Studies On The Xanthomonas, Oryzae Pv. Oryzae Of IS1112 And IS1113

Insertion Sequence (IS) is a normal constituent of bacteria's chromosome and plasmid, which has been proved well correlated with the evolutionary classification, pathogenic type and virulence of bacteria. IS is a small DNA fragment which can auto-transpose, and the movement of the IS could make the changing of the neighboring genes, altering the biological character. Therefore, the study of IS of Xanthomonas oryzae pv. Oryzae (Xoo) would facilitate the identification of various pathogenic types of Xoo and understand the their characters. With 12 types of Xoo in China as research objects, this study conducts a preliminary research of the copies number, target loci and flanking sequences of their IS1112 and IS1113, and leads a analysis of discrepancy of IS1112 and IS1113's complete genome sequence which is known by us. Moreover, we seek to develop a method of high celerity and accuracy in order to detect the copies number of IS.What we've done has important reference for the futher study of the IS1112和IS1113's distributing variance of loci and integrative property, and lays a good foundation for revealing the movement pattern and control mechanism of the IS in Xoo.The results of our study go as follows:1.Using the the method of LA-PCR, we've propagated the IS1112, IS1113 and their flanking sequences of 12 strains of China. The result of electrophoresis test demonstrates that C5 strain differs greatly from the other 11 strains, in which IS1112 contains one diversity band and IS1113 contains two, while nothing different could be found among the other 11 strains.2.the statistical result of MAFF,KACC,PXO99A中IS1112 and IS1113 indicates that, in this three strains, IS1112 has 10 target loci in common while the number of diversity target loci is 27. On the other hand, IS1113 has 8 target loci in common while the number of diversity target loci is 4.Since the insert location is chosen randomly, there's no specific identify loci, however, the target loci would develop repetition of 2-4 and 8 base when IS is inserted.3. Using of the overlapping PCR method, we construct vector pMD18-T with 16S rDNA fragments of Xoo and the insertion sequence IS1112 and IS1113 inserted into the vector and make it as the standard plasmid of the quantitative PCR. Then the copy numbers of IS1112 and IS1113 of twelve kinds of Chinese strains are identified via TaqMan real-time quantitative PCR. The results indicate that the standard curve derived from standard plasmid has relatively high amplification efficiency and good linear relationship, the copy numbers of IS1112 and IS1113are true and accurate, the method has advantage of simpleness, celerity and needing less DNA, non-radioactive pollution.