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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48h,诱导培养基中添加200μg/mL乙酰丁香酮,选择培养基添加100μg/mL潮霉素B、200μg/mL头孢噻肟钠、200μg/mL氨苄青霉素和200μg/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。
Melon blight is the current main hazard to melon and seriously affects the yield and quality of melon. However, the etiological study of Didymella bryoniae is still far behind. So far, there is no report on the functional gene of this melon. In this study, pBIG2RHPH2 harboring hygromycin B phosphotransferase gene (hph) was used as the transformation vector and Agrobacterium tumefaciens C58C1 as the transformation mediator to transform the strongly pathogenic strain DB11 of meliloti. The results showed that the optimum transformation system of A. meliloti was as follows: the concentration of conidia suspension of A. meliloti was 1 × 106 spores / mL, the Agrobacterium suspension OD600 was 0.15 and the co-culture time was 48h. The induction medium 200 g / mL acetosyringone, 100 g / mL hygromycin B, 200 g / mL cefotaxime sodium, 200 g / mL ampicillin and 200 g / mL tetracycline were added to the selection medium. About 1 × 105 strains of Bacillus subtilis could produce about 45 transformants. Three transformants were randomly selected for PCR and RT-PCR. Transformants were detected on PDA medium without hygromycin B After five generations of continuous culture, the hph gene still existed and transcribed stably. Southern blotting showed that T-DNA was inserted into the chromosomes of three transformants in a single copy. The transformation system established by this research will provide important technical support for the functional gene research and host-pathogen interaction research.