PEG 介导的苹果腐烂病菌原生质体转化

Development of Genetic Transformation system of Valsa mali of apple mediated by PEG

摘要:

摘要: 【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。

关键词:关键词: 原生质体，转化子，hph 基因，转化效率，稳定性

Abstract:

Abstract: ［Objective］The genetic transformation of Valsa mali var． mali was developed by PEG-mediated protoplasts transformation．［Method］It was transformed by PEG-induced fusion of protoplasts． The plasmid pBIG2RHPH2-GFPGUS carrying hph gene was used and Valsa mali var． mali 03-8 isolate was used as the host strain．［Result］At50 mg/mL driselase + 10 mg/mL lysing enzymes concentration，the mycelium of Valsa mali var． mali cultured in YEPD medium for 48 h was hydrolyzed in 10 mL enzymes liquid /0. 5 g wet mycelium for 2 h． The protoplast yield was 4×107 CFU /mg． The transformation efficiency was 44 per g DNA． Analysis of the transformants by PCR and Southern blotting showed that the selectable marker gene hph was integrated effectively into the genome of Valsa mali var． mali．After 5 subculturing on PDA，87. 5% transformants could grow． This stability test of transformants suggested that the foreign gene hph was stable in heredity．［Conclusion］This transformation system is a valuable and important tool for the further study of the pathogenic gene of Valsa mali．

Key words:Keywords: protoplast，transformant，hph gene，transformation efficiency，stability test