Preliminary Analysis On 16s-ITS Gene And DHPLC Polymophisim Of 5 BALOs

This study explored a non-sequencing detection and identification method for BALOs. 5 strains, namely FJZZ1, FJZZ2, FJZZ3, TP and TY, isolated from fresh water and had abilities to lyse Gram negative bateria, were preliminarily determined as BALOs. Genomic DNA from five BALOs was extracted by different methods and found the chemical kilit the best result and a freeze-and-thaw method the most convenient through comparison. 16s rRNA gene of 5 BALOs strains were amplified by bacterial univasal primer 63F and BALOs-specific primer(842R) and sequenced. Those strains were identified as Bdellovobrio bacteriovorus by phylegenic analysis, with 98% simlarity and above to this species. To obtain higher validity of the results, ITS(Internal Transcribed Spacer, or ISR,Intergenic Spacer Region) gene , which is more variale and identifiable, were amplified (by primer 842F and 4R) and commited to sequencing and phylegenic analysis. The results showed changes in phylegenic trees and provided more reliable revolutionary realations for the 5 strains. The amplicons were then cloned to pMD-T vectors and faciliate following studies as convenient, quantitative and reliable templates. To facilliate analysis, plasmid PCR products from 3 highly sequence-disimilarious BALOs strains(CHA1, CHA2, CHA3, preserved by our labaratory ) together with TY and FJZZ2 served as materials for dHPLC analysis. The optimal conditions for seprating different 63F-842R fragments by dHPLC were given: column temprature 60℃and detected under semi-denaturation mode with flow velocity 0.7mL/min and B2 solution gradient 66.7%-68.1% passing through in 3 minutes. Specific Retention Times of 5 fragment were determined: FJZZ2 5.31min, TY 5.20min, CHA2 5.46min, CHA1 5.44min and CHA3 5.38min. A function was fomulated tentatively to simulate the relationship between Dissimilarity of DNA fragmentsα(%) and their difference of Retention Time(δT) :δT(s) = 20.224lgα(%) + 7.1949(0<α<100) R2 = 0.967. The fomula had a higher corealation coeffient and was expected to provide a new method for BALOs polymorphism studies.