知识与财富的链接
构建植物病原菌突变体库是进行新基因发掘和功能基因组学研究的有效途径之一。为了快速准确地确定Tn5的插入位点和相应的功能基因,本研究采用TAIL-PCR和Tn5转座子质粒拯救两种技术对本实验室在筛选Tn5转座子插入水稻条斑病菌的突变体库过程中获得的8个在水稻上毒性减弱的突变体进行了鉴别。结果显示,TAIL-PCR和Tn5质粒拯救相互结合使用,可有效鉴别Tn5的插入位点和相应的功能基因。TAIL-PCR明确的5株突变体是Tn5分别插入在gspF、cAMP-regulatory protein、Integral membrane protease subunit、S-adenosylmethionine decarboxylase proenzyme和gpsJ 基因上从而导致水稻条斑病菌的毒性发生了改变;Tn5质粒拯救法明确的3株突变体是Tn5分别插入在pathogencity protein、conserved hypothetical protein和flagellum-specific ATP synthase 基因上。同源分析发现,8株突变体Tn5插入的基因与已基因组测序的BLS256菌株中的基因同源性达100%。这表明,TAIL-PCR和Tn5质粒拯救技术可有效应用于植物病原菌Tn5突变体库的鉴别和目标基因的分离。
Construction of a mutant library is a fundamental approach to new genes and functional genomics of a plant pathogen. To rapidly and accurately identify a Tn5 insertion position in a corresponding gene in Xanthomonas oryzae pv. oryzicola, the critical pathogen of bacterial leaf streak in rice, thermal asymmetric interlaced PCR (TAIL-PCR) and Tn5 transposon rescue were adopted to verify 8 virulence-reduced mutants screened from the Tn5 inserted library of the pathogen based on virulence assay in rice. TAIL-PCR revealed that 5 virulence-reduced mutants were due to that the Tn5 transposon was inserted in the general secretion protein F (gspF), cAMP-regulatory protein, integral membrane protease subunit, S-adenosylmethionine decarboxylase proenzyme, and gpsJ genes, respectively. Tn5 transposon rescue indicated that the genes in other three mutants were inserted by Tn5 were pathogencity protein, conserved hypothetical protein, and flagellum-specific ATP synthase genes, respectively. Homology analysis demonstrated that eight genes inserted by Tn5 were 100% identities to the corresponding genes in the genome sequence of the strain BLS256. The results above suggest that TAIL-PCR and Tn5 transposon rescue are effective tools for identifying Tn5 insertion position of a target gene of a plant pathogen and for isolating the target and can be used coordinatively and cooperatively.
