【作者】 李正和；

【导师】 周雪平；

【作者基本信息】 浙江大学 ， 植物病理学， 2005， 博士

【摘要】 双生病毒是一类具有孪生颗粒形态的植物单链DNA病毒,目前已在多个国家和地区的作物上造成毁灭性危害。近年来,我国华南各省作物上双生病毒病害的发生有逐年加重的趋势。本文对云南番茄上的双生病毒类群以及烟草曲茎病毒(TbCSV)的致病分子机理进行了研究。 从云南省采集了23个的番茄曲叶病的样品,TAS-ELISA和部分序列分析发现它们属于不同的种类。测定了四个分离物(Y25、Y41、Y72和Y161)的全序列,同源性比较和进化分析表明,Y25、Y41、Y72和Y161分别属于四种不同的病毒,即中国番茄黄化曲叶病毒(TYLCCNV)、TbCSV、泰国番茄黄化曲叶病毒(TYLCTHV)以及云南烟草曲叶病毒(TbLCYNV)。所有TYLCCNV和TYLCTHV的分离物都发现伴随有卫星DNAβ分子,而属于TbCSV和TbLCYNV的几个分离物都没有检测到卫星的存在。测定了Y25、Y72、Y77和Y79伴随的DNAp的全序列,比较发现,Y25p与其它TYLCCNV分离物伴随的卫星紧密相关,而Y72β、Y77β和Y79β属于一类新的卫星分子,与TbCSV卫星的同源性最高。 田间调查发现,在42个TbCSV侵染的样品中,仅13个分离物伴随有卫星分子。对其中一个伴随DNAB的分离物(TbCSV-Y35)进行的致病性分析表明,TbCSV-Y35单独能够在本氏烟和自然寄主普通烟上产生严重的上卷症状,且子代病毒能够被烟粉虱传播,表明TbCSV-Y35单独能够在田间维持病害的发生。当与卫星共同接种时,TbCSV-Y35能够在其烟属(Nicotiana)寄主上产生更为严重的下卷症状。然而在番茄寄主上,卫星对于TbCSV-Y35诱导的症状没有影响,且辅助病毒不能稳定的维持卫星的存在。与其它双生病毒/DNAβ病害复合体不同,卫星对于TbCSV-Y35的核酸积累、侵染效率、发病时间和寄主范围都没有影响。对另一个不伴随卫星的分离物(TbCSV-Y1)进行研究发现,TbCSV-Y1能引起和TbCSV-Y35相似的症状,且能与DNAp进行功能性的互作。这些结果表明,TbCSV DNAβ并非辅助病毒系统侵染所必需的,但能以寄主专化性的方式加重病害的症状。 利用Overlap Extension PCR的方法,将TbCSV-Y35 C4 ORF上2422和2440更多还原

【Abstract】 Geminiviruses are a group of plant DNA viruses characterized by their unique twinned particles, and are currently emerging as a major threat to crop production worldwide. In southern China, the incidence and severity of begomovirus diseases have increased rapidly during the past several years. The current study was carried out for identification of tomato begomoviruses in Yunnan and elucidating molecular mechanism of pathogenicity of Tobacco curly shoot virus (TbCSV).23 tomato leaf curl samples were collected in Yunnan. They fell into several groups according to their reaction with a panel of monoclonal antibodies in TAS-ELISA and partial viral DNA sequences. The complete DNA A sequences of 4 isolates, corresponding to the four groups, were determined. Sequence comparisons and phylogenetic analysis revealed that they are most closely related to Tomato yellow leaf curl China virus (TYLCCNV), TbCSV, Tobacco leaf curl Yunnan virus (TbLCYNV) and Tomato yellow leaf curl Thailand virus (TYLCTHV), respectively. In addition, all TYLCCNV and TYLCTHV isolates were found to be associated with DNAβ molecules, while satellite DNA was not found in virus isolates of TbCSV and TbLCYNV. The complete DNAβ sequences of TYLCCNV isolate Y25, TYLCTHV isolates Y72, Y77 and Y79 were determined. Sequence analysis showed that Y25 DNAβ is most closely related to satellites associated with other TYLCCNV isolates, whereas DNAβ of Y72, Y77 and Y79 seem to be different from other characterized DNAβ, sharing the highest nucleotide sequence identity with DNAβ of TbCSV.Field survey demonstrated that only 13 isolates were associated with DNAβ among 42 TbCSV infected, field-collected samples. Infectivity showed an infectious clone of TbCSV isolate Y35 (TbCSV-Y35, an isolate associated with DNAβ) alone induced severe upward leaf curling in Nicotiana benthamiana and its natural host tobacco. In addition, TbCSV-Y35 can be transmitted between plants by whitefly vector, regardless of the presence or absence of DNAβ, indicating TbCSV-Y35 alone is sufficient for disease maintenance in the field. Co-inoculation of TbCSV-Y35 and itscognate DNAβ alters the symptom to a more severe downward leaf curling on Nicotiana spp. When inoculated on tomato, however, DNAβ failed to exacerbate the symptom phenotype induced by helper virus. In a significant number of tomato plants co-inoculated with clones of TbCSV-Y35 and DNAβ, the virus does not steadily maintain the satellite. In contrast to other begomovirus/DNAβ complexes, the satellite has no discernible effect on the DNA accumulation, infectivity, onset of symptoms and host range of helper virus. For a TbCSV isolate not associated with DNAβ (TbCSV-Yl), it induced symptoms resembling those of TbCSV-Y35 and retained the ability to functionally interact with DNAβ. Our data indicates that TbCSV DNAβ. is not necessary for infection but intensify symptoms in a host-dependent manner.TbCSV-Y35 mutant was constructed in vitro by Overlap Extension PCR, in which T was replaced with C at nucleotides 2422 and 2440, respectively, within the C4 open reading frame (ORP). The mutations destroy the two possible initiator ATG codons for the C4 ORF without disrupting the coding capacity of the C1 ORF, which entirely overlaps the C4 ORF. The mutant retained the capability of autonomous replication in leaf disc of N. benthamiana and was able to infect this host systemically although symptom development was dramatically attenuated and DNA levels were greatly reduced. Co-inoculation with DNAβ significantly enhanced the symptom severity in N. benthamiana plants as well as DNA accumulation in systemically infected tissue and in leaf disc. When tobacco and tomato plants were inoculated, however, the mutants were unable to move systemically, regardless of the presence or absence of DNAβ. The results suggest that the TbCSV C4 ORF encodes a polypeptide involved in symptom development and host-specific movement, while satellite DNAβ, which also encodes a pathogenicity determinant, can partially compensated the functional defect of C更多还原