to optimize PCR-DGGE conditions and obtain fingerprints and sequencing.It is a guide for correlational reseach on community structure.With crude oil as sole carbon source the consortium is screened from the sediment of Tianjin Habor.The sample were taken once a week in experimental period.The total DNA of oil-degraders was extracted by different kinds of bacterial DNA extraction kits and its extraction effect was detected by agarose gel electrophoresis.PCR conditions were obtainedby the optimization of the amount of primer,the volumes of the DNA in PCR program,the annealing temperature and cycle numbers in PCR system,respectively.Meanwhile,DGGE conditions were obtained by optimizing the concentration of polyacrylamide gel,electrophoresis voltage and time,respectively.Results showed that the quality and purity of the total DNA were satisfied with the experimental requirements by the New Probe bacterial genome DNA extraction kit.The mixture of optimized PCR contained 25 ul of mixed Master buffer,3ul of the template DNA,1ul of each oligonucleotide primer,20 ul ddH2O,respectively.And after an initial temperature of 96 °Cfor 4 min and thermocycling at 94 °C for 1 min,the annealing temperature was set to 55 °C for 1 min with a 1 min elongation time at72 °C,25 cycles.Additional 6 min were performed temperatures at 72 °C.The PCR product from each sample was applied to a lane and analyzed on 8 % polyacrylamide DGGE gels containing gradients of 30–60% denaturant.DGGEwas performed at a constant voltage of 60 V for 16 h at 60 ?.According to DGGE fingerprint,bacterias needed different time to adapt to the new environ-ment during two months,and the community structure of petroleum degraders are relatively stable without distinct declining trend.In the light of the sequencing,Alpha deformation bacteria is dominant in the communities.Otherwise,Common oil degradation species include Roseobacter.sp.,Arthrobacter.sp,Pseudomonas sp.,Microbacterium sp.,respectively.