Protoplast Fusion and Genetic Analysis of Entomopathogenic Fungi

研究了昆虫病原真菌原生质体的分离、培养、融合及DNA分离成染色体大小分子的条件。用酵母酶和Novozyme 234处理几种昆虫病原真菌幼菌丝体，获得了较高的原生质体产量。玫烟色拟青霉营养缺陷型突变体间的原生质体融合互补频率为10 <-1>~ 10。<-2>为了更方便、更有效地利用虫生真菌的原生质体进行遗传学研究，需要采用合适的原生质体保存技术。然而，很少有人尝试保存昆虫病原真菌的原生质体。然后，我们测试了浓度为0、20、40、60和80%的甘油作为冷冻保护剂。在-80 ℃下保存20天后，根据原生质体的活力判断，20%的浓度获得最佳保护。以未处理对照为100%，贮藏21和48天的贮藏效果分别为71.3%和68.8%，昆虫病原真菌的遗传学是复杂的，甚至不能确定这些真菌含有多少染色体。我们测试了用脉冲场电泳将玫烟色拟青霉原生质体DNA分离成染色体大小的分子。玫烟色假单胞菌基因组被解析为六条带。按降序排列，这些波段估计为7.8、6.2、5.3、4.4、3.3和3.1mb。玫烟色拟青霉基因组的总大小超过30.1mb。

Conditions for isolation, culture, and fusion of protoplasts and separation of DNA into chromosome size molecular in entomopathogenic fungi were studied. High yields of protoplasts from young mycelia of some entomopathogenic fungi were obtained by treatment with Zymolyase or Novozyme 234. In protoplast fusion between the auxotrophic mutants of Paecilomyces fumosoroseus the complementation frequencies were 10^<-1>-10^<-2>To use the protoplasts of entomogenous fungi more easily and effectively for genetic purposes, proper techniques for protoplast preservation are needed. However, little attempt has been made to preserve protoplasts of entomopathogenic fungi. We then tested glycerol as a cryoprotectant at concentrations 0, 20, 40, 60, and 80%. After 2days of storage at -80^ﾟC, the best protection was obtained at the concentration of 20%, as judged by the viability of the protoplasts. When the value of the untreated control was taken as 100%, those of 21 and 48 days of storage were as 71.3% and 68.8%, respectively.The genetics of entomopathogenic fungi is in its fancy and it is not even certain how many chromosomes these fungi contain. We tested the separation of P. fumosoroseus protoplast DNA into chromosome size molecules by pulsed field electrophoresis. The P. fumosoroseus genome was solved into six bands. The bands were estimated, in descending order, to be 7.8, 6.2, 5.3, 4.4, 3.3 and 3.1mb. The Total P. fumosoroseus gennome is therefoe more than 30.1mb.