根癌农杆菌介导致病疫霉转化体系的建立

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Phytophthora infestans

为建立低成本、快速、高效的根癌农杆菌介导的致病疫霉转化体系，以菌株 HK0919为材料，从抗生素筛选浓度、共培养转膜方式、乙酰丁香酮（AS）浓度、共培养时间和共培养温度等方面对致病疫霉的转化体系进行了研究。综合各因素优化结果，建立的转化体系条件为：以1．0μg/mL的潮霉素B进行转化子筛选，采用玻璃纸作为共培养介质，AS浓度为200μmol/L，培养6 d，温度为22℃。在此条件下的转化效率为每106个游动孢子获得50～60个转化子。随机选取10个转化子，利用特异性引物对潮霉素抗性基因hph进行PCR扩增，转化子均能扩增出800 bp左右的预期条带；同时，利用根癌农杆菌vir基因特异引物对转化子进行 PCR扩增，排除了转化子被农杆菌污染所致的假阳性；转化子继代培养5代后，仍能在含潮霉素 B的黑麦培养基上生长。说明外源T DNA已成功整合到致病疫霉基因组中，并能稳定遗传。

The aim of this study was to establish a low-cost,rapid and efficient Agrobacterium tumefaciens-mediated transformation（ATMT ）system of Phytophthora infestans.The transformation conditions of acetosyringone concentration,coculture temperature,coculture time and membrane-transferring method were optimized using P.infestans isolate HK09-19.The optimized conditions for this ATMT system included 1.0 μg/mL hygromycin B for transformant-screening,using cellophane as transferring membrane,with 200μmol/L acetosyringone added,and taking 6 d for incubation at 22 ℃ at coculture stage.Under this condition,the transformation efficiency was 50-60 transformants per 106 zoospores.The obtained transformants showed high resistance to hygromycin B after five generations for propagation.An 800 bp band was amplified from ten transformants randomly selected by PCR using specific primers which were designed for the hph gene.Meantime,the vir gene of A.tumefaciens was tested by PCR in colonies of P.infestans transformants in order to eliminate the false-positivity caused by A.tumefaciens contamination.The results showed that exogenous T-DNA was successfully integrated into the genome of P.infestans and able to stably inherit.An efficient and time-saving ATMT system of P.infestans was established,which provided a convenient method for the P.infestans mutant library construction and gene function analysis.