蜜蜂球囊菌(Ascosphaera apis)原生质体制备及再生

Preparation and Regeneration of Ascosphaera apis Protoplasts

为建立高效稳定的蜜蜂球囊菌(Ascosphaera apis)遗传转化体系,构建具有不同表型特征的球囊菌转化子,本实验对影响蜜蜂球囊菌菌株原生质体制备及再生的因子进行了系统的研究,同时对蜜蜂球囊菌原生质体的释放及再生过程进行了显微观察。结果表明,采用液体培养基进行24 h培养的蜜蜂球囊菌菌体,在28℃条件下,应用50 mg/mL的崩溃酶,经4 h酶解所制备的原生质体释放量最大,达到34.00×105/mL。在上述条件下,采用0.8 mol/L柠檬酸与NaCl的混合液作为稳渗剂,原生质体的再生率也较高,达到53.06%。蜜蜂球囊菌以菌丝断裂方式释放原生质体,原生质体再生时表现为原生质体先是突出,其后延长,并最终发育成为正常菌丝。本实验首次优化了蜜蜂球囊菌原生质体制备实验流程及原生质体再生最佳条件,为深入研究蜜蜂球囊菌的致病机理及寻找蜜蜂球囊菌致病相关基因奠定基础。

In order to develop an efficient and reproducible protoplast isolation and regeneration protocol targeting to make A scosphaera apis amenable to genetic studies and transformation, different enzymolysis and osmotic pressure stabilizing agents along with different growth mediums, incubation periods, and temperature variants have been utilized. The fungus of Ascosphaera apis has demonstrated varying responses in terms of protoplast yield and regeneration rates to different factors tested. More specifically, incubation in liquid growth medium for 24 hours has yieldedthe highest number ofprotoplasts （34.00×10^5/mL）. The use of 50 mg/mL driselase was the best enzymolysis agent at 28℃, yielded the highest number of isolated protoplasts. Furthermore, 0.8 mol/L citric acid-monohydrate with NaCl as an osmotic stabilizer and 4 hours ofenzymolysis time has supported 53.06% proto- plast regeneration. From observation by microscope, we understood that Ascosphaera apis released protoplasts through mycelium rupture. Consequently, we found that the mode of regeneration ofA scosphaera apis protoplasts is with surface protrusion initially, and the whole body prolongs into a fungal hypha in the end. With this first time reported protocol, viable protoplasts were obtained and regenerated successfully from A scosphaera apis. Thus, we believe, it is an important foundation for genetic engineering in this fungus and potentially used for better under- standing in studies targeting pathogenicity and virulent genes identification and modification in the future.