【作者】 邓清超；

【导师】 王克荣；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 栗疫菌细胞中的低毒力病毒是一类没有衣壳的双链RNA病毒,可以使寄主栗疫菌的毒力、分生孢子和色素的产量降低,并引起雌性不育。低毒力病毒的发现为栗疫病的生物防治提供了一种全新的思路。低毒力病毒缺少细胞外的传播途径,可以通过感染菌株与未感染菌株间的菌丝融合进行传播,但是这种传播受到营养体不亲和性的限制。本研究通过在实验室条件下对3个CHV1低毒力病毒在栗疫菌各营养体亲和群菌株间的重复传播实验,量化分析了3个病毒在相差1-2个vic基因位点时传播效率的差异,首次直接证明了低毒力病毒CHV1在栗疫菌菌株间的水平传播受到病毒毒株的影响。结果表明,不同的CHV1低毒力病毒具有不同的水平传播能力,与其他两种已经测序并构建了全长可侵染性cDNA克隆的低毒力病毒CHV1-Euro7和CHV1-EP713相比,来自中国的CHV1-CN280具有最高的传播能力。而在国际上,之前的研究普遍认为关于低毒力病毒自身对其水平传播的没有影响。这一发现为选择高效果疫病生防病毒提供了新的指标,提示通过发现和使用传播能力更高的低毒力病毒,将会显著提高对栗疫病的生物防治效率。通过逆转录构建cDNA文库并结合RT-PCR,对来自中国的低毒力病毒CHV1-CN280进行了基因组全长测序。CHV1-CN280的基因组全长12730bp(包括polyd(A)的26bp),序列分析表明,CHV1-CN280与已经测序的3个CHV1病毒在基因组上具有相似的结构。在核苷酸水平水平上CHV1-CN280和CHV1-Euro7有61-93%的序列同源性；与CHV1-EP713有60-90%的同源性。在氨基酸水平上CHV1-CN280和CHV1-Euro7有59-95%的同源性,与CHV1-EP713有61-94%的同源性。CHV1-CN280的3,端与已经测序的3个CHV1病毒同源性较高,但在5’端,CHV1-CN280与已经测序的3个CHV1病毒在核酸和蛋白序列上差异巨大。已经测序的4个低毒力病毒CHV1-EP713、CHV1-Euro7、CHV1-EP721和CHV2-NB58,在ORFA和ORFB之间都有一个UAAUG五核苷酸的连接区,其中AUG作为ORFB的起始密码子,UAA作为ORFA的终止密码子；而在CHV1-CN280中,ORFA和ORFB之间的连接区是AUGUAUAA八个核苷酸。设计了一对RT-PCR引物(A2275/B2915)对本实验室保存的12个CHV1病毒的ORFA和ORFB的连接区的序列测定表明,这种AUGUAUAA八核苷酸的连接方式和UAAUG五核苷酸的连接方式一样常见。设计了另一对RT-PCR引物(A364／B1402)用于测定本实验室保存的CHV1病毒的5’UTR和ORFA的部分序列以研究CHV1病毒的生物多样性。结果表明,所设计的引物在所有12个CHVl病毒的cDNA中都能很好的扩增出特异性的目标片段。结合本实验室在之前研究中对其他CHV1病毒的部分测序结果,对总共36个CHV1病毒进行了群体遗传分析。分析结果表明CHV1病毒具有复杂的遗传多样性,36个CHV1病毒可以被划分为包含8个亚型的2个群体,而之前已经测序的3个低毒力病毒CHV1-EP713、CHV1-Euro7和CHV1-EP721在系统发育树上聚集在一个很小的区域内。鉴于南方群体和北方群体间的巨大遗传差异,以及北方群体的CHV1-CN80与南方群体的CHV1-Euro7、CHV1-EP713和CHV1-EP721的全基因组序列比较结果,建议CHV1病毒应该划分为两个亚种(subspecies).根据已经测序的低毒力病毒CHV1-CN280基因组全长cDNA序列信息,运用RT-PCR得到包含CHV1-CN280全部基因组序列的一系列cDNA片段的克隆,逐步通过酶切和连接,得到了CHV1-CN280全长12.7kb的cDNA克隆。体外转录成为单链RNA后在由电击转染栗疫菌原生质体,成功的在栗疫菌中再生出CHV1-CN280的病毒dsRNA,并表现出CHV1-CN280的一系列特性。CHV1-CN280感染不同宿主后表型相似,都和野生带毒菌株CN280表型一样菌体呈白色,可以产生少量分生抱子；在供体-受体菌株相差一个vic Aa基因时,再生出的CHV1-CN280病毒具有和野生CHV1-CN280病毒相似的传播能力,都显著高于CHV1-EP713. CHV1-CN280的侵染性克隆的获得,为进一步通过对CHV1-CN280、CHV1-Euro7和CHV1-EP713的区段互换,构建一些重组病毒来确定决定CHV1病毒水平播能力的关键位点、阐明CHV1-CN280高传播能力的分子生物学机制、设计和构建表型性状更好转基因生防工程菌株等研究打下了良好的基础。更多还原

【Abstract】 Hypoviruses are a group of unencapsidated, cytoplasmically replicating RNA viruses which can reduce virulence of Cryphonectria parasitica and alter host biological processes, resulting in suppressed pigmentation, reduced asexual production, and loss of female fertility. The discovery of the hypovirus presented a new avenue for biological control of chestnut blight. Hypoviruses lack an extracellular route of infection, they are transmitted from infected to non-infected fungal strains through hyphal anastomosis, but the transmission is limited by vegetative incompatibility (vic).The effects of virus strains on the virus transmission in C. parasitica were observed in this study. In laboratory assays by replicating vegetative incompatibility (vic) genotypes in independent fungal isolates, we quantified the transmission of three CHV1 viruses between isolates heteroallelic at one or two vic loci, and demonstrated that different CHV1 virus isolates determined their transmission abilities. The Chinese local hypovirus CHV1-CN280 showed higher transmission efficiency than the other two sequenced hypoviruses CHV1-EP713 and CHV1-Euro7 in this study.The results suggested that using the hypoviruses with higher transmission abilities could increase the effciencies of the biological control of chestnut blight.By constructing cDNA library and RT-PCR technology, the whole genome sequence CHV1-CN280 was determined. The virus showed extensive identities with other three sequenced hypoviruses:CHV1-EP713, CHV1-Euro7 and CHV1-Euro7, with an average of 61% and 94% identities at nuceotide level and 59% and 95% identities at amino acids level respectively. Compared with the other three sequenced hypovirus, the 3’end of CHV1-CN280 was relatively conservative, but the 5’end of CHV1-CN280 was very different from the other sequenced hypoviruses. The junctions of ORFA and ORFB were UAAUG in CHV1-EP713、CHV1-Euro7、CHV1-EP721 and CHV2-NB58, but in CHV1-CN280, it was AUGUAUAA:the UAA was the stop codon of ORFA, and the AUG was the initiation codon of ORFB. A pair of primers was designed to determine junctions of ORFA and ORFB in other 12 CHV1 hypoviruses. The results showed that The junction mode AUGUAUAA was very common as well as UAAUG. Another pair of primers was designed according to the conserved sequences in the 5’-UTR and ORFA region of CHV1 genome and the reverse transcription PCR (RT-PCR) method was developed for the population genetics analysis of CHV1. A total of 12 CHV1 hypoviruses was partially sequenced, combining the partially sequences of other CHV1 hypoviruses determined before, comparison analysis was performed. The results showed that all 36 CHV1 hypoviruses could be grouped into two groupes and eight subtypes. Considering the great differences between the two groupes and the enormous differences that CHV1-CN280 genomic sequence share with other three sequenced hypoviruses, it is suggested that the CHV1 hypoviruses similar to CHV1-CN280 should be classified into a new subspecies.The full-lenghth cDNA clone of the CHV1-CN280 was constructed by accurately joining viral cDNA fragments according to the whole genome sequence with aid of restriction enzymes. By transfection, CHV1-CN280 was successfully introduced into virus-free fungal host strains CN280(v-) and BB-1-5. All of the transfected strains showed similar phenotype to CN280 and the regenerated dsRNA showed similar transmission abilities to CHV1-CN280 which was much higher than CHV1-EP713, conforming the high transmission ability was the property of the virus.The availability of infectious cDNA clone of CHV1-CN280 makes it possible to dissect the molecular basis of a viral genome that controls the horizontal transmission ability and to understand other aspects of interaction between host and virus.更多还原