采用两种突变方法对甘蓝黑腐病菌XC_1348基因的功能解析

The Functional Analysis of the XC_1348 Gene of Xanthomonas campestris by Two Mutant Methods

为研究甘蓝黑腐病菌Xcc8004基因组中一个功能未知的假定蛋白XC_1348的基因功能,本研究用自杀质粒pK18mob和pK18mobSacB分别通过同源单交换和同源双交换构建XC_1348的非极性整合突变体1348nk和有标记的缺失突变体DM1348Gm,并对两种突变体的表型进行检测,结果显示该基因突变后不影响细菌的胞外多糖(EPS)产量、胞外酶活性以及细菌游动性。有趣的是该基因的两种突变体的致病力存在较大差异,原因可能是Xcc经两种方法定点突变后分别具有的不同基因组结构而影响到某些基因的表达所致。通过研究初步了解了XC_1348基因的功能并为今后改进定点突变方法提供现实依据。

To investigate the unknown function of the putative protein of XC_1348 gene of Xanthomonas campestris pv. campestris strain 8004, we used the suicide plasmid pK18mob and pK18mobSacB to construct a nonpolar integrated mutant 1348nk and a marked deletion mutant DM1348Gm by homologous single-crossover and homologous double-crossover integration. Phenotypes studies of these two mutants showed that the mutation on XC_1348 gene did not affect the yield of exopolysaccharides （EPS）, the extracellular enzymatic activity, and bacterial motility. However, interestingly, the two mutants showed a great variation in virulence. The reason might be that the two different mutation methods changed the genomic structure of Xcc 8004 and the expression of certain genes was affected accordingly. This study not only helps us understand XC_1348 gene preliminarily, but also provides us a basis to improve the site-directed mutation method for further study.