以携带潮霉素B磷酸转移酶抗性基因(hph)的pBHt1作为转化载体，根癌农杆菌AGL-1作为转化介体，实施转化异角状拟盘多毛孢菌。研究发现，异角状拟盘多毛孢菌的最优转化体系为：异角状拟盘多毛孢菌分生孢子悬浮液浓度为1×106个/mL，根癌农杆菌OD600为0.3，共培养时间48 h，共培养温度为25 ℃，诱导培养基中乙酰丁香酮（AS）浓度为200 μmol/L，选择培养基添加250 μg/mL潮霉素B、250 μg/mL头孢噻肟钠。1×106个异角状拟盘多毛孢菌分生孢子可以产生200～300个转化子，随机挑选10个转化子进行PCR检测，均能扩增出预期条带，且在不含潮霉素B的PDA培养基平板上转化子连续培养5代后，hph基因仍能稳定遗传，这表明T-DNA已经插入到异角状拟盘多毛孢菌的基因组中。此次建立的异角状拟盘多毛孢菌的转化体系可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有效的工具。
Abstract
In this study, we developed an Agrobacterium tumefaciensmediated transformation system for Pestalotiopsis heterocornis by using A. tumefaciens strain AGL1 which carried plasmid pBHt1 harboring the hygromycin B phosphotransferase gene(hph). The results showed a total of 200-300 transformants were obtained per 1×106 spores when the spore suspension were cocultivated with A. tumefaciens cells,the optimal conditions were OD600=0.3, 25 ℃,and 48 h cultivation in the presence of induction medium containing acetosyringone at 200 μg/mL. Selection medium performed best with concentrations of 250 μg/mL of hygromycin B and 250 μg/mL of cefotaxime sodium in PDA. 〖JP2〗To detect the heredity stability, ten random transformants were cultivated on the PDA medium for five generations, colony PCR of each generation obtained expected amplification bands, which showed that the transformants exhibit high heredity stability. The transformation system will be useful for further studies of functional genes and hostpathogen interaction in P. heterocornis.
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基金
收稿日期：2011-10-28修回日期：2011-11-15基金项目：国家重点基础研究发展计划(2009CB119205);国家自然科学基金项目(31000303);江苏省自然科学基金项目(BK2010566)；国家林业局林业公益性行业科研专项项目(200704026)；江苏省高校优势学科建设工程资助项目第一作者：张林平，讲师，博士生。*通信作者：叶建仁，教授。Email: jrye@njfu.com.cn。
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