【作者】 钱亚娟；

【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】 双生病毒是一类世界范围内广泛发生的具有孪生颗粒形态的植物单链DNA病毒,近年来已在多种作物上造成毁灭性危害,给作物生产造成了严重损失。卫星DNAβ是与一些单组份双生病毒伴随的并是病毒在自然寄主上引起典型症状所必需的致病相关分子。迄今发现的所有DNAβ分子在互补链都含有一个位置和大小保守的βC1基因。为了弄清双生病毒DNAβ分子参与致病的机理,本论文对缺失βC1基因的DNAβ分子的致病性及稳定性进行了研究,并利用缺失βC1基因的DNAβ分子进行了外源基因的表达及基因沉默研究。 对中国番茄黄化曲叶病毒Y10分离物(TYLCCNV-Y10)DNAβ上的βC1基因进行了功能鉴定。构建了一个βC1基因完全缺失的突变体Y10 DNA△C1β,侵染性测定表明在和TYLCCNV-Y10共同侵染寄主植物时,突变体Y10 DNA△C1β能系统侵染本氏烟、心叶烟、矮牵牛,但不能侵染普通烟、三生烟、番茄,突变体DNA△C1β在寄主上不诱导明显症状。而野生型Y10 DNAβ和TYLCCNV-Y10共同侵染后能够系统侵染以上的所有寄主并且诱导严重的病害症状,侵染效率也高于Y10 DNA△C1β。推测βC1基因是一个参与症状诱导的关键因子并且可能和TYLCCNV-Y10/DNAβ复合体的侵染效率有关。Southern印迹分析显示βC1基因不是TYLCCNV和Y10β复制所必需的,但是它的存在可以增加病毒及卫星在植物组织中的积累量。免疫捕获PCR检测表明,βC1基因缺失的Y10 DNA△C1β(1.0kb)能够包裹在Y10编码的外壳蛋白中。PCR和Southern印迹检测都表明βC1基因缺失的突变体Y10 DNA△C1β在寄主植物中非常稳定,并且能利用多种异源双生病毒复制。 对烟草曲茎病毒Y35分离物(TbCSV-Y35)DNAβ上的βC1基因也进行了功能鉴定。构建了一个βC1基因完全缺失的突变体Y35 DNA△C1β。侵染性测定表明在和TbCSV-Y35共同侵染寄主植物时,突变体Y35 DNA△C1β能够系统侵染本氏烟、心叶烟、矮牵牛、三生烟、普通烟和番茄等寄主,但是与野生型DNAβ相比,TbCSV/DNA△C1β复合体引起的症状减轻,并且缺失βC1基因后TbCSV/DNAβ复合体的侵染效率会有不同程度的下降。Southern印迹分析显示βC1基因缺失对TbCSV-Y35的复制没有明显影响,但是它的缺失使DNAβ在植物组织中的积累水平明显降低。在Y35与Y35 DNA△C1β共同侵染的本氏烟、心叶烟、三生烟、矮牵牛中发更多还原

【Abstract】 Geminiviruses are a group of plant viruses occurred worldwide and characterized by their unique twinned particles, which encapsidate a circular single-stranded DNA genome. In recent years, they cause destructive diseases in many important crops and have caused significant yield losses for crop production. A novel satellite DNA, referred to as DNAβ is found to be associated with monopartite begomoviruses and is found to be required for inducing typical symptoms in natural hosts. All reported DNAβ possesses a βC1 gene on the complementary strand, which has a conserved position and size. In order to further investigate the role of DNAβ in pathogenicity, the pathogenicity and stability of βCl truncated DNAβ molecules was investigated, furthermore, the βC1 truncated DNAβ was used for expression of foreign genes and inducing gene silencing.The function of βC1 gene of DNAβ associated with Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10) was studied. A βC1 truncated Y10 DNAβ was constructed. Co-agroinoculation with TYLCCNV-Y10 showed the truncated DNAβ was infectious in Nicotiana benthamiana and N. glutinosa plants producing asymptomatic symptoms but not in N. tabacum Samsun, N. tabacum and Lycopersicon esculentum plants. The wild type Y10 DNAp co-agroinoculated with TYLCCNV-Y10 caused systemic infection in all the above hosts and induced severe disease symptoms. These results suggest that βC1 gene is a pathogenicity determinant and contributes to the infectivity of the disease complex of TYLCCNV and the satellite DNAβ. Results of Southern blot analysis indicate that βC1 gene is not required for TYLCCNV and DNAβ replication. However, the presence of βCl gene in DNAβ can increase both TYLCCNV and DNAβ accumulation in infected plants. The immunotrapping-PCR demonstrated the truncated Y10 DNAβ (1.0kb) was encapsidated by Y10 coat protein. The PCR and Southern blot analysis showed that the truncated Y10 DNAP was stable in N. benthamiana and N. glutinosa plants and could replicate when co-agroinoculation with other geminiviruses.The function of βC1 gene of DNAβ associated with Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) was studied. A βCl truncated Y35 DNAβ was constructed. The truncated Y35 DNAβ was infectious in N. benthamiana, N. glutinosa, Petunia bybrida, N. tabacum Samsun, N. tabacum and Lycopersicon esculentum plants when co-infected with更多还原