中国水稻科学 ›› 2018, Vol. 1 ›› Issue (1): 111-118.DOI: 10.16819/j.1001-7216.2018.7094

• 研究报告 •    下一篇

水稻纹枯病菌RsPhm基因的克隆及其表达分析

江绍锋, 王陈骄子, 舒灿伟, 周而勋*()   

  1. 华南农业大学农学院/广东省微生物信号与作物病害防控重点实验室, 广州510642;
  • 收稿日期:2017-08-10 出版日期:2018-01-10 发布日期:2018-03-10
  • 通讯作者: 周而勋
  • 基金资助:
    国家自然科学基金资助项目(31271994, 31470247)

Cloning and Expression Analysis of RsPhmGene inRhizoctoniasolani AG-1ⅠA of Rice Sheath Blight Pathogen

Shaofeng JIANG, Chenjiaozi WANG, Canwei SHU, Erxun ZHOU*()   

  1. Guangdong Province Key Laboratory of Microbial Signals and Disease Control/College of Agriculture, South China Agricultural University, Guangzhou 510642, China;
  • Received:2017-08-10 Online:2018-01-10 Published:2018-03-10
  • Contact: Erxun ZHOU

摘要:

【目的】为了阐明苯酚2-单加氧酶基因(RsPhm)在水稻纹枯病菌(Rhizoctoniasolani 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(qRT-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,RsPhm基因的DNA和cDNA全长序列分别为2628 bp和1983 bp,编码660个氨基酸。系统进化树分析显示,RsPhm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qRT-PCR技术分析了在不同浓度儿茶酚胁迫下水稻纹枯病菌RsPhm基因的转录表达情况。外源儿茶酚能提高RsPhm基因的表达量,在12.5 µg/mL浓度下表达量最高,极显著上调35.7倍,在25 µg/mL和50 µg/mL浓度下表达量分别上调19.1倍和28.4倍,但在100 µg/mL浓度下表达量仅上调2.1倍。【结论】获得了RsPhm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RsPhm基因调控黑色素形成机制奠定了理论基础。

关键词: 水稻纹枯病菌, 苯酚2-单加氧酶基因, 基因克隆, 表达分析

Abstract:

【Objective】In order to elucidate the functions of phenol 2-monooxygenase(RsPhm)gene in melanization of RhizoctoniasolaniKühnAG-1ⅠA, the causal agent of rice sheath blight,【Method】the gene was cloned by routine PCR andRT-PCR techniques, and the bioinformatics analysis of this gene was conducted; furthermore, therelative expression levelunder catecholstresswas determinedby using fluorescence quantitative real-time PCR (qRT-PCR) technique.【Result】Bioinformatics analysis showed that the full-lengthDNA and cDNA sequences of RsPhmgene were 2628 bp and 1983 bp, respectively, which encode660 amino acids. The phylogenetic tree analysis showed thatRsPhm gene hada close relationship in different anastomosis groups (AGs) ofR.solani, and a certainevolutionary conservation amongdifferent fungal species. Results of qRT-PCR indicated that the exposure to exogenous catecholcould improvethe expression level ofRsPhm gene, and which peaked at 12.5 µg/mL of catechol,with a significant increase of 35.7 times, 19.1times and 28.4 timesup-regulated at 25 µg/mL and 50 g/mL,respectively, but only 2.1 times up-regulated at 100 g/mL.【Conclusion】The full-length sequence ofRsPhm gene was obtained, its basic biological information was understood, and its expression pattern under catechol stress was clarified.Thesefindings will lay abasis for the scientific and systematic elucidation of regulatorymechanism of melanin formation byRsPhmgene ofR. solaniAG-1ⅠA.

Key words: RhizoctoniasolaniKühnAG-1ⅠA;, RsPhm gene, gene clone, expression analysis

中图分类号: