水稻纹枯病菌原生质体制备与再生条件的优化  

Optimized Conditions for the Preparation and Regeneration of Protoplasts from Rhizoctonia solani Kühn AG-1 IA

作　　者：杨迎青[1] 李明海[1] 杨媚[1] 李勇[1] 周而勋[1] 

机构地区：[1]华南农业大学资源环境学院,广州510642

出　　处：《华中农业大学学报》2010年第5期546-551,共6页Journal of Huazhong Agricultural University

基　　金：国家2007年公益性行业(农业)科研专项(nyzx3-16;原nyzx07-049);教育部留学回国人员科研启动基金项目(教外司留[2006]331号)资助

摘　　要：为获得进行水稻纹枯病菌遗传转化所需的高质量原生质体,选用该病菌强致病力菌株GD-118,分别从酶种类、菌龄、酶解时间、酶解温度、酶液pH值和渗透压稳定剂及其浓度6个方面优化了原生质体的制备条件,从酶解时间和渗透压稳定剂及其浓度2个方面优化了原生质体的再生条件。结果表明:优化后的水稻纹枯病菌原生质体制备的最佳条件组合是"纤维素酶+溶菌酶+崩溃酶"组合酶、总酶终质量浓度10mg/mL、菌丝菌龄15h、酶解时间3h、酶解温度35℃、酶液pH5.6、以0.6mol/LMgSO4.7H2O为渗透压稳定剂,此最佳条件组合所制备的原生质体产率高达4.00×107/g;原生质体最佳的再生条件是酶解3h的原生质体在含1.0mol/L甘露醇的再生培养基上26℃时进行培养,在此条件下可获得最佳的再生效果,再生率为39%。In order to obtain high quality protoplasts for the transformation of Rhizoctonia solani Kühn AG-1 IA,isolate GD-118 of this pathogen was used for the protoplast preparation and regeneration by optimizing the conditions in six aspects for the preparation of protoplast,including enzymes,mycelial ages,digest times,digest temperature,digest pH and osmotic stabilizers and their concentrations,and in two aspects for the regeneration of protoplast,including digest time and osmotic stabilizers together with their concentrations.The results showed that the best optimized combination conditions for the preparation of protoplast were ＂cellulase ＋ lysozyme ＋ driselase＂ at final total enzyme concentration of 10 mg/mL,15 h of mycelial age,3 h of digestion,0.6 mol/L of MgSO4·7H2O as osmotic stabilizer,35 ℃ of digest temperature and pH 5.6,which gave the protoplast yield as high as 4.00×10^7/g fresh mycelia;whereas 3 h of digestion and 1.0 mol/L of mannitol were the best optimized conditions for the regeneration of protoplast in this study,and the regeneration rate was 39%.

关 键 词：水稻纹枯病菌 原生质体 制备 再生 

分 类 号：S435.111.42[农业科学—农业昆虫与害虫防治]