水稻黄单胞菌hrp基因簇中致病相关基因hpaB的鉴定  

Identification of a hrp-associated gene hpaB in the role of pathogenicity of Xanthomonas oryzae pv.oryzae

作　　者：曾申艳[1] 胡军[1] 黄贵修[2] 何朝族[1] 

机构地区：[1]中国科学院微生物研究所植物基因组国家重点实验室 [2]中国热带农业科学院环境与植物保护研究所,儋州571737

出　　处：《微生物学报》2007年第3期402-406,共5页Acta Microbiologica Sinica

基　　金：中国科学院创新工程方向项目(KSCX2-YW-005)~~

摘　　要：由水稻黄单胞菌引起的水稻白叶枯病是水稻最严重的细菌性病害。通过筛选18000个XooTn5转座子插入突变体,得到其中一个致病力缺失的突变体XOG11。TAIL-PCR方法分离该突变体中插入转座子的侧翼序列,发现转座子插入到位于hrp基因簇的hpaB基因中。对该基因进一步的分析表明该基因编码一个含有156个氨基酸,等电点为4.28,亮氨酸含量为14.4%的蛋白HpaB。Southern blot和PCR验证表明Tn5在该突变体中为单拷贝插入且未发生转座子携带侧翼序列的转移。将hpaB克隆到具有广泛寄主的质粒pHM1中,转化重组质粒进入突变体后,突变体恢复了在其寄主水稻IR24上的致病力,而转化空质粒pHM1后的突变体仍然表现为致病力缺失。证实了水稻黄单胞菌中hpaB基因与该细菌的致病力相关,在侵染水稻的过程中起着不可缺失的作用。Xanthomonas oryzae pv. oryae （ Xoo ）, a Gram-negative bacterium, is the causal agent of rice bacterial blight disease, which can cause severe yield loss of rice worldwide. To identify genes contributing to virulence and explore the possible mechanism of pathogenicity, transposon mutagenesis was used to isolate nonpathogenic mutants. By screening of a high-quality Tn5-like transposon （EZ： ：TN） insertional mutant library of Xoo PXO99 against a host plant （rice cuhivar IR24）, one virulence-deficient mutant, XOGII, was identified. Genomic fragment flanking the insertion site of the mutant was amplified by thermal asymmetric interlaced polymerase chian reaction （TAIL-PCR） and sequenced. The result of NCBI blast homologue searching of the fragment shows that the transposon was inserted into a hrp associated gene, hpaB. Xoo hpaB gene is one of the hrp gene cluster members that encode a type Ⅲ secretion system （TTSS） and locates at the downstream of hrpE. The product of hpaB in Xoo is a small （Molecular Weight, 17.6kDa）, acidic （PI, 4.28） and Leucine-rich （14.4 % ） protein and shares high homology with corresponding proteins in other Xanthomonas. It suggests that HpaB may play as a TI＇SS chaperone. Mutant XOGI 1 was confirmed both by PCR and Southern blotting： The PCR result by using primers upstream and downstream of hpaB respectively verified Tn5 insertion in hpaB and excluded the rare case of second transfer of the transposon associated with flanking sequence; Southern blot of digested genomic DNA with the probe of Km resistance gene aph proved that XOGI 1 was inserted by a single-copy transposon, indicating that the loss of pathogenicity in XOGI 1 was due to the Tn5 insertion in hpaB gene. Genetic complementation by cloning hpaB in the wide host range plasmid pHMI and transferring the recombinant plasmid into XOGI 1 restored its pathogenicity in IR24. These results suggest that the pathogenicity deficiency of XOGI 1 is due to the mutation of hpaB gene.

关 键 词：水稻黄单胞菌 TAIL-PCR hrp基因簇 hpaB基因 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]