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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。
In order to elucidate the regulatory mechanism of flagellar biosynthesis and motility in Xanthomonas oryzaepv. Oryzae (Xoo), we first detected the expression of the transcriptional regulatory factors fleQxoo and its coding by gene cloning, sequence analysis, deletion mutation and phenotyping. The σ54 gene, rpoNxoo, was molecularly identified. By gene-specific amplification, fleQxoo and rpoNxoo were successfully cloned from Xoo strain PXO99A. Its gene sequence is highly conserved among homologous sequences in other X. pathogen. FleQxoo is a member of the NtrC family of activating proteins with a domain that interacts with sigma54 and a DNA binding conserved domain (HTH). The △ fleQxoo and △ rpoNxoo gene deletion mutants were constructed by the marker exchange method. Compared with PXO99A, △ fleQxoo and △ rpoNxoo flagella lost their ability to produce exercise, weakened and complemented by genes could recover them. However, their extracellular cellulase and xylanase activities and sensitization to tobacco leaf tissue were not significant change. Therefore, FleQxoo and sigma54 are mainly involved in the regulation of flagella biosynthesis and their motility.