水稻纹枯病菌多聚半乳糖醛酸酶基因RsPG5的克隆与功能分析

Cloning and function analysis of polygalacturonase gene RsPG5 in Rhizoctonia solani, the pathogen of rice sheath blight

摘要:

【背景】纹枯病是水稻最重要的病害之一，目前对其病菌致病机制的研究还较少。【目的】鉴定更多水稻纹枯病菌致病基因，为纹枯病的防治提供理论依据。【方法】采用3'-RACE方法获得RsPG5基因全长，并使用ExPASy等在线软件对其编码产物的结构及生物学特性进行分析，测定其编码产物的致病功能。【结果】RsPG5具有7个外显子和6个内含子，编码区全长1 263 bp，可编码420个氨基酸。编码产物为糖苷水解酶GH28家族成员，具有真菌多聚半乳糖醛酸酶特有的保守序列NTD、DD、GHG和RF(I)K，并且有一个含15个氨基酸的信号肽；二级结构由α-螺旋、β-折叠和随机卷曲螺旋构成，并且可形成4个二硫键；三级结构为由α-螺旋、β-折叠和随机卷曲螺旋按右手螺旋规则形成的具有裂隙区的特定空间结构，裂隙区可能负责着其酶活功能。生物学性质预测表明，RsPG5为稳定、易溶于水的外泌性蛋白，主要定位于细胞壁、液泡和线粒体。RsPG5具有明显的多聚半乳糖醛酸酶活性，可分解果胶，破坏水稻叶鞘细胞；针刺接种分蘖末期水稻叶鞘，72 h后可形成明显的褐色坏死斑；将纹枯病菌接种至水稻叶鞘，在病菌致病过程中RsPG5可上调表达。【结论】RsPG5是一个典型的多聚半乳糖醛酸酶蛋白，为水稻纹枯病菌的重要致病因子。

关键词:功能分析;多聚半乳糖醛酸酶基因RsPG5;纹枯病菌;水稻

Abstract:

[Background] Sheath blight, caused by Rhizoctonia solani, is one of the major devastated rice diseases in the world, while little is known about the pathogenic mechanism of the pathogen. [Objective] To identify more virulence genes from R. solani and provide a theoretical basis for the control of sheath blight. [Methods] The full-length sequence of RsPG5 was obtained by 3'-RACE, and the structure and biological properties of the deduced protein were predicted by ExPASy online. The pathogenic function of RsPG5 was then determined. [Results] RsPG5 harbored seven exons and six introns, with the coding region of 1 263 bp, which encoded 420 amino acid residues. RsPG5, one member of the glucoside hydrolase family 28, contained a signal peptide of 15 residues and NTD, DD, GHG and RF(I)K domains conserved in the polygalacturonases from fungi. The secondary structure of the deduced protein contained 4 disulfide bonds, α-helix, β-sheet, and random coil, which arranged according to right-handed helix and formed a cleft that was responsible for the enzyme activity. RsPG5 was a stable, water-soluble, exocrine protein localized in cell wall, vacuole, and mitochondria. The eukaryotic expression products of RsPG5 had the polygalacturonase activity to hydrolyze pectin and destroy the sheath cells of rice. Distinct brown necrotic spots appeared 72 h after the expression products were inoculated in the rice sheathes by a needle. The expression level of RsPG5 was up-regulated in the infection course of R. solani. [Conclusion] RsPG5 is a typical polygalacturonase and a major pathogenic factor of R. solani.

Key words:function analysis;polygalacturonase gene RsPG5;Rhizoctonia solani;rice