The Sclerotinia sclerotiorum pac1 Gene Is Required for SclerotialDevelopment and Virulence(Rollins, 2003)
PEG介导的转化
（1）制备真菌原生质体【现学现卖】实验-制备真菌原生质体，并且用STC：PEG为4：1的溶液（含有1%DMSO以及0.3mg/ml的肝素）重悬，终浓度为1×108 protoplasts/ml 。
Protoplasts were resuspended at aconcentration of 1×108 protoplasts per ml in four parts STC(1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl2) one part PEG(40% polyethylene glycol 3350, 50 mM Tris-HCL, pH 7.5, 50 mM CaCl2)solution, plus 1% dimethyl sulfoxide and 0.3 mg of heparin per ml. Theseprotoplast stocks were routinely stored at –80C.
（2）将5 µg的DNA材料与2µl亚精胺（50 mM），5 µl肝素（2.5mg/ml）混合。然后立刻加入到100µl原生质体溶液中。冰浴30min然后与1mlPEG溶液混合，室温下放置20min。
For fungal transformation, 5 µg ofDNA was mixed with 2 µl spermidine (50 mM stock) and 5 µl heparin (2.5 mg perml in STC stock) and, then, immediately added to 100 µl of the protoplast stocksolution. This mixture was incubated on ice for 30 min and, then, gently mixedwith 1 ml of PEG solution and incubated at room temperature for 20 min.
（3）混合液均匀涂布在25ml的RM下层培养基，室温黑暗培养15-18h，然后上层覆盖4ml的RM上层培养基（含抗生素）。
Each suspension was evenly spreadon the surface of 25 ml RM (regeneration medium 239.6 g of sucrose, 0.5g of yeast extract per liter) bottom agar plates (15 g of agar per liter).Plates were incubated 15 to 18 h in the dark at room temperature, then overlaidwith four ml of molten RM top agar (8 g of agar per liter) containing2.9 mg of hygromycin B.
（4）原生质体再生在RM上，然后转移到含有100μg/ml潮霉素的PDA培养基。
Colonies that regenerated throughthe top agar were transferred to PDA media containing 100 µg of hygromycin per milliliter.
（5）抗性转化子的菌丝尖端在含有抗生素的PDA上进行次代培养，至少三代。
Hygromycin-resistant transformantswere hyphal-tip transferred a minimum of three times on PDA containing 100 µgof hygromycin per milliliter.
A geminivirus-related DNA mycovirus that confers hypovirulence to aplant pathogenic fungus(Yu et al., 2010)
另一篇文献对上述方法进行改进和简化【现学现卖】那些DNA真菌病毒
第（3）步，原生质体在整合的RM培养基上直接培养，20℃培养3-4d。而且在转化到PDA时，也没有刻意转移菌丝尖端部分，而是随机打菌饼。
Protoplasts were regenerated inregeneration medium at 20 °C for 3–4 days. Mycelial plugs were cut at randomfrom the regenerated colonies and transferred to fresh PDA plates.
参考文献
Rollins, J.A.(2003) The Sclerotinia sclerotiorum pac1 gene is required for sclerotialdevelopment and virulence. Molecularplant-microbe interactions 16:785-795.
Yu, X., Li, B., Fu, Y., Jiang,D., Ghabrial, S.A., Li, G. et al. (2010) A geminivirus-related DNA mycovirusthat confers hypovirulence to a plant pathogenic fungus. Proceedings of the National Academy of Sciences 107: 8387-8392.
