【作者】 张成玲；

【导师】 李向东；

【作者基本信息】 山东农业大学 ， 植物病理学， 2010， 博士

【摘要】 烟草脉带花叶病毒(Tobacco vein banding mosaic virus,TVBMV)、马铃薯Y病毒(Potato virus Y,PVY)和芜菁花叶病毒(Turnip mosaic virus,TuMV)都属于马铃薯Y病毒属(Potyvirus)。TVBMV和PVY是烟草上的重要病毒,在我国烟草上的危害呈上升趋势;TuMV是十字花科植物上的重要病毒。它们侵染作物后造成作物产量和品质的下降。本研究分别分析了TVBMV、TuMV和PVY三种病毒的群体结构。从中国烟草主产区采集到40个TVBMV分离物,测定了其HC-Pro、P3、6K1和CP基因,通过比较GenBank上其它分离物的相应序列分析了TVBMV的遗传多样性和种群结构。根据不同基因的分析结果,将TVBMV分离物划分为两个(6K1和CP)或三个组(P3和HC-Pro)。中国云南分离物单独成簇,基因交流频繁,但与其它省份分离物之间的基因交流不频繁。TVBMV的4个基因片段都处于负向选择,但不同的基因片段承受的选择压力不同。分析的42个分离物中有13个发生重组。通过RT-PCR扩增得到中国的56个TuMV萝卜分离物的3′-末端基因组序列,分析其群体变异。中国105个分离物(含GenBank上49个)中,共有12个分离物存在明显重组。系统进化树结果表明,中国TuMV可分为Asian-BR、basal-BR和world-B三个组,有50.5%的分离物位于basal-BR组中。中国basal-BR组分为3个亚组,I和II亚组与日本的basal-BR I、II亚组一致,但是III亚组单独成簇,日本还存在IV亚组。潍坊和泰安的basal-BR分离物是新出现的谱系,处于爆发状态。TuMV每个组或亚组都处于负向(纯化)选择。泰安和潍坊地区的分离物基因交流频繁。从中国烟草主要种植区采集PVY分离物,接种到苋色藜(Chenopodium amaranticolor)上,经过多次单斑分离获得25个生物纯的分离物。测定了这25个分离物的HC-Pro和CP序列及另外4个分离物的CP序列。25个分离物中有22个能引起烟草叶脉坏死,3个引起烟草花叶或脉明。19个分离物用PVYO CP单克隆抗体检测呈现阳性反应。血清学和分子进化分析表明,25个分离物中有3个为O株系、2个为N株系、2个为NTN株系、16个为N-Wi株系和2个为N-HCO株系。Feixian8和Mengyin60属于N-HCO株系,他们具有类似PVYO株系的HC-Pro与类似PVYN株系的CP基因,但是能引起烟草叶脉坏死,为新的株系。HC-Pro和CP编码区处于负向选择,但是HC-Pro序列的7个氨基酸和CP序列的6个氨基酸处于正向选择。PVY的亚种群不具有寄主特异性,不同亚种群承受的选择压力不同。马铃薯X病毒(Potato virus X,PVX)是常见的植物病毒之一,主要侵染茄科植物。本研究测得了一株1985年的PVX分离物(PVX-1985)的全基因组序列。PVX-1985不包括Poly(A)尾巴共6 435个核苷酸(nucleotides,nt),含有5个开放阅读框(ORF)。ORF1编码复制酶蛋白,ORF2、3和4为重叠的三联基因块,ORF5编码外壳蛋白。系统进化分析表明,PVX分离物可以分为两个组:欧亚组和美洲组,PVX-1985分离物位于欧亚组。PVX的所有开放阅读框处于负向(纯化)选择,但是依赖于RNA的RNA聚合酶基因(RdRp)中间区域处于正向(多样化)选择。更多还原

【Abstract】 Tobacco vein banding mosaic virus (TVBMV), Turnip mosaic virus (TuMV) and Potato virus Y (PVY) belong to the genus Potyvirus, the family Potyviridae. TVBMV and PVY infect plants of the family Solanaceae and TuMV infects the family Cruciferae in nature and they cause great economic losses to crop production. The genetic structures of TVBMV, TuMV and PVY were analyzed in this study.TVBMV is one of the most economically important viruses infecting tobacco in China. In this paper, we determined the HC-Pro, P3, 6K1 and CP gene sequences of 40 TVBMV isolates collected from different tobacco-growing areas in China and compared them with those sequences available to study its genetic diversity and population structure. TVBMV isolates could be divided into two (6K1 and CP) or three (P3 and HC-Pro) groups, depending on which gene was analyzed. The populations from most part of China showed high genetic identities and had frequent gene flow. The population from Yunnan, a southern western province of China, formed a separate group and had frequent gene flow within this region. However, the gene flow between TVBMV isolates from Yunnan and other parts of China was infrequent. TVBMV genes were under strong negative selection, but the pressures were different for different genes. Recombination was common in the evolution of TVBMV isolates. Our data also suggested that the most recent outbreak of TVBMV in China was not due to the emergence of new TVBMV subtype(s).The 3′- terminal genome of 56 TuMV isolates were determined and analyzed with other Chinese and Japanese isolates available in the GenBank to understand the genetic structure of this virus. Twelve of 105 isolates were clear recombination. Isolates from Radish in China formed three groups: Asian-BR, basal-BR and world B. Group basal-BR consisted of 50.5% isolates and were divided into three subpopulations. Subpopulations I and II were genetical identical with Janpanese subpopulations but subpopulation III was not, while subpopulation IV only existed in Japan. Isolates in the group basal-BR from Tai′an and Weifang were new emergent and in a state of expansion. All populations and subpopulations were under negative pression. Gene flow between Tai′an and Weifang was frequent.Genetic variability of PVY isolates infecting potato has been characterized, but little is known about genetic diversity of PVY isolates infecting tobacco crops. In this study, PVY isolates were collected from major tobacco-growing areas in China and single-lesion isolates produced by serial inoculation on Chenopodium amaranticolor. Twenty-two of 25 isolates caused systemic veinal necrosis symptoms and three isolates caused mosaic in tobacco. Nineteen of 25 isolates were detected by PVYO CP specific antibodies. Serological and phylogenetic analyses showed that, among the 25 isolates, three isolates belonged to group O, two isolates belonged to group N, two isolates belonged to group NTN, sixteen isolates belonged to group N-Wi and two isolates belonged to group N-HcO. Isolates Feixian8 and Mengyin60 contained a PVYN-like coat protein (CP) and PVYO-like helper componentproteinase (HC-Pro) but caused systemic veinal necrosis symptoms in tobacco, and hence designated as PVYN-HcO recombination. Seven amino acids in HC-Pro and six amino acids in CP were under positive selection. Selection pressures differed between the subpopulations of PVY distinguished by phylogenetic analysis of HC-Pro and CP sequences. When PVY isolates from potato were included, no host-specific clustering of the PVY isolates was observed in phylogenetic and nucleotide diversity analyses. Potato virus X (PVX) is one of the most common plant viruses that cause great economic losses to solanaceous plants. The complete genome of PVX-1985 is 6 435 nucleotides (nt) exinclude Poly (A) and contains five open reading frames (ORFs). ORF1 encodes a protein of 166 kDa that functions as RNA-dependent RNA polymerase (RdRp). The overlapping ORFs 2, 3 and 4 encode triple gene block proteins (TGBp) of 25, 12 and 8 kDa, respectively. ORF 5 encodes the 25 kDa coat protein (CP). The phylogenetic trees of the full genomic sequences and the CP gene demonstrated that PVX isolates were clustered into two groups: Eurasia and America, and PVX-1985 fell within the group Eurasia. All the open reading frames of PVX were under negative (or purifying) selection, but the central region of RdRp was under positive or diversifying selection.更多还原