【作者】 青玲；

【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】 本论文对中国番茄黄化曲叶病毒(TYLCCNV)Y10分离物(Y10)和烟草曲茎病毒(TbCSV)Y35分离物(Y35)及其伴随的卫星DNAβ(Y10β及Y35β)分子之间的互作及DNAβ分子的变异进行了研究。 将Y35+Y35β、Y35+Y10β、Y10+Y10β及Y10+Y35β分别接种本氏烟、心叶烟及三生烟,Southern印迹分析表明,Y10β及Y35β均可被异源辅助病毒复制,但Y10β或Y35β作为异源DNAβ分子时的积累量明显低于伴随其同源辅助病毒时的积累量。症状观察表明,无论Y10还是Y35伴随同源或异源DNAβ时诱导的症状明显不同,Y10β及Y35β作为异源卫星时与其辅助病毒诱导的症状也不同于与其同源辅助病毒引起的症状,且Y10β伴随异源病毒Y35时致病性更强,Southem印迹显示,病毒及卫星的积累量并不完全与症状严重程度直接相关。 当Y10β及Y35β同时伴随Y10或Y35侵染本氏烟及心叶烟时,PCR及Southem印迹显示,异源DNAβ到侵染后期均丢失。当两种病毒同时伴随Y10β或Y35β共同侵染本氏烟时诱导的症状比一种病毒伴随一种卫星引起的症状轻,表明有拮抗作用发生,但侵染心叶烟仅在前期观察到拮抗现象,PCR及Southem印迹显示Y10在侵染后期已丢失,而本氏烟中两种病毒及卫星均存在。当两种病毒伴随两种卫星共同侵染本氏烟及心叶烟时,在整个侵染过程中两种病毒及卫星在本氏烟中始终存在,而在心叶烟中仅在侵染前期能检测到Y10及Y10β。 对Y10β及Y35β伴随不同辅助病毒及在不同寄主中的种群变异的研究表明,与伴随同源辅助病毒时相比,Y10β及Y35β伴随异源辅助病毒时其种群结构具有更强烈的变异趋势及更丰富的种群遗传多样性,且Y35β及Y10β种群在两种不同寄主中的变异水平存在差异,表明病毒及寄主在卫星DNAβ种群进化中可能具有重要作用。变异数据分析显示,Y10β及Y35β种群基因组内在SCR区及βCl编码区内均没有任何位点发生突变,而突变区域主要集中在A-Rich区。Y35β及Y10β种群中突变类型是多样化的,包括碱基替代、插入或缺失,还发现有多个克隆在同一核苷酸位点发生相同类型的突变,这种突变积累现象的生物学意义及其在病害流行中的作用有待进一步研究。 Y10β及Y35ββCl基因缺失突变体(Y10△Clβ及Y35△Clβ)能介导对同源更多还原

【Abstract】 The interactions between Tomato yellow leaf curl China virus (TYLCCNV) Y10 isolate (Y10) and Tobacco curly shoot virus (TbCSV) Y35 isolate (Y35) with their associated DNAβ molecules (YlOp and Y35P) and molecular variations among YlOp and Y35β populations were investigated in this study.Y35+Y35β, Y35+Y10β, Y10+Y10β and Y10+Y35β were agroinoculated to Nicotiana benthamiana, N. glutinosa plants and N. tabacum Samsun nn, respectively Southern blot analysis showed that both YlOp and Y35P could be trans-replicated by heterogenous geminivirus, but DNAβ molecules associated, with heterogenous helper virus accumulated in plants at a lower level as compared with those associated, with cognate helper virus. Symptoms induced by Y10 or Y35 with their cognate DNAβ were different from those induced by co-infection with heterogenous DNAβ, and high pathogenicity was found in plants inoculated with Y10β and Y35. Southern blot analysis indicated that no direct co-relation existed, between symptom severity and virus and satellite accumulation levels in plants.PCR and Southern blot analyses showed that the heterogenous DNAβ. could not be detected in N. benthamiana and N. glutinosa plants agroinoculated with Y10+Y10β+Y35β or Y35+Y35β+Y10β in later stage of infection. In N. benthamiana plants agroinoculated with Y35+Y10+Y35β or Y35+Y10+Y10β, both viruses and DNAP molecules were detectable in the whole growing period, and the inoculated plants showed milder symptoms as compared with those induced by Y35 or Y10 plus cognate or heterogenous DNAβ. In contrast, symptom antagonism was observed in N. glutinosa inoculated with the same combinations in the early stage of infection, but not observed in later stage of infection, and PCR and Southern blot indicated that Y10 was undetectable at later stage of infection. Presence of viruses and associated DNAβ . molecules could be detected in the whole growing period in N. benthamiana plants agroinoculated with Y35+Y10+Y35β+Y10β, however, Y10 and Y10β only existed in N. glutinosa in the early stage of infection.The population diversity of DNAβ was compared by DNA sequencing of progenies of Y10β and Y35β molecules in N. benthamiana and N. glutinosa plants agroinoculated withY35+Y35β, Y35+Y10p, Y10+Y10β and Y10+Y35β, respectively. Comparisons showed that Y10β and Y35β populations associated with heterogenous helper virus had stronger mutation tendency and higher population diversity than associated with cognate helper virus. Moreover, Y10β and Y35β populations had different levels of mutation frequency in N. benthamiana and N. glutinosa, indicating that hosts may play important role in evolution of DNAβ populations. Most mutations generated in Y10β and Y35β populations were located in A-rich region. No mutation was found in βCl ORF and SCR. Various mutation types including substitution, deletion and insertion occurred in Y10β and Y35β populations, and the same type of mutation in the same nucleotide position was observed in several DNAp clones. The biological significance of the mutation accumulation in disease epidemic is unclear.Pre-inoculation of βCl gene defective mutant of Y10β or Y35β (Y10ΔC1β and Y35AC1β) with cognate virus could mediated cross-protection to challenge-infection of cognate or heterogenous helper viruses and associated DNAp. All N. benthamiana and N. glutinosa plants pre-inoculated with Y10+Y10ΔC1β or Y35+Y35AC1β exhibited mild symptoms after challenge inoculation with Y10+Y10P or Y35+Y35β, but better protection effects were found for cognate helper viruse and associated DNAp, and protection effects in N. benthamiana were better than those in N. glutinosa. The above results will have important role for control of TYLCCNV and other begomo viruses.Y128 isolate was obtained from tobacco in Baoshan, Yunnan province. The PCR amplification proved that Y128 is an isolate of TbCSV. Satellite DNA molecule (Y128β) was found to be associated with Y128 and the complete nucleotide sequence of Y128β was determined to be 1350 nucleotides. Y128p has 85更多还原