水稻纹枯病菌RsPhm基因的克隆及其表达分析

Cloning and Expression Analysis of Rs Phm Gene in Rhizoctonia solani AG-1ⅠA of Rice Sheath Blight Pathogen

【目的】为了阐明苯酚2-单加氧酶基因(Rs Phm)在水稻纹枯病菌(Rhizoctonia solani AG-1ⅠA)黑化中的功能,【方法】采用常规PCR和RT-PCR技术对该基因进行克隆和生物信息学分析,通过荧光定量PCR(q RT-PCR)技术检测在儿茶酚胁迫下该基因的相对表达量。【结果】生物信息学分析结果表明,Rs Phm基因的DNA和c DNA全长序列分别为2 628 bp和1 983 bp,编码660个氨基酸。系统进化树分析显示,Rs Phm基因在立枯丝核菌(R.solani)不同融合群中具有较近的亲缘关系,在不同真菌之间在进化上具有一定保守性。通过q RT-PCR技术分析了在不同浓度儿茶酚胁迫下水稻纹枯病菌Rs Phm基因的转录表达情况。外源儿茶酚能提高Rs Phm基因的表达量,在12.5μg/m L浓度下表达量最高,极显著上调35.7倍,在25μg/m L和50μg/m L浓度下表达量分别上调19.1倍和28.4倍,但在100μg/m L浓度下表达量仅上调2.1倍。【结论】获得了Rs Phm基因全长序列,了解了其基本生物学信息,明确了其在儿茶酚胁迫下的表达模式。研究结果为科学、系统地阐明水稻纹枯病菌Rs Phm基因调控黑色素形成机制奠定了理论基础。

【Objective】In order to elucidate the functions of phenol 2-monooxygenase(RsPhm)gene in melanization of Rhizoctonia solani Kühn AG-1ⅠA,the causal agent of rice sheath blight,【Method】the gene was cloned by routine PCR and RT-PCR techniques,and the bioinformatics analysis of this gene was conducted;furthermore,the relative expression level under catechol stress was determined by using fluorescence quantitative real-time PCR(qRT-PCR)technique.【Result】Bioinformatics analysis showed that the full-length DNA and cDNA sequences of RsPhm gene were 2 628 bp and 1 983 bp,respectively,which encode 660 amino acids.The phylogenetic tree analysis showed that RsPhm gene had a close relationship in different anastomosis groups(AGs)of R.solani,and a certain evolutionary conservation among different fungal species.Results of qRT-PCR indicated that the exposure to exogenous catechol could improve the expression level of RsPhm gene,and which peaked at 12.5μg/mL of catechol,with a significant increase of 35.7 times,19.1 times and 28.4 times up-regulated at 25μg/mL and 50 g/mL,respectively,but only 2.1 times up-regulated at 100 g/mL.【Conclusion】The full-length sequence of RsPhm gene was obtained,its basic biological information was understood,and its expression pattern under catechol stress was clarified.These findings will lay a basis for the scientific and systematic elucidation of regulatory mechanism of melanin formation by RsPhm gene of R.solani AG-1ⅠA.