荧光假单胞菌7-14 flhA基因的克隆与功能的初步分析

Cloning and primary analysis of flhA gene function of Pseudomonas fluorescens strain 7-14

为了探索flhA基因在荧光假单胞菌7-14(Pf7-14)鞭毛合成的作用,以铜绿假单胞菌UCBPP-PA14的鞭毛蛋白F lhA(NC_008463.1)为诱饵,在荧光假单胞菌Pf-5(NC_004129)、Pf0-1(NC_007492)和SBW 25(NC_012660)的全基因组中进行BLASTP比对分析,结果在Pf-5、Pf0-1和SBW 25的全基因组搜索到flhA的同系物,分别命名为flhAPf-5f、lhAPf0-1和flhASBW25。根据三者的核酸序列,设计简并引物flhA-F/flhA-R,以荧光假单胞菌7-14(Pf7-14)基因组DNA为模板进行PCR扩增,并对得到的片段测序比对,发现该片段2130 bp为一完整的开放阅读框,命名为flhAPf7-14。flhAPf7-14在氨基酸水平上与flhAPf-5和flhAPf0-1的同源性分别达到93%和94%。Southern杂交证明flhA在Pf7-14基因组中为单一拷贝。通过同源重组单交换的方式构建了Pf7-14flhA基因插入失活突变株,结果表明flhA突变体缺失了鞭毛装置并丧失了游动性,而互补菌株则恢复了鞭毛合成能力和游动能力。本研究为今后进一步研究Pf7-14鞭毛合成、菌体游动性与该菌定殖能力及生防能力的关系奠定基础。

Pseudomonas aeruginosa belongs to Pseudomonas spp..The flhA mutant and motility of P.aeruginosa was shown to be nonmotile.To explore the role of flhA in P.aeruginosa 7-14（Pf7-14） flagella synthesis,a BLASTP search was performed in the genome of P.fluorescens strains Pf-5（NC_004129）,Pf0-1（NC_007492） and SBW25（NC_012660） using flhA protein from P.aeruginosa UCBPP-PA14（NC_008463.1） as a bait.The flhA homologs,named flhAPf-5,flhAPf0-1 and flhASBW25,were obtained from their genomes,respectively.According to the nucleic acid sequences of flhAPf-5,flhAPf0-1,and flhASBW25,a pair of degenerate primers flhA-F/flhA-R,were designed and used to amplify the flhA gene from Pf7-14.By PCR amplification and sequencing,a 2 130 bp DNA fragment,a complete ORF,named flhASBW25 was obtained from Pf7-14.The result of sequence analysis revealed that flhAPf7-14 shared 93% and 94% amino acid similarities with those from Pf-5 and Pf0-1,respectively.The result of Southern blot suggested that flhA had a single copy in Pf7-14 genome.A flhA mutant was constructed by single crossover and the flhA mutant was deficient in flagella assemble and swimming motility.The study paves the way for further analyzing the effect of flagella synthesis and swimming motility on colonization and biocontrol ability of Pf7-14.