摘要
十字花科黑腐病菌(Xcc8004)中的一个转录调控因子XC_2736(HpaR1)在致病过程中具有重要的作用。前期研究发现该转录调控因子可能调控胞外纤维素酶的合成。为了解HpaR1对纤维素酶的转录调控机理,本研究对HpaR1进行原核表达纯化,并与488bp的包含XC_0639的启动子区DNA片段进行凝胶电泳迁移率试验,发现HpaR1与XC_0639启动子可以发生结合。将488bp的XC_0639的启动子DNA片段与报告基因gus融合,构建XC_0639的报告质粒pGUS0639r,分别导入野生型8004菌株和缺失突变体DM2736中,分析发现在突变体背景下GUS的表达水平比野生型背景明显降低。表明HpaR1正调控XC_0639的表达。构建XC_0639的极性整合突变体PK0639,检测发现PK0639几乎丧失胞外纤维素酶的活力;通过功能反式互补构建的互补菌株CPK0639可以恢复纤维素酶活性。研究结果表明HpaR1通过调控纤维素酶基因XC_0639的表达来调控细胞的纤维素酶活性。本研究为更深入地了解HpaR1如何调控细菌生理生化功能奠定了基础。
The transcriptional regulator XC_2736 （HpaR1） of X. campestris pv. campestris （Xcc） 8004 plays important roles in regulating bacterial diverse biological processes. Previous work showed that this transcriptional regulator might play role on extracellular cellulase. To illustrate the regulatory mechanism, HpaR1 was over expressed, and electrophoretic mobility shift assays （EMSA） were performed using HpaR1 protein and the promoter fragment end XCA-p of XC_0639. Results showed that HpaR1 could specifically bind to the promoter fragment ofXC_0639. The reporter plasmid pGUS0639r was constructed by fusing the promoter ofXC_0639 with gus gene, and them been introduced into wild type strain 8004 and the HpaRl mutant DM2736, respectively. The expression level of GUS decreased significantly in the mutant, compared to the wild type background, suggesting HpaR1 positively regulates the expression ofXC_0639. Mutation in XC_0639 reduces the cellulose activity greatly in Xcc. The results revealed HpaR1 regulates the cellulase activity by regulating the expression of the cellulase gene XC_0639. This work expends our horizons on the underlying regulatory mechanism and the physiological and biochemical function ofHpaR1.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2012年第4期333-340,共8页
Genomics and Applied Biology
基金
国家自然科学基金(31171206)项目资助
关键词
十字花科黑腐病菌
转录调控因子
HpaR1
纤维素酶
X. campestris pv. campestris, Transcriptional regulator, HpaR1, Extracellular cellulose
