水稻条斑病菌gacA基因的克隆及功能初析

Cloning, sequencing and fuctional study of gacA gene from Xanthomonas oryzae pv. oryzicola

摘要:

根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacA_Xooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacA_Xooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。

Abstract:

A gacA homologue, designated gacA_Xooc, was cloned from Xanthomonas oryzae pv. oryzicola (Xooc), a bacterium that causes leaf streak of rice, with degenerated primers by polymerase amplification reaction (PCR). NCBI blast search indicated that GacA_Xooc had a similar structure to that of other GacA proteins, and had a CheB（Chemotaxis response regulator containing a CheY-like receiver domain）domain. Sequence comparison showed that the gacA_Xooc was conserved in the Xanthomonas genus. Homology search revealed that the gacA_Xooc was 99.7% similarities to gacA（AY870457, this lab）of Xanthomonas oryzae pv. oryzae (Xoo). A gacA_Xooc disruption mutant was successfully generated by a single cross-over event, and confirmed by PCR and Southern blot. But the mutant still had strong pathogenicity，and its virulence was not obviously different from that of wild type strain. The gacA did not globally regulate metabolism in Xooc, which was different from DC3000 of P. syringae pv. tomato, CHA0 of P. fluorescens and IC1270 of Serratia plymuthica. Chemotaxis to 0.1% tryptone of the mutants was reduced compared to wild type strain. The results suggest that gacA_Xooc is involved chemotaxis of Xooc. Nevertheless, how gacA to regulate chemotaxis of Xooc, transcription and expression of genes involved in regulation still need to be further studied.