农杆菌介导的甜瓜蔓枯病菌遗传转化体系的建立

Transformation of Didymella bryoniae mediated by Agrobacterium tumefaciens

摘要:

甜瓜蔓枯病是当前危害瓜类的主要病害，严重影响甜瓜的产量和品质，但是蔓枯病菌Didymella bryoniae病原学研究还非常落后，关于该菌功能基因的研究还未见报道。本研究以携带潮霉素B磷酸转移酶基因(hph) 的pBIG2RHPH2作为转化载体，根癌农杆菌C58C1作为转化介体，转化甜瓜蔓枯病菌的强致病菌株DB11。研究发现，甜瓜蔓枯病菌的最优转化体系为：甜瓜蔓枯病菌的分生孢子悬浮液浓度为1×106个孢子/mL，农杆菌悬浮液OD600为0.15，共培养时间48 h，诱导培养基中添加200 μg/mL乙酰丁香酮，选择培养基添加100 μg/mL潮霉素B、200 μg/mL头孢噻肟钠、200 μg/mL氨苄青霉素和200 μg/mL四环素。1×105个蔓枯病菌分生孢子可以产生45个左右的转化子，随机挑取3个转化子进行PCR和RT-PCR检测发现，在不含潮霉素B的PDA培养基平板上转化子连续培养5代后，hph基因仍能稳定存在和转录，Southern blotting检测发现，T-DNA都是单拷贝插入3个转化子的染色体内。本研究建立的甜瓜蔓枯病菌的转化体系将为该病菌的功能基因研究和寄主与病原菌的互作研究提供重要技术支撑。

Abstract:

Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. The disease can seriously reduce melon yield and quality. However, little information is available on the genetics and functional genomics of the fungal pathogen. In this study, we developed an Agrobacterium-mediated transformation system for D. bryoniae by using a universal pathogenic isolate DB11 and the Agrobacterium tumefaciens strain C58C1 carriing plasmid pBIG2RHPH2 harboring the hygromycin B phosphotransferase gene (hph). Total 45 transformants could be obtained per 1×105 spores when 1×106 spores per milliliter of D. bryoniae spore suspension were cocultivated with Agrobacterium cells at OD600=0.15 for 48 h in the presence of induction medium (pH 5.2) containing acetosyringone at 200 μg/mL and selection medium contained 100 μg/mL of hygromycin B and 200 μg/mL of cefotaxime sodium, ampicillin and tetracycline, respectively. The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe. The transformation system will be useful for further studies of functional genes in D. bryoniae.