论文部分内容阅读
【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 purpose of this study was to clone and express Rspg1 gene of Rhizoctonia solani and clarify the biological characteristics of its coding product, providing a theoretical basis for studying the role of Rspg1 in pathogenicity and host-host interaction. 【Method】 According to the related sequences provided by GenBank, specific primers were designed to amplify the target gene and analyze the prokaryotic expression and bioinformatics. 【Result】 A 1395 bp fragment was amplified from genomic DNA of Rhizoctonia solani. RT-PCR analysis showed that the fragment was a complete open reading frame of Rspg1 gene and contained 5 introns (278-334, 57 bp; 545-601, 57 bp; 657-715, 59 bp; 1090-1155, 66 bp; 1244-1304, 61 bp) with a coding region of 1 095 bp encoding a protein with 364 amino acids. Prokaryotic expression of Rspg1 gene, the expression product size of about 40 kDa, with significant polygalacturonase (Polygalacturonase, PG) activity, the activity value of 277.78 U / mg. Bioinformatics analysis showed that RsPG1 contained strictly conserved sequences of all biological PGs such as 180NTD, 202DD, 223GHG and 255RIK, and an 18-amino acid signal peptide molecule existed. The secondary structure consisted of α-helix, β-sheet and random curl Its basic structural unit, six cysteine residues to form three disulfide bonds, transmembrane structure is predicted to be mainly from the intracellular to extracellular secretion; tertiary structure of the 10 repeated β-sheet ring according to the right-handed spiral rules Arrange the spiral structure formed, forming an open and active fracture structure. 【Conclusion】 The product encoded by Rspg1 is a 40 kDa protein with obvious activity of PG (PG), which is mainly secreted by cells and has an open and active cleft structure.