【作者】 李彦鹏；

【导师】 邱立友；

【作者基本信息】 河南农业大学 ， 微生物学， 2007， 硕士

【摘要】 本研究利用dsKNA技术,从平菇天达300菌丝体中提取出平菇dsRNA病毒的基因组,共有4个片段,大小分别为8.2kb,2.6kbp,2.0kbp,1.1kbp(根据BioCaptMW软件计算);酶切验证4个片段均为dsRNA,其中8kbp为doublet。利用一步RT-PCR对dsRNA病毒的部分基因组进行了克隆、测序,从8.2kb、2.6kb中扩增出的目的片段,大小分别为为556bp和351bp,经过blast、保守序列比对分析,来自2.6kp的556bp序列与Pleurotus ostreatus virusⅠ(PoVⅠ)编码的RdRp基因有很高的相似性,核苷酸的相似性为86%,翻译为蛋白质的相似性为94%,其中10个不同的氨基酸中有9个不在RdRp保守区域内,只有一个在V保守区。在这10个氨基酸中,有7个是保守替代残基。因此2.6kb序列编码RdRp与双分体基因组分病毒科(Partitiviridae)的成员相似,提取的dsRNA病毒可能属于双分体基因组病毒科(Partitiviridae)。通过CsCl梯度离心,提取到两种大小、形状不同的病毒颗粒:直径为27～28nm,形状为等轴状;直径为22～24nm,形状为球形颗粒。平菇直径22～24nm球形颗粒病毒属首次报道。采用高温33℃培养重复切取尖端菌丝的方法对平菇天达300进行脱毒,结果表明,脱毒菌株日平均生长速度是出发菌株的1.2倍,摇瓶培养菌丝生物量显著提高,是出发菌株的2.8倍。脱毒菌株第一茬菇产量与出发菌株基本相同,第二茬菇产量比出发菌株提高10.9%。脱毒菌株子实体形状与出发菌株相比有明显改观,菇盖宽大,平均宽度为13cm,长度为8cm,子实体肥厚,外观整齐、白洁,而出发菌株菇盖较小,平均宽度为10cm,长度为6cm,子实体较薄,外观有些散乱,大小不均一。更多还原

【Abstract】 ABSTRACT The genomes of dsRNA viruses infecting Pleurotus ostreatus were isolated from the mycelia of Pleurotus ostreatus and had 4 segments by the dsRNA technique.The sizes of the dsRNAs were 8.2kbp,2.0kbp,1.1kbp(according to the calculation BioCaptMW sorftware).The sample was treated with DNaseⅠ,S1 Nuclease and RNAase A,and was proved to be dsRNA.The 8.2kbp of the dsRNA genomes was doublet.The partial dsRNA genomes were cloned and sequenced by one-step RT-PCR. The sizes of RT-PCR were 556bp and 351bp from the 2.6kb and 8.2kb,respectively. The deduced amino acid sequence of the 556bp was very similar to Pleurotus ostreatus virusⅠ(PoVⅠ)by blast and alignment of conserved motifs in RdRp.The nucleotide identities was 86%and the amino acid identities was 94%.9 amino acids among the difference 10 amino acids in which 7 amino acids were positive were not in the conserved motifs and only one was in the V tentative superpositions of conserved motifs in the RdRp.As a result,the 2.6kb code the RdRp,which was similar to Partitiviridae.So the dsRNA genomes might belong to Partitiviridae.Though CsC1 gradient centrifugation the purified virus preparations contained isometric particle of 27～28nm in diameter and 22～24nm spherical particle.The 22～24nm spherical particle was first reported.Base on those studies the virus-free prparation experiment which repeatedly cut the 3-4mm tender mycelia of the edge of the colony to be cultured in the 32-34℃was performed.The average growth rate of the virus-free strain was 1.2 times that of the control strain and the biomass of the virus-free strain was 2.8 times that of the control strain.In contrast to the control strain the product of the first flush of the virus-flee strain was almostly equal and the one of the second flush of the virus-free strain increased by 10.9%.The virus-free strain developed in the shape of sporophores.The caps of the virus-free strain were bigger and the average width was about 13cm and 8cm in length.The Fruitbodies were fleshy and neat.But the caps of the control strain were smaller and the average width was about 10cm and 6cm in length.Fruitbodies were thinner and different in the sizes.更多还原