Optimization of PCR-DGGE conditions for analyzing the diversity of rhizosphere bacteria in wetland

In order to make better use of PCR-DGGE in analyzing the bacterial diversity of plant rhizosphere soil samples in wetland,3 pairs of universal primer were selected to optimize the conditions for PCR 16S rDNA amplification and for the denaturing gradient and the electrophoresis time of DGGE.The results showed that touch-down PCR procedure for annealing temperature could improve the specific amplification.The ideal denaturing gradients for target sequences of primer 357f/518r and 357f/907rM consisted of 50% to 80% and 40% to 70% denaturant,respectively.And the ideal electrophoresis time for targeted sequences of primer 357f/518r and primer 357f/907rM were 17 h and 14 h,respectively.Moreover,throughout comparing profile analysis of DGGE patterns,it was found that either the number of bands or Shannon diversity index,the result of primer 357f/518r was the best,followed by the one of primer 357f/907rM.In order to fully reflect the bacterial diversity in plant rhizosphere soil by DGGE,the joint use of primer pair 357f/518r and primer pair 357f/907rM was more effective.