Protoplast Fusion and Genetic Analysis of Entomopathogenic Fungi

研究了昆虫病原生菌原生质体的分离、培养和融合以及将DNA分离到染色体大小分子中的条件。用酵母酶或Novozyme234处理昆虫病原真菌幼龄菌丝体，获得了较高的原生质体产量。在烟粉拟青霉营养缺陷型突变体的原生质体融合中，互补率为10^-lt；-1&gt；-10；-lt；-2&gt；为了更容易和有效地利用虫生真菌的原生质体进行遗传，需要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。原生质体在-80ﾟC下保存2天后，以20%的浓度保存效果最好。当未经处理的对照为100%时，贮藏21天和48天的分别为71.3%和68.8%。昆虫病原真菌的遗传学很受欢迎，甚至不确定这些真菌含有多少染色体。采用脉冲场电泳法对烟曲霉菌原生质体DNA进行了染色体大小分子的分离。Fumosoroseus基因组被分成6条带。从大到小依次估计为7.8、6.2、5.3、4.4、3.3和3.1mb。烟粉潜蝇基因组总长度超过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.