油茶果生炭疽菌小分子GTP酶Rab7的功能研究

Function of small GTPase Rab7 in Colletotrichum fructicola

摘要:

【目的】炭疽病是油茶的一种重要病害，果生炭疽菌是油茶炭疽病的主要致病菌。本文对果生炭疽菌小分子GTP酶Rab7进行研究，为油茶炭疽病的防控治理提供依据。【方法】构建CfRAB7基因敲除载体，通过PEG介导的原生质体转化、抗性筛选和PCR电泳验证获得果生炭疽菌突变体菌株∆Cfrab7和互补菌株∆Cfrab7/CfRAB7。进一步分析CfRAB7基因敲除突变体∆Cfrab7的生长、产孢、附着孢的形成、胁迫应答、液泡融合和致病力等生物学表型。【结果】在PDA和MM培养基上，突变体∆Cfrab7的菌落直径显著减小，产孢量和附着孢形成率显著降低，且不能穿透玻璃纸；在10 mmol/L H₂O₂条件下，∆Cfrab7生长受到明显抑制；进一步研究发现突变体∆Cfrab7液泡无法正常融合，在油茶有伤和无伤的幼叶上均不发病。【结论】CfRAB7基因参与调控果生炭疽菌生长产孢、附着孢形成、H₂O₂胁迫应答、液泡融合和致病力。

Abstract:

[Objective] Anthracnose, a major disease of tea-oil tree (Camellia oleifera), is mainly caused by Colletotrichum fructicola. In this study, we investigated the biological function of the small-molecule GTPase Rab7 of C. fructicola, aiming to provide a theoretical basis for the prevention and control of anthracnose. [Methods] The CfRAB7 gene knockout vector was constructed based on the principle of homologous recombination. After PEG-mediated protoplast transformation, resistance screening, and verification by PCR and electrophoresis, the mutant strain ∆Cfrab7 and the complementary strain ∆Cfrab7/CfRAB7 were obtained. The growth, sporulation, appressorium formation, stress response, and other biological characteristics of ∆Cfrab7 were explored. [Results] On the PDA and MM plates, ∆Cfrab7 showcased significantly decreased colony diameter, spore production, and appressorium formation. ∆Cfrab7 failed to penetrate cellophane. The oxidative stress (H₂O₂) had higher inhibition rate on the growth of ∆Cfrab7 than on that of WT and ∆Cfrab7/CfRAB7. ∆Cfrab7 did not cause disease spot on the leaves of Ca.oleifera. Furthermore, CfRab7 was required for homotypic vacuole fusion, which was essential for pathogen invasion. [Conclusion] Our findings reveal that CfRAB7 gene plays a vital role in the growth, sporulation, appressorium formation, oxidative stress response, homotypic vacuole fusion, and pathogenicity of C. fructicola.