CAJ | 학술논문

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。
위건립저성본、쾌속、고효적근암농간균개도적치병역매전화체계，이균주 HK0919위재료，종항생소사선농도、공배양전막방식、을선정향동（AS）농도、공배양시간화공배양온도등방면대치병역매적전화체계진행료연구。종합각인소우화결과，건립적전화체계조건위：이1．0μg/mL적조매소B진행전화자사선，채용파리지작위공배양개질，AS농도위200μmol/L，배양6 d，온도위22℃。재차조건하적전화효솔위매106개유동포자획득50～60개전화자。수궤선취10개전화자，이용특이성인물대조매소항성기인hph진행PCR확증，전화자균능확증출800 bp좌우적예기조대；동시，이용근암농간균vir기인특이인물대전화자진행 PCR확증，배제료전화자피농간균오염소치적가양성；전화자계대배양5대후，잉능재함조매소 B적흑맥배양기상생장。설명외원T DNA이성공정합도치병역매기인조중，병능은정유전。
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.