【作者】 王向阳；

【导师】 周雪平；

【作者基本信息】 浙江大学 ， 植物病理学， 2004， 博士

【摘要】 近年来，我国西南地区番木瓜(Carica papaya)曲叶病有加重的趋势。为此我们对广西和广东的番木瓜曲叶病样本开展了研究，并对当地的杂草胜红蓟(Ageratum conyzoides)的黄脉病也进行了研究。 利用三抗体夹心ELISA(TAS-ELISA)及PCR检测的方法对采自广西的7个番木瓜曲叶病样品和广东1个番木瓜曲叶病样品，及采自广西胜红蓟的5个黄脉样品进行了检测，表明均为粉虱传双生病毒。PCR扩增产物克隆后进行序列测定，结果表明这些样品主要存在两类病毒，少数存在多种双生病毒复合侵染现象 克隆并测定了G2和GD2 DNA-A全序列。序列比较分析表明，G2和GD2相互之间的同源性只有73.4％，因此是两个不同的病毒。经NCBI检索和序列比较分析表明，G2与胜红蓟曲叶病毒Ageratum yellow China vein virus-[Hn2](AYVCNV-[Hn2])的同源性最高，同源性达到83.4％；GD2与胜红蓟曲叶病毒Ageratum yellow vein virus(AYVV)的同源性最高，同源性达到75.2％。根据双生病毒分类与命名原则，我们将G2命名为中国番木瓜曲叶病毒，英文名为(Papaya leaf curl China virus，PaLCuCNV)；将GD2命名为广东番木瓜曲叶病毒，英文名为(Papaya leaf curl Guandong virus，PaLCuGV)，并已经得到国际病毒分类委员会的确认。PaLCuCNV-[G2]和PaLCuGV-[GD2]的外壳蛋白与辣椒曲叶病毒-马来西亚分离物(Pepper leaf curl virus-[Malaysia]，PepLCV-[MY])的外壳蛋白的氨基酸序列同源性最高，分别有97.7％和94.2％同源性，说明这三种病毒的外壳蛋白可能来自同一个祖先。 从胜红蓟样本G8和G10中克隆并测定了两个DNA-A全序列，序列比较分析发现，G8和G10之间的同源性达到93.2％，认为是同一病毒的不同分离物。经NCBI检索和序列比较分析发现，G8和G10与中国番木瓜曲叶病毒G2的序列最接近，同源性分别达到92％和89.2％，因此认为G8和G10是中国番木瓜曲叶病毒的分离物。G8和G10的外壳蛋白的氨基酸序列与PepLCV-[MY]的外壳蛋白序列的同源性达到98.4％，说明它们有共同的起源。G8和G10的AC1(rep) AC2，AC3，AC4蛋白序列与中国胜红蓟黄脉病毒相应蛋白序列的同源性比较，显示关系密切。因此，G8和G10可能是一个重组的菜豆金色叶病毒属病毒。 用几种粉虱传双生病毒的特征引物，对G7进行了复合侵染的检测，发现存浙江大学博士学位论文侵染番木瓜和胜红蓟的双生病毒研究在多种病毒的复合侵染现象。得到部分G7的DNA一A序列，经NCBI检索和序列比较分析发现了5种菜豆金色叶病毒属病毒的部分DNA序列，分别是中国番木瓜曲叶病毒、中国胜红蓟黄脉病毒、中国番茄曲叶病毒、一品红曲叶病毒、云南烟草曲叶病毒。更多还原

【Abstract】 Leaf curl symptom of papaya (Carica papaya) occurred severely in recent years in south-west part of China. The papaya leaf curl samples from Guangxi and Guangdong provinces were collected and studied, and ageratum yellow vein sampleswere also collected and studied.7 papaya leaf curl samples collected from Guangxi province and 1 fromGuangdong province, and 5 ageratum yellow vein samples from Guangxi province were tested with triple antibody sandwich ELISA (TAS-ELISA) and PCR, Results showed all the virus samples were infected by whitefly-transmitted geminiviruses. The products of PCR were cloned and sequenced. Sequence analyses showed 2 types of viruses existed among those samples, a few samples were infected by several geminiviruses.The full-length DNA-A molecules from papaya sample G2 and GD2 were cloned and sequenced. Sequence analyses and comparisons showed that DNA-A between G2 and GD2 shared only 73.4% nucleotide sequence identity. So G2 and GD2 were 2 different begomovirus species. Sequence comparisons and phylogenetic analysis showed that DNA-A of G2 was most related to that of Ageratum yellow vein China virus-[Hn2] (AYVCNV-[Hn2] with 83.4% nucleotide sequence identity, and GD2 were most related to that of Ageratum yellow vein virus (AYVV) , with 75.2% nucleotide sequence identity. According to geminivirus taxonomic criteria, the name Papaya leaf curl China virus (PaLGuCNV) for G2 and Papaya leaf curl Guangdong virus (PaLCuGV) for GD2 were proposed, and the two viruses were approved by International Committee on Virus Taxonomy. Compared with deduced ORFs showed the coat protein of G2 and GD2 shared highest amino acid sequence similarity (97.7% and 94.2% respectively) with that of Pepper leaf curl virus-[Malaysia] (PepLCV-[MY]), suggesting that CP of these three isolates may evolved from identical ancestor.The full-length DNA-A molecules from ageratum sample G8 and G10 were cloned and sequenced. Sequence analyses and comparisons showed DNA-A of G8and G10 shared 93.2% identity to each other, So G8 and G10 were isolates of a same begomovirus. Comparisons with other geminiviruses showed that the DNA-A of G8 and G10 were most closely related to that of PaLCuCNV-[G2] (92% and 89.2% sequence identity, respectively), so G8 and G10 were regarded as virus isolates of PaLCuCNV. Comparisons with encoded amino acid sequences of CP show that G8 and G10 may have a common ancestor with PepLCV-[MY] (98.4% sequence identity), and the amino acid sequences of AC 1 (rep) AC2, AC3 AC4 of G8 and G10 had highest identities with that of AYVCNV, indicated that they may have evolved from the same ancestry. The molecular data showed that G8 and G10 might be a recombination Begomovirus species.Mixed infection was tested in G7 with primers specific for DNA-A of several geminiviruses. The results showed that mixed infection of begomo viruses occurred in G7. Partial DNA-A sequence comparison with other geminiviruses showed that G7 were infected with 5 begomoviruses, these viruses were Papaya leaf curl China virus, Ageratum yellow vein china virus, Tomato leaf curl China virus, Euphorbia leaf curl virus and Tobacco leaf curl Yunnan virus.更多还原