The Dominant Intestinal Bacteria Flora In Apriona Germari Adult

By traditional culture and 16S rDNA sequence analysis,the intestinal bacteria flora in Apriona germari adult was analyzed and their predominant flora was found out.Meanwhile, we studied the method of genomie DNA extraction and optimization of PCR amplification and DGGE analysis conditions.The aim was to establish a set of technology conditions for studying the diversity of Apriona germari bacteria and to provide a theoretical and technological support for the research of Apriona germari and other pest control.The main results were showed as follows:(1) Different bacterial strains,which belonged to 6 different genus,were obtained from Apriona germari adult by traditional culture methods.They were Klebsiella,Escherichia,Pseudomonas,Acinetobacter,Serratia and Enterobacter.(2) The results showed that extracting genomie DNA by modified CTAB method can remove impurities such as protein and intestinal tissue effectively,to ensure high grade genome DNA were obtained.(3)Several important factor including MgCl2, dNTP,random primer,Taq polymerase and template DNA were optimized through L16(45)orthogonally designed experiment. Annealing temperature and number of amplification cycle were optimized through single factor experiment.The results showed that the optimal reaction system for PCR analysis was as follows:a total volume of 25μL PCR reaction system contained 10×Buffer 2.5μL, MgCl22.0 mmol/L, dNTP 0.2 mmol/L, random primer 0.48μmol/L, Taq polymerase0.75 U, template DNA 75ng, and annealing temperature was 60℃for 30 cycles amplification.(4)The range of denaturant concentration in vertical polyacrylamide gel was 15~45%, the concentration of polyacrylamide gel was10%, the denaturant temperature was 60℃,the tension was 130V and the electrophoresis time was 5h. The range of denaturant concentration in horizontal polyacrylamide gel was 27~38%, the concentration of polyacrylamide gel was10%, the denaturant temperature was 60℃,the tension was 130V and the electrophoresis time was 7h.(5)The six different bacteria strains,which were abtained through traditional culture from Apriona germari gut,were analyzed by technology of 16S rDNA-PCR.It was showed that they were Klebsiella oxytoca, Escherichia coli, Pseudomonas fluorescens, Acinetobacter,Serratia and Enterobacter. They were consistent with the results of traditional culture methods. All their 16S rDNA sequences similarity rates in GenBank were more than 90%.(6) Different bacterial strains,which belonged to 8 different genus,were obtained from Apriona germari adult by technology of 16S rDNA-PCR-DGGE. 5 genus consistent with the results of traditional culture methods.It revealed that Klebsiella oxytoca and Serratia marcescens were the real predominant flora in Apriona germari adult gut.