尖镰孢菌人参专化型FoeIF1a基因的功能

Functions of FoeIF1a in Fusarium oxysporum f. sp. ginseng

摘要:

【背景】 尖镰孢菌(Fusarium oxysporum)是引起人参根腐病的优势菌株，其致病范围广泛且致病力极为严重，对农田栽参造成毁灭性危害。在尖镰孢菌中，FoeIF1a是真核生物翻译起始的关键因子，其研究对理解真菌致病机制具有重要意义。【目的】 探究翻译起始因子FoeIF1a对尖镰孢菌人参专化型(Fusarium oxysporum f. sp. ginseng)菌株0083在菌丝生长、孢子产量、高渗及胁迫应答、毒素含量、致病力等方面的影响，明确其基因功能。【方法】 对基因FoeIF1a进行生物信息学预测分析；构建基因FoeIF1a的敲除及回补载体，通过根癌农杆菌介导的转化(Agrobacterium tumefaciens-mediated transformation, ATMT)后，经分子生物学验证筛选敲除和回补菌株的转化子，观察野生型菌株Fo0083、敲除菌株及回补菌株的表型，对结果进行分析。【结果】 FoeIF1a基因全长为408 bp，编码135个氨基酸。敲除菌株ΔFoeIF1a较野生型菌株Fo0083的生长速率及生物量显著降低；孢子产量、孢子萌发率显著降低；对pH 5.0、pH 7.0、pH 9.0、真菌细胞壁胁迫因子800 μg/mL刚果红、0.01%十二烷基磺酸钠、不同碳源(1 mol/L葡萄糖，1 mol/L蔗糖)、氧化胁迫5 mmol/L H₂O₂、渗透胁迫(1 mol/L NaCl，1 mol/L KCl，1 mol/L山梨醇)及酸碱指示剂0.15%溴酚蓝均表现敏感；镰刀菌酸含量显著增加；致病力有一定程度降低。回补菌株ΔFoeIF1a-C在各方面均基本趋于野生型水平。【结论】 尖镰孢菌中FoeIF1a基因可以降低菌丝生长速率、生物量、孢子产量及萌发率，在高渗及胁迫中敏感，影响镰刀菌酸含量及致病力。

Abstract:

[Background] Fusarium oxysporum is a dominant pathogen for the root rot of ginseng, which has a wide spectrum of hosts and strong pathogenicity, causing devastating harm to cultivated ginseng in farmland. FoeIF1a of F. oxysporum is the key factor in the initiation of eukaryotic translation, and its research is of great significance for understanding the pathogenic mechanism of this fungus. [Objective] To explore the influences of FoeIF1a on the mycelial growth, spore yield, hyperosmotic and stress responses, toxin content, and pathogenicity of F. oxysporum f. sp. ginseng 0083 and clarify the gene functions. [Methods] We employed bioinformatics tools to predict the properties of FoeIF1a, and then constructed the knockout and complementary vectors of this gene. After the vectors were introduced by Agrobacterium tumefaciens-mediated transformation, the transformants were screened by molecular biology verification. We then analyzed the results by observing the phenotypes of the wild-type strain Fo0083, knockout strain, and complementary strain. [Results] The full-length FoeIF1a was 408 bp, encoding 135 amino acid residues. The growth rate, biomass, spore yield, and spore germination rate of ΔFoeIF1a were significantly lower than those of the wild-type strain Fo0083. ΔFoeIF1a was sensitive to pH 5.0, pH 7.0, pH 9.0, 800 mg/mL CR, 0.01% SDS, different carbon sources (1 mol/L glucose and 1 mol/L sucrose), oxidative stress (5 mmol/L H₂O₂), osmotic stress (1 mol/L NaCl, 1 mol/L KCl, and 1 mol/L sorbitol), and acid-base indicator (0.15% bromophenol blue). In addition, ΔFoeIF1a showed significantly increased content of fusarium acid and attenuated pathogenicity. The complementary strain ΔFoeIF1a-C approached the wild-type strain in the indicators above. [Conclusion] FoeIF1a in F. oxysporum can reduce the mycelium growth rate, biomass, spore yield, and spore germination rate, and it is sensitive to hyperosmotic stress, affecting the fusarium acid content and pathogenicity.