【作者】 吴剑丙；

【导师】 周雪平；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 双生病毒是植物病毒中唯一一类具有孪生颗粒形态的单链DNA病毒,已在多个国家和地区的多种重要经济作物上造成毀灭性危害。在我国的云南、广东、广西、海南和台湾等地的作物及杂草上已相继发现了多种双生病毒。为进一步了解双生病毒在我国的种类、分布及危害,本论文对我国上海、广东和广西地区发生的双生病毒进行了分子鉴定及致病性研究。从上海孙桥农场采集了2份表现为黄曲叶症状的番茄样品(SH1和SH2),利用双生病毒特异性引物PA/PB扩增得到约500 bp片段的相似性为97.4%。随机选择SH2进行全序列测定(登录号AM282874)及检测分析后发现,样品中只含有单组份双生病毒,并且与日本报道的番茄黄化曲叶病毒(Tomato yellow leafcurl virus,TYLCV)分离物相似性最高,为99.8%,因此认为SH1和SH2是TYLCV的分离物,TYLCV是一类全世界广泛发生并引起严重危害的双生病毒,但这是我国首次报道有该病毒。从广东省表现为黄脉、叶脉外突和矮化等症状的稀莶样品(GD11、GD12、GD13、GD16、GD21、GD22、GD24、GD25、GD27和GD30)上分离到了双生病毒。利用简并引物PA/PB扩增得到约500 bp的片段,其相似性为95.4-100%。随机选择GD13、GD24和GD27进行全序列测定(登录号AM183224、AM230634-5),序列比较表明,它们相互之间的相似性为99.7-99.8%,与中国番木瓜曲叶病毒(Papaya leaf curl China virus,PaLCuCNV)的相似性最高,为76.8%。根据双生病毒命名原则,将其命名为稀莶黄脉病毒(Siegesbeckia yellowvein virus,SbYVV)。利用Southern blot和PCR检测,发现所有样品中都含有卫星分子DNAβ。对GD13、GD24和GD27 DNAβ全序列测定(登录号AM230643-5)及分析后发现,他们相互间的相似性为98.3-98.7%,并与其它已报道的DNAβ的相似性都很低,其中与泽兰黄脉病毒(Eupatorium yellow vein virus,EpYVV)卫星分子DNAβ最高,相似性分别为44.0、43.9和45.6%。随后,在这些样品中又分离到了两类DNA1分子。这两类DNA1分子的全长核苷酸序列相似性为66.2%,分别与赛葵黄脉病毒(Malvastrum yellow vein virus,MYVV)和烟草曲茎病毒(Tobaco curly shoot virus,TbCSV)DNA1的相似性最高,分别为为72.6%利69.1%。对所有样品的进一步分析发现,GD13、GD21、GD22、GD24和GD27中存在这两类DNA1分子的复合侵染。从广东地区表现曲叶症状的赛葵样品(GD6-10)上分离到了双生病毒。利用简并引物PA/PB扩增得到约500 bp的片段,其相似性为92.8-97.1%。随机选择GD6和GD9进行全基因组序列测定,全长均为2767 nts(登录号AM236779-80),两者间的相似性为98.9%,与已报道的PaLCuCNV相似性最高,为85.2%,因此将其命名为广东赛葵曲叶病毒(Malvastrum leaf curl Guangdongvirus,MLCGdV)。重组分析发现,GD6可能是一个重组分子,由PaLCuCNV、菲律宾番茄曲叶病毒(Tomato leaf curl Philippines Virus,ToLCPV)和其它未知病毒重组形成的。利用PCR和southern blot检测方法,均未在样品中发现DNA-B、DNAB或DNA1分子的存在。为进一步验证MLCGdV的致病性,构建了MLCGdV分离物GD6的侵染性克隆,发现农杆菌单独接种MLCGdV即可在本氏烟、心叶烟和矮牵牛等寄主植物上诱导产生典型的双生病毒侵染症状。而利用粉虱传毒试验,MLCGdV能在赛葵和普通烟上引起曲叶症状。此外,与中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)卫星分子DNAβ的互作研究发现,MLCGdV能支持TYLCCNV DNAβ的复制。从广东地区表现为曲叶、叶片边缘上卷、脉突和耳突等症状扶桑样品(GD33-38)中分离到了双生病毒。利用简并引物PA/PB对6个分离物扩增的约500 bp片段的相似性为97.3-99.0%。随机选择GD35和GD37分离物扩增病毒全长基因组DNA和DNAβ,序列分析表明,两者基因组的相似性为99.7%,DNAβ的相似性为99.8%,并分别和广东地区扶桑上已发现的木尔坦棉花曲叶病毒(Cotton leaf curl Multan virus,CLCuMV)和DNAB的相似性最高,为99.3%和99.5%及97.8%和98.0%。因此,认为GD33-38是CLCuMV的分离物。CLCuMV是一类在巴基斯坦广泛发生并引起严重危害的双生病毒,在中国属于检疫性病毒。构建了CLCuMV DNA-A和DNAβ的侵染性克隆并对其致病性进行测定。CLCuMV单独接种寄主植物不能诱导产生病毒侵染症状,但在接种的本氏烟、心叶烟、番茄和矮牵牛样品中能检测到病毒DNA的存在,说明CLCuMV能在这些寄主中自我复制。CLCuMV和DNAβ混合接种,能在番茄、木氏烟、普通烟、三生烟、矮牵牛和棉花等实验寄主中诱导典型的病害症状。从广西地区表现为植株矮化和黄脉等症状的稀莶样品(G110-112)分离到了双生病毒。利用简并引物PA/PB对3个分离物扩增的约500 bp片段的相似性为95.0-97.3%。随机选择G111分离物扩增病毒全长基因组DNA,序列测定表明全长为2784 nts(登录号AM238692),与SbYVV相似性最高,为84.5%,是一类新型的双生病毒,命名为广西稀莶黄脉病毒(Siegesbeckia yellow vein Guangxivirus,SbYVGxV)。从G111中发现有DNAβ,全长1355 nts(登录号AM238695),G111DNAp可能是由SbYVV DNAβ和其它未知基因重组而来。构建了已鉴定的一品红曲叶病毒(Euphorbia leaf curl virus,ELCV)G35分离物的侵染性克隆并进行致病性测定。发现ELCV单独接种寄主植物能诱导典型症状,并能与TYLCCNV DNAβ互作,扩大寄主范围,诱导产生更严重的症状。同时,ELCV与TYLCCNV DNAβ能侵染通常双生病毒较难侵染的寄主植物,如大豆、棉花、辣椒和黄瓜等,但不产生症状。更多还原

【Abstract】 Geminiviruses are plant viruses characterized by their unique twinned particles, which encapsidate a circular single-stranded DNA genome They have caused significant yield losses in many economically important crops worldwide. In China, several begomoviruses have been reported infecting crops and weeds in Yunnan, Guangdong, Guangxi, Hainan and Taiwan. In an attempt to provide a better understanding of the species, distribution and spread of begomoviruses in China, molecular identification of begomoviruses collected from Shanghai, Guangdong and Guangxi were carried out, and virus pathogenicity was studied in this dissertation.Two viral samples (SH1 and SH2) showing yellow leaf curl symptoms were collected from tomato plants in Sunqiao Farm of Shanghai and were confirmed to be infected by begomoviruses with PCR detection using universal primer pair PA/PB specific for the genus Begomovirus. The two amplified 500-bp fragments shared 97.4% nucleotide sequence identity. The complete sequence of SH2 isolate was determined (Accession No. AM282874). SH2 had the highest sequence identity (99.8%) with Tomato yellow leaf curl virus (TYLCV) isolate reported in Japan. Therefore, SH1 and SH2 were considered to be isolates of TYLCV. TYLCV has been reported to be widely distributed in the world, but this is the first report of TYLCV in China.Leaf samples of Siegesbeckia glabrescens (isolates GD11, GD12, GD13, GD16, GD21, GD22, GD24, GD25, GD27 and GD30) showing yellow vein, enation and stunting symptoms were collected in Guangdong province, China. The 500-bp fragments amplified by PA/PB shared 95.4-100% nucleotide sequence identity. Isolates GD13, GD24 and GD27 (Accession No. AM183224 and AM230634-5) were selected for determining the complete. The complete nucleotide sequences of GD13, GD24 and GD27 shared 98.3-98.7% identity and had the highest sequence identity (76.8%) with that of Papaya leaf curl China virus (PaLCuCNV). The molecular data showed that GD13. GD24 and GD27 were isolates of a distinct begomovirus species, for which the name Siegesbeckia yellow vein virus (SbYVV) was proposed. All isolates were found to be associated with DNAβmolecules by Southern blot and PCR. The complete DNAβsequences of isolates GD13, GD24 and GD27 were determined (Accession No. AM230643-5). Sequence analysis showed that they had relatively lower sequence identity with other reported DNAβs with the highest (44.0%, 43.9% and 45.6%, respectively) with Eupatorium yellow vein virus (EpYVV) DNAβ. In addition, two DNA 1 species were identified. The sequence identity between these two DNAls was 66.2%, and they had the higest nucleotide sequence identities with DNA1 of Malvastrum yellow vein virus (MYVV, 72.6%) and Tobaco curly shoot virus (TbCSV, 69.1%), respectively. Furthermore, mixed infection of these two DNA1 molecules were identified in GD13、GD21、GD22、GD24 and GD27 samples.Virus isolates GD6-10 were obtained from Malvastrum coromandelianum showing leaf curl symptoms in Guangdong Province of China. The 500-bp fragments were amplified by the degenerate primer pair PA/PB, and they shared 92.8-97.1% nucleotide sequence identity. The complete nucleotide sequences of both GD6 and GD9 were found to be 2767 nucleotides (Accession No. AM236779-80), with all the characteristic features of begomovirus genome organization. The two isolates have less than 85.2% nucleotide sequence identity with other reported begomoviruses. Consequently, GD6 and GD9 are considered to be isolates of a novel begomovirus species, for which the name Malvastrum leaf curl Guangdong virus (MLCuGdV) is proposed. Sequence analyses suggest that MLCuGdV may have arisen by recombination between viruses related to Papaya leaf curl China virus, Tomato leaf curl Philippines virus and other undiscovered virus ancestors. Neither the DNA-B component nor the DNAβmolecule associated with these begomovirus isolates was found. An infectious clone of MLCuGdV isolates GD6 was constructed. MLCuGdV efficiently infected test plants such as Nicotiana benthamiana, N. glutinosa and Petunia hybrida by agro-inoculation inducing typical begomoviral symptoms. MLCuGdV could effectively infect Malvastrum coromandelianum and N. tabacum by whitefly transmission. The interaction between MLCuGdV and Tomato yellow leaf curl China virus (TYLCCNV) DNAβwas studied and MLCuGdV could support the replication of TYLCCNV DNAβ. Virus isolates GD33-38 were obtained from Hibiscus rosa-sinensis showing leaf curl and enation symptoms in Guangdong Province of China. The 500-bp fragments were amplified by the degenerate primer pair PA/PB, and they shared 97.3-99.0% nucleotide sequence identity. The complete sequences of viral DNA and its associated DNAβof GD35 and GD37 were determined with 99.7% identity for viral DNA and 99.8% for DNAβ. Both the viral DNA and DNAβshared the highest sequence with Cotton leaf curl Multan virus (CLCuMV) collected in Hibiscus rosa-sinensis plant in Guangdong with 99% sequence identity for viral DNA and 98% for DNAβ. Therefore, GD33-38 were isolates of CLCuMV. CLCuMV have been widely distributed in Pakistan, and it is a quarantinable in China. An infectious clone of CLCuMV isolate GD37 and DNAβwere constructed. CLCuMV could replicate in test plants such as N. benthamiana, N. glutinosa, S. lycopersicum and P. bybrida without any symptoms. Co-inoculation of CLCuMV and DNAβcould induce typical begomoviral symptoms in these plantsVirus isolates G110-112 were obtained from Siegesbeckia glabrescens plants showing stunting and yellow vein symptoms in Guangxi, China. The 500-bp fragments were amplified by the degenerate primer pair PA/PB, and they shared 95.0-97.3% nucleotide sequence identity. The complete G111 DNA sequence is 2784 nucleotides (Accession No. AM238692) and shares the highest nucleotide sequence identity (85.4%) with SbYVV. The molecular data showed that G110-112 were isolates of a new begomovirus for which the name Siegesbeckia yellow vein Guangxi virus (SbYVGxV) is proposed. DNAβmolecule was indentified in G111 with 1355 nucleotides (Accession No. AM238695) and G111 DNAβmay have arisen by recombination between SbYVV DNAβand other unknown DNA molecule.An infectious clone of Euphorbia leaf curl virus (ELCV) isolate G35 was constructed and its infectivity was tested in host plants. ELCV alone could systemically infect tested plants and induce symptoms, while it could infect more tested plants and induce more severe symptoms when co-inoculated with TYLCCNV DNAβ. Moreover, ELCV and TYLCCNV DNAβcould systemically infect Glycine max, Gossypium hirsutum. Capsicum annuum and Cucumis sativus although symptoms were not observed.更多还原