大丽轮枝菌微丝荧光标记载体构建及应用

Construction and application of actin fluorescent marker in Verticillium dahliae Kleb.

摘要:

微丝在真菌生长发育、胞质分裂等生命过程中具有重要功能。通过农杆菌介导遗传转化方法，将荧光mCherry标记微丝的表达载体pSULPH-Lifeact-mCherry转入大丽轮枝菌 (Verticillium dahliae Kleb.) 野生型V592，获得稳定的微丝荧光标记菌株V592/Lifeact-mCherry，并检测了其生物学表型和孢子萌发、菌丝生长等过程中的微丝荧光动态变化。结果表明：微丝荧光标记菌株的菌落形态、生长速率、产孢量、萌发率等表型与野生型没有显著差异；且可以观察到微丝荧光信号在分生孢子和菌丝的顶端及隔膜都有清晰定位，同时对该菌株隔膜形成过程微丝动态观察发现，微丝参与胞质分裂进程中肌动球蛋白收缩环CAR (Contractile actomyosin ring) 的形成。微丝荧光标记菌株可用于微丝在真菌发育中的动力学研究，这为深入研究微丝在真菌发育及致病过程中的作用机制提供理论与实践支撑。

Abstract:

Actin filaments play an important role in fungal life processes such as growth, development and cytokinesis. The expression vector pSULPH-Lifeact-mCherry of fluorescent mCherry-labeled actin was transferred into Verticillium dahliae Kleb. wild type V592 by the genetic transformation system mediated by Agrobacterium tumefaciens to obtain the stable fluorescent labeled actin strain V592/Lifeact-mCherry. Then we detected its biological phenotype and the dynamic changes of actin fluorescence during the process of spore germination, mycelial growth and development. There was no significant difference in the colony morphology, colonial growth rate, sporulation and germination rate between the fluorescent labeled actin strain and the wild type. The actin fluorescence signal was observed at the tip of the conidia and hyphae and the septum clearly. Actin participated in the formation of the contractile actomyosin ring (CAR) during cytokinesis by observing the dynamic behavior of the actin in the process of hyphal septum formation. The fluorescent labeled actin strain can be used to study the dynamics of actin in fungal development to provide theoretical and practical support for further study of the mechanism of actin in fungal development and pathogenesis.