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【目的】建立PEG介导的苹果腐烂病菌原生质体遗传转化体系。【方法】本文利用带有hph基因的质粒,以苹果腐烂病菌(Valsa mali var.mali)03-8为受体菌株,通过PEG融合法对其原生体进行转化。【结果】于YEPD内培养48 h的菌丝,在酶解液浓度为50 mg/mL Driselase+10 mg/mL Lysing Enzymes情况下,按10 mL酶液/0.5 g湿菌体比例,酶解2 h时可以释放出4×107个/mL原生质体,其转化效率为44个/μg DNA。对转化子的PCR检测和Southern杂交分析表明,hph基因已经整合进苹果树腐烂病菌的基因组中。转化子在PDA培养基中继代5次后,87.5%的转化子仍能正常生长,表明外源基因hph能在苹果树腐烂病菌中稳定遗传。【结论】该转化体系的建立为苹果树腐烂病菌致病相关基因的深入研究奠定了基础。
【Objective】 PEG-mediated protoplast genetic transformation system of apple rot pathogen was established. 【Method】 In this study, the hph gene was used to transform the protoplast into Valsa mali var. Mali 03-8 by PEG fusion. 【Result】 The mycelia cultured in YEPD for 48 h were digested by 10 mL enzyme solution / 0.5 g wet bacterial cells at a concentration of 50 mg / mL Driselase + 10 mg / mL Lysing Enzymes h can release 4 × 107 / mL protoplasts, the transformation efficiency of 44 / μg DNA. PCR analysis of the transformants and Southern hybridization analysis showed that the hph gene has been integrated into the genome of apple tree rot pathogens. Transformants were subcultured in PDA medium for 5 times, and 87.5% of transformants could still grow normally, indicating that hph gene can be stably inherited in apple tree rot fungi. 【Conclusion】 The establishment of this transformation system lays the foundation for the further study of the pathogenicity-related genes of apple tree rot pathogens.