Molecular Ecology Analysis And Biosafety Assessment Of Genetically Engineered Pseudomonas Fluorescens Strains Released In The Fields

In this study, firstly, one method of soil total DNA extraction has been ascertained which was the best for the following denaturing gradient gel electrophoresis (DGGE) with high extractive efficiency (3.15 mg DNA/g soil), low extractive cost and good quality. The main process was pretreating of soil sample, extracted the crude DNA and then purified the crude DNA;following, amplified the bacteria 16S rDNA V3 region;furthermore, loaded the PCR product on the polyacrylamide gel to operate the denaturing gradient gel electrophoresis. At last, combined the pattern observed method and cluster analysis to analyze the results. In this study, no significant difference on DGGE pattern was be found between the soil samples of blank control and treated by BioP8, 8'P303, and 12P303. Associated the software ImageJ and Mathworks Matlab v7.1 with PAST to analyze the DGGE gels pattern. The reults indicated: first, among the samples inoculated with BioP8, the CK and treated samples of 7th, 15th, 45th were all clustered together, the similarity of bacterial community was 88% or so;the four samples of 0 day and lst,30th and 45th, 60th and 90th clustered together, respectively;the similarity of bacterial community the corresponding soil samples of CK and inoculated with BioP8 was about 85% on average;second, among the samples inoculated with 8'P303,there was a there main clusters, the first cluster included the all the samples from 1st to 15th ,the similarity of bacterial community was about 80%;the second cluster included the samples of 30th and 45th, the similarity was 90%;the third cluster included the samples of 60th and 90th, the similarity was 80%. Moreover, among all the soil samples, the similarity of bacterial community came to 60%;third, among the samples inoculated with 12P303, the samples from 1st to 15th ,the similarity of bacterial community was about 80%;the samples of 30th and 45th ,the similarity was 85%;the samples of 60th and 90th, the similarity was 80%. All in all, on the perspective of microbial molecular ecology, BioP8, 8'P303, 12P303 were all compatible to environment ,all of them were safety to soil aboriginal bacterial. In this study, the microbial molecular ecological technology was adopted to offer the scientific basis for industrialization production and application of the engineered bacterial strain. It will have important theory significance and practical value.