【作者】 吴承春；

【导师】 王国平；

【作者基本信息】 华中农业大学 ， 植物病理学， 2009， 博士

【摘要】 藠头(Allium chinense G.Don)为百合科葱属多年生植物,其起源地为亚洲,是一种具有极高的经济价值的传统蔬菜作物。而藠头病毒病是致使藠头产量和品质大幅降低的主要因素。本文在田间病害调查的基础上,利用生物学、血清学以及病毒粒子形态和感病植物细胞病理特征观察,结合分子生物学方法,对来自武汉江夏区的藠头病毒样品进行了种类鉴定,并对所获病毒克隆基因组部分序列特性进行了分析,确定了该藠头产区侵染的病毒至少包括三个属4种病毒,主要研究结果如下:1.明确发生在江夏区藠头存在复合感染现象。通过在湖北省武汉市江夏区藠头田间病害调查,发现田间藠头植株表现为花叶、褪绿条纹、植株扭曲、黄化等症状;通过汁液摩擦接种到6种指示植物上,确定部分藠头病毒病的繁殖寄主为昆诺藜;利用4个不同感病样品的粗提液,经负染后电镜观察,发现有3种不同的病毒粒子形态,包括杆状、线型(歪曲和直线型);直接采取感病藠头叶片制作超薄切片,经电镜观察发现感病寄主细胞质内含有大量的马铃薯Y病毒属(Potyvirus)成员特异的风轮状(横切)或柱状(纵切)内含体。2.首次发现侵染我国藠头的马铃薯Y病毒属OYDV病毒分离物。利用马铃薯Y病毒属内成员基因组简并引物,结合通用引物M4,获得该属基因组3′端序列大小约1.7kb的扩增片段,对该片段进行了克隆和序列测定,其全长为1625个核甘酸,包括部分NIb(636bp)、全长CP基因(771bp)和3′端UTR序列。将所获克隆基因组3′端核甘酸序列在数据库比对分析,发现所获病毒克隆基因组3′端区域与洋葱黄矮病毒(Onion yellow dwarf virus,OYDV)相同区域的核甘酸同源性最近,最高达99%。外壳蛋白基因的核甘酸和推测氨基酸序列与已报道的OYDV分离物CP相应区域同源性分别为80%-99%和87%-99%;构建了CP基因核甘酸和编码外壳蛋白氨基酸序列进化树,结果显示,所获克隆基因组CP序列与来自大蒜上的OYDV云南分离物OYDV-YN2(GenBank登录序列号AJ409313)、两个江苏分离物OYDV-JS2(登录序列号AJ409310)、OYDV-JS1(登陆序列号AJ293278)及日本分离物OYDV-Gzm(登录序列号AB000840)相同区域同源性均很高(≥97%),聚成一簇。将该分离物暂命名为OYDV-WH,这是OYDV病毒侵染我国藠头的首次报道。3.首次发现侵染我国藠头的香石竹潜隐病毒属7个不同克隆。利用香石竹潜隐病毒属内成员基因组简并引物,结合引物M4,获得该属基因组3′端核甘酸序列,共9个克隆,大小为1808-1812个核甘酸,序列特性分析表明,其包含病毒RNA基因组全长的第3-6共4个阅读框(ORF)。序列比对显示,这些克隆与现有报道的大蒜潜隐病毒(Garlic latent virus,GLV)基因组3′端相同区域的核苷酸序列最为接近,同源性分别在75%-82%之间,9个克隆中有7个克隆在该区域的核苷酸差异比较大,同源性在77%-93%之间。CP序列比对分析表明,所获得克隆基因组CP序列与目前报道的GLV基因组CP核苷酸和氨基酸同源性分别在75%-87%和88%-97%之间,其中克隆P2和carl-5基因组序列结构最相似,与日本藠头上的分离物GLV-Rjpn(登录序列号AB004565)氨基酸同源性最接近,达97%。本研究获得的具有较大序列差异的7个克隆之间基因组CP核苷酸和氨基酸的同源性分别76%-89%和88%-100%。外壳蛋白核甘酸序列进化树分析表明,7个克隆分别与其他GLV分离物共形成4个不同进化族,其中P4克隆CP核甘酸进化树上形成独立一族,且其核甘酸数量也有较大差异,因此,推测该克隆很可能为香石竹潜隐病毒属的一个新种。将所获的克隆基因组序列第3个ORF(编码蛋白TGB2)、第4个ORF(编码蛋白TGB3)和第6个ORF(编码核酸结合蛋白NABP)分别与已知报道的GLV基因组对应区域序列进行比对分析表明,氨基酸序列同源性分别为71%-90%、59%-85%、82%-95%。ORF3、ORF4、ORF6基因变异比ORF5基因变异要大些,其中ORF4变异最大,而ORF5变异主要发生在其编码氨基酸的N末端。根据Carlavirus属分类标准-不同病毒CP氨基酸同源性小于68%,同一病毒不同株系间为75%-90%。因此推断本研究所获得的克隆是属于GLV不同的分离物。这是我国藠头上的首次发现侵染GLV病毒。利用所获病毒克隆基因组RNA3′端序列设计RNA基因组部分中间片段下游特异性简并引物pCarl-2dW(-)和上游简并引物pCare-2(+),RT-PCR扩增获得包含病毒GLV基因组的第2个ORF2及部分ORF1的片段。序列结构分析发现,ORF1和ORF2之间有30个核甘酸的非编码区。ORF2与ORF3有23个核苷酸重叠区域。ORF2共含有708个核苷酸,编码TGB1蛋白,将所获病毒克隆基因组ORF2区域序列片段与现有报道的相关病毒相同区域比对分析,结果显示该病毒基因组ORF2也存在较大的变异,核甘酸和氨基酸同源性分别在72%-78%和76%-83%。4.首次发现侵染我国藠头的利用葱X病毒属2个不同病毒。利用葱X病毒属内成员基因组简并引物,结合引物M4,获得该属基因组3′端序列约1.0kb扩增片段,共8个克隆。其中克隆pGTdw-8基因组3′端序列含有939个核甘酸,其它7个克隆均为949个核甘酸(代号为pGTdw-15),包括部分CP基因(441个核甘酸)、完整NABP全长基因381个核甘酸和3′端非编码区。经数据库Blast序列分析表明,克隆pGTdw-8基因组3′端序列与现有报道的葱X病毒属我国大蒜上发现的大蒜病毒E(GarV-E,AJ292230)分离物相同区域核甘酸序列最相似,同源性达94%。另一个克隆pGTdw-15与大蒜病毒X韩国分离物(GarV-X,U89243)相同区域核甘酸序列最相似,具有91%的同源性,序列系统进化树表明,本研究所获葱X病毒属相关病毒克隆基因组3′端的核甘酸进化树与完整的NABP核甘酸进化树存在较大的进化族相关性和一致性。同时,基因组3′端UTR非翻译区序列的比对结果显示,本研究所得克隆pGTdw-8基因组3′端UTR与分离物Garv-E相同区域同源性达94%,另一类病毒克隆pGTdw-15基因组3′端UTR与分离物Garv-X相同区域同源性高达98%,根据国际病毒分类委员会发表的Allexivirus属分类标准是CP氨基酸同源性小于90%,3′端UTR核甘酸同源性小于90%。因此,认为本研究获得的2种克隆pGTdw-8和pGTdw-15分别属于葱X病毒属两种病毒Garv-E和Garv-X的分离物,这是我国藠头上的首次发现这两种葱X病毒属病毒。更多还原

【Abstract】 Allium chinense(G.Don),the genus Allium,family Liliaceae,is an economically important vegetable that has been considered to be of Asian origin.It can be usually used as edible bulb or medicinal herb.However,virus diseases of A.chinense are sever,causing the serious losses in bulbs production and poor quality.Some plants of cultivated A.chinense showing mosaic,chlorotic streak,twist,and crinkle on leaves were collected from fields at Jiangxia,a suburban district of Wuhan, Hubei Province,China,and transplanted into pots in a greenhouse.Crude sap from naturally infected plants were mechanically inoculated to six indicator plants.Results showed that the indicator chenopodium quinoa was the best host for virus propagation indoor.Three types of negatively stained virus particles from 4 infected sample leaves, including rod shape,lightly straight and curved filamentous shape,were observed under an electron microscope.Pinwheel or cylindrical inclusions,typical of a potyvirus infection,were also examined in the electron microscope by using ultrathin sections of diseased leaves.A potyvirus was detected in diseased A.chinense using degenerate primer sprimer and M4.The 3’-termined part(1625nts) of its genome was cloned and sequenced. Sequence analysis showed it contained the partial NIb gene(636 bp) and the complete coat protein(CP gene;771 bp),excluding its poly(A) tails.The 3’-termined part of obtained virus genome was closely similar to Onion yellow dwarf virus(OYDV)(highest identity 99%).The CP gene had 80%-99%identity at nucleotide level and 87%to 99% identity at the amino acid level with corresponding regions of known OYDV isolates from other hosts.Analysis of CP phylogenetic tree indicated the isolate,named tentatively OYDV-WH,showed high identity(no less than 97%) at nts level with Yunnan isolate (OYDV-YN2,GenBank accession number AJ409313),Jiangsu isolates(OYDV-JS1, OYDV-JS2,GenBank accession number AJ293278 & AJ409310,respectively) and Japan isolate(OYDV-Gzm,AB000840),and became closely the same group.It was first report of OYDV infecting A.chinense in China.Nine clones of carlavirus were obtained using carlavirus specific degenerate primer(pCar-1(|)) and p(?)imer M4,the 3’-terminal part(1808-1812 nts,respectively) of its genome RNA were amplified and sequenced,excluding its poly(A) tails.Sequences analysis showed that the partial genome contained ORF3-6 and 3’-UTR of the RNA genome of carlavirus genus.Sequence comparison indicated that the 3’-termined part of obtained virus clones were closely similar to Garlic latch t virus(GLV),showing 75%-82%identity to other known GLV.7 clones out of obtained clones were great variable in the 3’-termined same region of genome,showing 77%-93%identity at nucleotide level between themselves.The CP gene had 75%-87%identity at nucleotide level and 88%to 97%identity at the amino acid level with corresponding regions of known GLV isolates from other hosts,and had 76%-89%identity at nucleotide level and 88%to 100%identity at the amino acid level between the 7 clones from this paper.The isolates P2 and carl-5 had high identity(97%) at the amino acid level with the Japan isolate GLV-Rjpn(GenBank accession number AB004565) from the same host A. chinense.Analysis of CP phylogenetic tree suggested that the clone P4 was a distant group,and other clones were grouped respectively with other reported GLV isolates. More ORFs sequence comparison indicated that the ORF3,ORF4,ORF6 of its genome had 71%-90%,59%-85%and 82%-95%identity at the amino acid level between the clones and the reported GLV strains,respectively.Based on ORF3(TGB2),ORF4(TGB3) and ORF6(nucleic acid binding protein,NABP) amino acid sequences respectively,the ORF5(coat protein) gene was most conserved and the ORF4(TGB3 protein) gene was most great variable.The variable region of CP was mainly N-end of its amino acid sequences.According to the strains and species demarcation criteria for the genus carlavirus formulated by ICTV including CP amino acid identity(＜65%for distinct species and 75%-90%for distinct strains),the clones from A.chinense,in this paper were different GLV strains.This was first report of GLV occurring in A.chinense in China.Based on the obtained sequences above,a specific virus downstream primer pCarl-2dw(-) with another upstream degenerate primer pCarl-2(+) were designed for PCR. the fragment including partial ORF1 and ORF2(708nts) of GLV genome was amplified and sequenced.There was a non-translated region(30nts) between ORF1 and ORF2,and a overlapping region(23nts) between ORF2 and ORF3.Comparing with other known isolates showed that the ORF2 gene was also variable,and had 72%-78%identity at nucleotide level and 76%-83%identity at the amino acid level.Two Allexivirus were detected from the infected leaves in A.chinense using the degenerate primer pGV-3t(+) and M4.The 3’-end of its clone pGTdw-8 RNA genome had 939nt long,including partial CP(441nts) and complete NABP gene(381nts) and 3’-UTR,excluding its poly(A) tails.The same region of another deputy clone pGTdw-15 RNA genome had 949nt long,also including partial CP(441nts) and complete NABP gene(381nts) and 3’-UTR,excluding its poly(A) tails.Sequence comparisons showed that the two clone from A.chinense in this paper had 94%and 91%respectively nucleotides identity with the published China isolate of GarV-E(AJ292230) in garlic and the Korean isolate of GarV-X(U89243) in garlic.Analysis of NABP and 3’-UTR gene phylogenetic tree suggested that the isolates in this paper had close relationship with the reported garlic China isolate(AJ292230) and Korean isolate(U89243),and closely grouped respectively.Moreover,their 3’-UTR gene had 94%identity to GarV-E and 98%identity to GarV-X.According to the species demarcation criteria for the genus Allexivirus formulated by ICTV including CP amino acid identity(＜90%) and 3’-UTR nucleotide identity(＜90%),the two clones pGTdw-8 and pGTdw-15 in this paper were different strains of GarV-E and GarV-X respectively.This was first report of Allexivirus occurring in A.chinense in China.更多还原