【作者】 程家森；

【导师】 姜道宏；

【作者基本信息】 华中农业大学 ， 植物病理学， 2003， 硕士

【摘要】 盾壳霉(Coniothyrium minitans)是核盘菌(Sclerotinia sclerotiorum)的重寄生真菌，在世界范围内广泛分布，在菌核病的生物防治中有着非常重要的作用。本论文对盾壳霉中的真菌病毒(Coniothyrium minitans RNA virus，CmRV)dsRNA基因组的分子生物学特性进行了研究。通过cDNA克隆和RT-PCR技术获得了该病毒dsRNA基因组的全长序列，共4975个碱基。序列分析表明该病毒仅有一条dsRNA片段，含有两个开放阅读框架(ORF)。ORF1位于该dsRNA的正链上62—2389nt，编码一个分子量为80344Da的外壳蛋白(Coat Protein, CP)；ORF2位于正链上2386—4875nt，编码一个分子量为82511Da的依赖于RNA的RNA聚合酶(RdRp)；位于2386—2389nt的四个核苷酸AUGA为两个阅读框架所共有，ORF1的终止密码子与ORF2的起始密码子相重叠。根据基因组的组成结构及序列分析，盾壳霉Chy-1菌株中的病毒(CmRV)可以归属于Totiviridae科，CmRV的dsRNA基因组结构和外壳蛋白及RdRp氨基酸序列同源性比较表明该病毒与寄生丝状真菌的Totivirus属的病毒之间的亲缘关系要更为接近。 本文同时还对CmRV的稳定性作了分析。从盾壳霉菌株Chy-1获得了100个单孢分离物，对它们的菌落形态进行了观察，发现在这100个单孢分离物与原始菌株Chy-1之间的菌落形态非常相似，随机挑选32个分离物进行检测，结果表明这些分离物中均含有与Chy-1菌株中大小相同的dsRNA片段。将盾壳霉菌株Chy-1的孢子埋于土壤中，两个月后用菌核为诱饵从土壤中分离到了8个分离物，这8个分离物都具有与Chy-1相似的菌落形态，并可检测到同样的dsRNA片段，Northern杂交证实这些dsRNA片段是CmRV的基因组。以上实验结果说明CmRV可以在盾壳霉稳定存在。 对28个盾壳霉菌株(其中5个菌株来自于英国，3个菌株为Chy-1经紫外线诱变而得)进行dsRNA检测，在其中12个菌株中检测到了dsRNA。Northern杂交结果显示在这12个菌株中的dsRNA病毒相互之间具有一定的同源性，这些病毒有可能属于同一个种，实验结果表明真菌病毒在盾壳霉中有可能普遍存在。更多还原

【Abstract】 Coniothyrium minitans, an important sclerotial mycoparasite of Sclerotinia sclerotiorum with worldwide distribution, has great potential for biological control of plant diseases caused by S. sclerotiorum and other Sclerotinia spp. The complete nucleotide sequence, 4975bp, of the double-stranded RNA (dsRNA) mycovirus infecting the sclerotial parasite Coniothyrium minitans (CmRV) was determined. Sequence analysis revealed the occurrence of two overlapping open reading frames (ORFs): the 5’ -proximal large ORF (ORF1; nucleotide positions 62-2389) encodes a putative coat protein (CP) with a predicted molecular mass of 80344 Da, and the 3’-proximal ORF (ORF2, nucleotide positions 2386-4875) encodes a putative RNA dependent RNA Polymerase (RdRp) with a predicted molecular mass of 82551 Da. The tetranucleotide AUGA at nucleotide positions 2386-2389 includes the predicted start codon of ORF2, which overlaps with the stop codon for ORF1. Based on genome organization and sequence analysis encoded proteins, the virus infecting C. minitans strain Chy-1, designated C. minitans RNA virus (CmRV), belongs to the family Totiviridae. Pairwise sequence comparisons of the deduced amino acid sequences encoded by CmRV as well as phylogentic analysis indicated that it is more closely related to the totiviruses that infect filamentous fungi than to those infecting protozoa, yeast and smut fungi.To test the stability of CmRV in C. minitans, 100 single-conidium-isolation cultures were obtained and observed for development of colony morphology, all of the 100 cultures had a similar colony morphology compared to the original strain Chy-1, among them 32 cultures randomly selected were proved to carry dsRNA genome of CmRV. Spores of Chy-1 were buried into field soil for two months, and 8 cultures were recovered from soil with sclerotial baiting. All the 8 cultures had the same phenotype of Chy-1, and viral dsRNA was extracted from all the 8 cultures. Northern blottings showed that all the viral dsRNA of the 8 cultures were the viral genome of CmRV. These results suggested that CmRV in C. minitans was stable.Twenty-eight C. minitans strains (including 5 from United Kingdom and 3 UV-treated Chy-1) were used to check the dsRNA of mycovirus, among them 12 strains were proved infected by mycovirus. Northern blotting analysis revealed that the viral dsRNA in all these ten strains could be hybridized with the probe made from a cDNA clone derived from CmRV dsRNA, suggesting that those viruses might be in the same group (species). It inferred that virus in C. minitans might be very common.更多还原