水稻纹枯病菌Rspg1基因的克隆、表达及其编码产物生物信息学分析

Cloning,prokaryotic expression and bioinformatics of Rspg1 gene of Rhizoctonia solani

【目的】为克隆、表达水稻纹枯病菌Rspg1基因,明确其编码产物的生物学特性,为研究该基因在病菌致病过程及与寄主互作中的作用提供理论依据。【方法】据GenBank提供的相关序列设计特异引物扩增目的基因,并对之进行原核表达和生物信息学分析。【结果】从水稻纹枯病菌基因组DNA中扩增出一1395 bp的目的片段。RT-PCR分析表明,该片段为Rspg1基因的完整开放阅读框,含有5个内含子(278-334,57 bp;545-601,57 bp;657-715,59 bp;1090-1155,66 bp;1244-1304,61 bp),编码区为1 095 bp,编码一含有364个氨基酸的蛋白。原核表达Rspg1基因,表达产物大小约为40 kDa,具明显的多聚半乳糖醛酸酶(Polygalacturonase,PG)活性,活性值为277.78 U/mg。生物信息学分析表明,RsPG1中含有所有生物PG特有的严格保计序列180NTD、202DD、223GHG和255RIK,存在一18个氨基酸的信号肽分子;二级结构以α-螺旋、β-折叠和随机卷曲为其基本结构单元,6个半胱氨酸残基形成3个二硫键,跨膜结构预测以从胞内向胞外分泌为主;三级结构为10个重复的β-折叠环按右手螺旋规则排列形成的螺旋结构,形成一个开放的有活性的裂隙结构。【结论】Rspg1基因编码产物为一40 kDa蛋白,具明显的PG(Polygalacturonase,PG)活性,以胞外分泌为主,且有一个开放的有活性的裂隙结构。

[ Objective] The study was aimed at understanding the roles of polygalacturonases in the pathogenicity and the interaction between Rhizoctonia solani and rice. [ Methods] According to the sequences of Rspgl of R. solani deposited in GenBank, a pair of specific primers was designed. The gene Rspgl was cloned and expressed using prokaryotic expression tool to elucidate its biological characteristics. The structures of the protein RsPG1 were predicted using bioinformatics tools. [ Results] A 1395-bp fragment including an open reading frame （OFR） of Rspgl was amplified from the genomic DNA of the pathogen. Compared with RT-PCR results, it was found that this sequence fragment contains five introns （positions 278-334, 545-601, 657-715, 1090-1155 and 1244-1304） and one 1095 bp ORF. The ORF was predicted to encode 364 amino acids. Bioinformatics analysis showed that RsPG1 contains an 18-amino acid signal peptide and 4 conserved sequence segments （180 NTD, 202DD, 223 GHG and 255 RIK） characteristic of all the polygalacturonases. The main structural elements of the secondary structure are a-helix, β-sheet and random coil. Six cysteines form three disulfide bonds （Cys24-Cys40, Cys204-Cys220 and Cys329-Cys333）. Transmembrane prediction analysis suggested that RsPG1 could be secreted outside the cell. Tertiary structure is a right-handed helix which consisted of ten repeated β-sheet, forming an opening activity cleft. [ Conclusion] RsPG1 is tentatively a 40 kDa protein with polygalacturonase enzyme activity at 277.78 U/mg. It is probably a secreted protein and has characteristics of all the polygalacturonases. The results can help to further understand the roles that R. solani polygalacturonases play during the pathogenicity and how the pathogen interacts with the host.