Isolation and characterization of insertion sequence (IS) from Xanthomonas campestris pv. dieffenbachiae, the casual agent of anthurium blight

• Main Authors: Chia-Jung Lee, 李佳蓉
• Other Authors: Chan-Pin Lin
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/91813490001221383703

碩士 === 國立臺灣大學 === 植物病理學研究所 === 88 === A new insertion sequence was successful isolated from Xanthomonas campestris pv. dieffenbachiae（XCD）by applying the entrapment plasmid, pUCD800. The plasmid pUCD800 was introduced into XCD via electroporation. In the presence of sucrose, levansucrase produced by sacB of pUCD800 plasmid and caused XCD population decreased. Restriction enzyme analyses of these plasmids from 186 sucrose-resistant colonies revealed that the majority recovered that in the manner contained insertions in sacBR element of pUCD800（18.8﹪）or show no noticeable change in the structure of pUCD800（38.2﹪）, and 46.77﹪ probably involved gross rearrangement of pUCD800. The new insertion sequence, ISXCD1, ( accession no. AF263433 ) is 1,203 bp in size and contains 38 bp inverted repeats with 11 bp mismatches at its termini. It generated 4-bp target site duplications in sacBR regions of pUCD800 harbored in 15-4 and 27-1 transformants, and carried two overlapping ORFs（orfA and orfB）, with an A7T motif within the overlapping region. The deduced amino acid sequences of orfA and orfB contained a potential helix-turn-helix motif and a D, D(35)E domain of transposases, respectively. ISXCD1 is present about 9-11 copies in the XCD genome according to Southern hybridization. Southern hybridization analyses the distribution of the new IS is widely existed G（-）and G（+）. Sequence analysis of 5 independent genomic copies of IPCR products indicated no preferred insertion site for the new insertion sequence. In H-2 transformant revealed that ISXCD1 exactly inserted into IS1051, and caused a 4 bp target site duplication. For the detection purpose, a PCR primer pair F570/ R1006 was designed based on the nucleotide sequence of the new IS. A minimum of 1.5 pg DNA of purified genomic DNA of XCD was sufficient to amplified a 436 bp PCR-product.