果生炭疽菌转录因子CfHac1的BRLZ结构域生物学功能研究

Functional analysis of BRLZ motif of the transcription factor CfHac1 in Colletotrichum fructicola

【目的】炭疽病是油茶的主要病害,导致巨大的经济损失。果生炭疽菌是油茶炭疽病优势致病菌。对果生炭疽菌的转录因子CfHac1的结构域进行分析,为进一步解析转录因子CfHac1调控病菌致病的分子机制奠定基础,对挖掘油茶炭疽病防控新途径具有重要的理论意义。【方法】采用点突变技术构建转录因子CfHac1结构域缺失载体,通过PEG介导法将该回补载体转化至CfHAC1基因完全缺失突变体的原生质体中,通过博来霉素抗性和荧光筛选获得结构域缺失回补菌株,进一步研究BRLZ结构域在果生炭疽菌中的生物学功能。【结果】结果发现转录因子CfHac1含有一个碱性亮氨酸拉链结构域(BRLZ),该结构域包含58个氨基酸残基;与野生型菌株和完全互补菌株相比,BRLZ结构域缺失突变株ΔCfhac1^(ΔBRLZ)生长速率显著降低,分生孢子产量显著减少,不能形成附着胞,对内质网压力胁迫更敏感,丧失对油茶叶的致病力,其表型与ΔCfhac1突变体一致。【结论】上述结果表明,BRLZ是转录因子CfHac1重要的结构域,对CfHac1在果生炭疽菌中行使正常的生物学功能具有重要的调控作用。

[Objective]Colletotrichum fructicola is a major pathogen causing anthracnose on Camellia oleifera,and leads to substantial losses annually.The domain analysis of transcription factor CfHac1 will help us understanding the CfHac1-regulated pathogenic mechanism of the pathogen and provide new insights to control this disease.[Method]We constructed the domain deletion vector using point mutation technology and introduced it into theΔCfhac1 mutant through the PEG-mediated protoplast transformation.The strains were selected by bleomycin and fluorescence,then the function was analyzed in C.fructicola.[Result]The structure prediction revealed that CfHac1 contained one basic region-leucine zipper motif(BRLZ),which contained 58 amino acid residues.Comparing to the wide type and complemented strains,the mycelial growth rate,conidiation and appressorium formation rate of the Cfhac1 ^(ΔBRLZ) were significantly reduced,and the Cfhac1^(ΔBRLZ) strain was sensitive to dithiothreitol.The pathogenicity test showed that the Cfhac1^(ΔBRLZ) lost the ability to infect C.oleifera leaves,whose phenotype is consistent with ΔCfhac1 mutant.[Conclusion]The results show that BRLZ is an important domain of CfHac1 and is essential for normal function of CfHac1 in C.fructicola.