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引起玉米纹枯病的立枯丝核菌(Rhizoctonia solani K(u|¨)hn)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rDNAITS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rDNAITS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。
Rhizoctonia solani K (u | ¨) hn), which is mostly caused by sheath blight of corn, is a multinuclear heterotrophic fungus. It is also difficult to homogenize its nucleus through sexual behavior. The nuclei of JN nucleus of R. solani were homonuclearized by protoplast regeneration. Comparing rDNAITS sequences obtained from single protoplast regenerating bacteria, we found that there was a difference between the sequences, indicating that the nucleus did not reach the expected purification effect. And then amplify the rDNAITS sequences of Rhizoctonia solani and find their own rDNA-ITS sequences are also different, so that the Rhizoctonia solani this rDNA-ITS sequence may be the difference between the long-term without The result of the sex stage is independent of the number of nuclei. Transformation of Rhizoctonia solani with a vector transformed with filamentous fungi did not produce a stable transformant, and thus the vector was modified. A hygromycin gene was constructed using the promoter and terminator of the actin gene of Rhizoctonia solani (AG-3) pRsA. Transformation of Rhizoctonia monocytogenes was achieved using PEG-mediated protoplast transformation. Three transformants were confirmed by PCR. However, the hygromycin resistance of the transformants disappeared after five consecutive generations of PDA medium. The results have reference significance for the improvement of the transformation method of Rhizoctonia.