十字花科黑腐病菌中调控致病相关基因hpaS的XC_2486基因鉴定

十字花科黑腐病菌hpaS影响致病性、HR其编码产物分别与HpaR2、HrpG形成双组分系统。为了深入了解hapS的表达调控机制,本研究将hpaS的启动子与蔗糖敏感基因sacB融合,构建了hpaS的表达报告质粒pL6sacB3670并导入十字花科黑腐病菌野生型菌株8004中,获得了报告菌株8004/pL6sacB3670。然后利用转座子EZ：Tn5对该报告菌株进行诱变,筛选到一株转座子EZ：Tn5插入基因组的克隆。通过测序定位分析发现该克隆是由转座子EZ：Tn5插入到编号为XC_2486的ORF所产生的。然后将hpaS启动子与报告基因gusA融合的报告质粒pL6GUS3670分别导入野生型8004和XC_2486的突变体239C02中,测定比较pL6GUS3670的GUS表达水平,结果显示在突变体背景下GUS表达水平明显比在野生型背景下降低。这表明XC_2486正调控hpaS的表达。对XC_2486突变体239C02进行致病性和过敏反应检测发现,XC_2486与致病性相关,与HR无关。

Identification of Regulation Gene of Pathgennicity-related Gene hpaS in Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris （Xcc） is the causal agent which cause the black rot disease in cruciferous plants worldwide. In the previous work, we had found hpaS is involved in virulence, HR. In this work, the reporter plasmid pL6sacB3670 was constructed with the promoter region ofhpaS fused to the coding region of sacB, and transferred into Xcc wild-type strain 8004. The reporter strain 8004/pL6sacB was mutagenized randomly by the transposon EZ：Tn5 and one clone was identified to be inserted in the Xcc genome by transposon EZ：Tn5. Then we identified that the clone was discruption of in the open reading frame XC_2486, which was annoated as galactose-binding protein regulator. To futher confirm the regulation of XC_2486, the reporter plasmid pL6GUS3670 was constructed with hpaS fused to the coding region ofgusA and was introduced into the wild-srain 8004 and XC_2486 mutant srain 239C02 respectively. The GUS activity of pL6GUS3670 in the XC_2486 mutant background was significantly slower than in the wild-type background. These results show that XC2486 regulate the expression ofhpaS positively. The pathogenicity and hypersensitive response （HR） of XC_2486 mutant 239C02 were determined and the result show that XC_2486 is involved in pathogenicity but not involved in HR.