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应用Mu转座重组技术研究铜绿假单胞菌 (Pseudomonasaeruginosa)蹭行运动 (Twitchingmotility)的相关基因。通过转座突变、表型筛选 ,得到 8个Twitchingmotility缺陷或减弱的突变子。经过基因克隆、核苷酸测序研究 ,鉴定转座子插入到基因组中的位置。结果表明 ,在其中 4个突变子中 ,转座子分别插入到与IV型菌毛生物合成和功能相关的 3个已知基因中 (其中有两个突变子转座子插入到同一基因的不同位置 ) ,它们是pilV ,pilQ ,algR。另外 4个突变子中 ,有 3个是转座子分别插入到基因pilL基因的前端 ,中部和后端 ,均引起Twitchingmotility功能缺失。另一个突变子中 ,转座子插入到基因PA1 82 1中 ,引起Twitchingmotility功能减弱。PilL和PA1 82 1的编码产物均属于 3 类蛋白质 ,它们的功能是根据其保守的氨基酸基序或基因序列与已知功能基因的相似性推测得出的。但缺乏详细的试验证据。研究结果为pilL控制Twichingmotility提供了有力的证据。并证实基因PA1 82 1与Twitchingmotility有关。将Mu转座重组技术应用到假单胞菌的研究中 ,国内外均未见报道。由于该技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点。保证了表型的改变与转座子插入位点的基因突变的一一对应关系。为进一步研究铜绿假
Mu transposing recombination technique was used to study the related genes of Pseudomonas aeruginosa Twitching motility. By transposon mutagenesis and phenotypic screening, eight Twitchingmotility deficient or attenuated mutants were obtained. After gene cloning, nucleotide sequencing studies identified the insertion of the transposon into the genome. The results showed that of the four mutants, the transposons were inserted into three known genes related to type IV pili biosynthesis and function (two of them were inserted into different genes of the same gene Position), they are pilV, pilQ, algR. Of the other four mutants, three were transposons inserted into the front, middle and back of the gene pilL, respectively, causing loss of Twitching motility. In another mutant, the transposon was inserted into the gene PA1 82 1, resulting in weakened Twitching motility. The coding products of PilL and PA1 82 1 belong to class 3 proteins and their function is based on the similarity of their conserved amino acid motifs or gene sequences to known functional genes. But lack of detailed experimental evidence. The results provide strong evidence for pilL’s control of Twichingmotility. And confirmed that the gene PA1 82 1 and Twitchingmotility related. Mu transposing recombinant technology applied to the study of Pseudomonas, both at home and abroad have not been reported. Due to the advantage of random single-point insertion, this technique overcomes the shortcomings that traditional transposons can migrate on chromosomes. Which ensures the one-to-one correspondence between the change of the phenotype and the gene mutation of the transposon insertion site. For further study of copper green leave