玉米纹枯病菌细胞核纯化与原生质体转化的探讨

 引起玉米纹枯病的立枯丝核菌(Rhizoctonia solani Kühn)多为多核杂核真菌，也难以通过有性态使其细胞核进行单一化。利用原生质体再生的方法对单核玉米纹枯病菌JN的细胞核进行同核纯化，比较从单个原生质体再生菌获得的rDNA|ITS序列，发现序列间存在差异，说明其细胞核没有达到预期纯化的效果。继而扩增了双核和多核玉米纹枯病菌的rDNA|ITS序列，发现它们自身的rDNA|ITS序列也存在差异，因此认为立枯丝核菌的这种rDNA|ITS序列的差异可能是长期未经过有性阶段的结果且与其细胞核数目无关。使用转化丝状真菌的载体转化纹枯菌未获得稳定的转化子，进而对载体进行了改造，用纹枯菌(AG|3)actin基因的启动子和终止子控制潮霉素基因构建了载体pRsA。利用PEG介导的原生质体转化方法，实现了对单核纹枯菌的转化，PCR验证得到3个转化子。但在PDA培养基连续继代5代后，转化子的潮霉素抗性消失。结果对改进纹枯菌的转化方法有借鉴意义。

A glimpse of preparing homokaryotic and transforming Rhizoctonia solani through protoplasts

Rhizoctonia solani Kühn, the causal agent of corn sheath blight, are mostly multinuclear and he|terokaryotic fungi, and it is difficult to obtain the simplifying nucleus during the perfect stage. In the study, the nucleus of mononuclear R. solani strain JN was purified by protoplast regeneration method and the rDNA|ITS sequences of the cultures that each was regenerated from single protoplast was compared. Since the rDNA|ITS sequences among the cultures were still different, the expected nucleus purification effect was not obtained. Therefore, amplification of the rDNA|ITS sequences of the dicaryon and polykaryon was conducted. How|ever, the difference among their autologous sequences were also observed. Based on the results, it was consi|dered that the difference among rDNA|ITS sequences in R. solani might be caused by the absence of the perfect stage during the evolution but irrelevant to the number of nucleus. In addition, the stable transformants of R. solani strain JN were unable to obtain by conventional vectors suitable for filamentous fungal transformation. A vector pRsA that hph gene expression was regulated by the promoter and terminator of R. solani AG|3 actin gene was constructed. The pRsA was then transformed into the protoplasts of R. solani strain JN by PEG|mediated transformation, and three transformants were identified by PCR amplification. However, the transformants lost hygromycin resistance after reculturing for five generations on PDA medium. The results might have a positive on the improvement of the R. solani transformation method.