【作者】 卫冬妹；

【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】 本文对核盘菌(Sclerotinia sclerotiorum)Ep-1PN菌株弱毒相关dsRNA进行了cDNA克隆，分析了该序列的结构特性，并与其它相近病毒的基因组进行了同源性比较，在分子水平上进一步探讨真菌病毒与核盘菌弱毒现象的关系。研究结果报道如下： 1 采用RT-PCR及cDNA克隆技术对弱毒菌株Ep-1PN的6.4kb dsRNA片段进行了cDNA克隆，得到了多个阳性克隆，通过序列测定及拼接，获得了该dsRNA基因组含有4629bp的核苷酸的序列，并已在GenBank登录注册(登录号为AY147260)。 2 4629bp的RNA序列中包含一个推定的不完整的阅读框架(ORF)，编码含有1440个氨基酸残基的Helicase-RdRp融合蛋白，在已获得的核苷酸序列中没有发现编码病毒外壳蛋白性质的基因。 3 对核酸序列同源性进行比较(Blast)发现，核盘菌弱毒相关dsRNA序列与植物病毒Potexvirus组中的病毒有较高的同源性，这些植物病毒包括BaMV(Bamboo mosaic virus)、PVX(Potato vires X)、CIYMV(Clover yellow mosaic virus)和TuVX(Tulip virus X)等。对推定的Helicase和RdRp的氨基酸序列进行同源性比较也证实了核盘菌弱毒相关dsRNA与植物病毒有较高的同源性，而与典型的真菌病毒存在较大的差异。因此，我们推定弱毒菌株Ep-1PN感染了类似植物病毒的真菌病毒，并将其命名为核盘菌弱毒相关病毒，缩写为SsHV(S.sclerotiorum hypovirulence-associated virus)。 4 根据SsHV已知的核苷酸序列设计特异引物，对Ep-1PN及其恢复菌株进行RT-PCR，这些恢复菌株包括有性后代Ep-1PNAa、Ep-1PNAb、Ep-1PNAe，原生质体再生菌株Ep-1PNP14、Ep-1PNP94、Ep-1PNP131，以及单菌核分离物Ep-1PNSSB9。结果在原生质体再生菌株Ep-1PNP131和单菌核分离物Ep-1PNSSB9中均检测到相同的特异性扩增片段，大小为871bp，与前面所得到的Ep-1PN核苷酸序列大小相符。这两个菌株在表型上与核盘菌正常菌株相比具有一定的差异，属于部分恢复菌株。而在形态、生长速度及毒力恢复正常的另外两个原生质体再生菌株Ep-1PNP14、Ep-1PNP94及子囊孢子单孢分离物Ep-1PNAa、Ep-1PNAb、Ep-1PNAe中均没有扩增到特征性片段。结果表明，核盘菌在进行有性遗传时可能激活其体内某种未知的机制，摆脱弱毒相关真菌病毒的影响，通过原生质体再生技术也可获得不携带真菌病毒的核盘菌衍生后代。与Ep-1PN相比，采用常规dsRNA提取方法从Ep-1PNP94和Ep-1PNSSB9的菌丝样品中难以检测到dsRNA，据此可推定SsHV的浓度可能会影响核盘菌的表型。更多还原

【Abstract】 In this dissertation, the 6.4kb dsRNA genome of the mycovirus infecting the hypovirulent isolate Ep-lPN of Sclerotinia sclerotiorum (SsHV) was studied using techniques of cDNA synthesis, cloning, and sequencing. The relationship between hypovirulence of Sclerotinia sclerotiorum Ep-lPN and the dsRNA mycovirus was further confirmed at the molecular level. Results achieved so far include:1 Positive cDNA clones of the 6.4kb dsRNA of Ep-lPN were obtained. After sequencing the insertial cDNA fragments, in the plasmid partial cDNA sequence of the 6.4kb dsRNA genome of 4629 nucleotides was aligned with DNAMAN program. The sequence (4629bp) was submitted to the GenBank with the accession number AY147260.2 A incomplete open reading frame (ORF) encoding a fusion protein of 1440 ammo acid residues of helicase and RNA dependent RNA Polymerase(RdRp) of RNA virus was revealed. The gene coat protein amino acids sequence was not included in this sequence. Based on the characteristics of putative fusion protein, the 6.4 kb dsRNA element could be regarded as mycovirus, and named as Sclerotinia sclerotiorum hypovirulence-associated virus(SsHV).3 Both the nucleotide sequence and putative amino acid sequence were subjected to do homology search with Blast Program on http://www.ddbj.nig.ac.jpT the result revealed that the obtained nucleotide sequence had the characteristic of plant Potexvirus, subsequently the putative protein coded by the 6.4kb dsRNA was highly similar to those proteins coded by Potexvirus, such as BaMV(Bamboo mosaic virus), PVX(Potato virus X), ClYMV(Clover yellow mosaic virus) and TuVX(Tulip virus X) et al. The result suggested SsHV might derive from plant virus or showed the same ancestor with plant virus.4 Based on the cDNA sequence of SsHV, specific primers was designed to detect dsRNA in Ep-lPN and its derivatives using RT-PCR. the recovered cultures includ thee ascospore progenies (Ep-1PNAa, Ep-1PNAb and Ep-1PNAe), thee protoplast-regeneration cultures (Ep-1PNP14, Ep-1PNP94 and Ep-lPN131) and one single-sclerotium-isolationculture (Ep-1PNSSB9). The PCR product of 871bp in size occurred in Ep-lPNSSB9 and Ep-1PNP131, both cultures exhibited some atypical colony morphology, compared to health strains of S. sclerotiorum. This result suggests that S. sclerotiorum may activate some unknown mechanism to eliminate the hypovirulence associated mycovirus during sexual reproduction.更多还原