水稻白叶枯病菌转录调控因子基因fleQxoo和σ^54因子基因rpoNxoo的分子鉴定

Molecular identification of the transcriptional regulator fleQxoo and the alternative σ^(54) factor gene rpoNxoo of Xanthomonas oryzae pv. oryzae,the casual pathogen of bacterial blight in rice

为了阐明水稻白叶枯病菌(Xanthomonas oryzaepv.oryzae,简称Xoo)鞭毛生物合成及运动性的调控机理,本研究首先通过基因克隆、序列分析、缺失突变及表型测定,对转录调控因子fleQxoo和编码σ54因子的基因rpoNxoo进行了分子鉴定。通过基因特异性扩增,成功地从Xoo菌株PXO99A中克隆了fleQxoo和rpoNxoo。其基因序列与其它黄单胞病原菌中的同源序列高度保守。FleQxoo是NtrC家族激活蛋白成员之一,具有与σ54作用的结构域和DNA结合保守结构域(HTH)。用标记交换法构建了△fleQxoo和△rpoNxoo基因缺失突变体。与PXO99A相比,△fleQxoo和△rpoNxoo鞭毛产生能力丧失,运动性减弱,基因互补可以使之恢复;但其胞外纤维素酶和木聚糖酶活性以及对烟草叶片组织的致敏性无明显改变。因此,FleQxoo和σ54主要参与了鞭毛生物合成及其运动性的调控。

To better understand the regulation of single and polar flagellum biogenesis in Xanthomonas oryzae pv.oryzae（Xoo）,the casual pathogen of bacterial blight in rice,molecular identification of the transcriptional regulator fleQxoo and the alternative σ^54 factor gene rpoNxoo was performed through gene cloning,sequencing and deletion analysis.fleQxoo and rpoNxoo cloned from genomic DNA of the wild-type PXO99^A were found to be highly conserved in plant-pathogenic Xanthomonas spp..FleQxoo,an NtrC family activator protein and a cognate activator of σ^54,was structurally featured in σ^54-interacting domain and DNA-binding domain with a helix-turn-helix motif.△fleQxoo and △rpoNxoo,the gene deletion mutants were constructed after a double crossover recombination event between Gentamycin resistance gene（Gm^R）and fleQxoo or rpoNxoo through the marker exchange,and validated by PCR assay.Both mutants were non-flagellated and attenuated in flagellar motility on the semi-solid medium,which could be restored through complementation of mutants by introducing fleQxoo and rpoNxoo,respectively.Moreover,no significant changes in production of extracellular cellulase and xylanase in vitro and induction of hypersensitive response（HR）on non-host tobacco were observed in both mutants compared to PXO99^A.Therefore,both FleQxoo and σ^54 as the master regulators were involved in regulation of flagellation of Xoo.