西瓜花叶病毒2号南瓜分离物的鉴定及其外壳蛋白基因序列分析

 利用电镜和酶联免疫吸附测定法(ELISA)在黑龙江省采集的南瓜病样中检测到西瓜花叶病毒2号(WMV-2)。再利用免疫PCR (IC-PCR)和反转录PCR (RT-PCR)方法,扩增获得其外壳蛋白(CP)基因片段,并克隆到pGEM-T载体中。核苷酸序列测定表明,该分离物CP基因全长为852个核苷酸,编码由284个氨基酸组成的31.8 kDa蛋白。与国外已报道的WMV-2 CP基因相比,其核苷酸序列同源性为92.2%~94.0%,由此推导的氨基酸序列同源性为94.5%~98.1%。与国内2个分离物相比,和山西分离物核苷酸和氨基酸的同源性都达到98.5%,和郑州分离物核苷酸和氨基酸的同源性分别为91.5%和95.0%。     

小麦黄色花叶病毒不同分离物外壳蛋白(CP)基因序列的比较分析

 核苷酸序列分析结果表明,小麦黄色花叶病毒(W YMV)不同分离物的外壳蛋白基因存在一定的差异。邓州分离物CP基因在其31~33nt处均缺失了3个核苷酸,其余分离物与潢川分离物及日本分离物长度一致,均为882nt。不同分离物CP基因核苷酸序列同源性为97.3%~98.9%,由此推导的氨基酸序列同源性为97.6%~99.3%,外壳蛋白N末端的110个氨基酸和C末端的55个氨基酸在各个分离物间是高度保守的。潢川分离物有5个氨基酸与其它5个分离物明显不同。WYMV不同分离物外壳蛋白序列分析结果进一步确认了WYMV与WSSMV为Bymovirus属的2种不同病毒。     

番木瓜环斑病毒南瓜分离物外壳蛋白基因的克隆及序列分析

利用电镜和酶联免疫法在云南省采集到的5份南瓜病样中检测到番木瓜环斑病毒(Papayaring spot virus,PRSV)。为了进一步从分子水平确定云南省南瓜病毒病原种类,并为下一步转基因育种提供抗性基因,采用反转录PCR(RT-PCR)方法扩增了5个分离物的外壳蛋白(coat protein,CP)基因片段,并克隆到pGEM-T载体中。核苷酸序列测定表明,番木瓜环斑病毒石屏分离物(PRSV-SP)和番木瓜环斑病毒蒙自分离物(PRSV-MZ)的CP基因长873nt,编码290个氨基酸,番木瓜环斑病毒峨山分离物(PRSV-ES)、番木瓜环斑病毒版纳分离物(PRSV-BN)和番木瓜环斑病毒宾川分离物(PRSV-BC),3个分离物CP基因长867nt,编码288个氨基酸。PRSV5个分离物核苷酸序列的同源性在94%以上,氨基酸序列的同源性在96%以上。与国内外17个分离物相比,核苷酸序列同源性为89.6%~98.7%,氨基酸序列同源性为86.5%~99.6%。其中PRSV-SP和来自于越南分离物PRSV-V47无论是核苷酸序列,还是氨基酸序列同源性都达到了最高,而5个分离物与来自于巴西(PRSV-BR)、美国(PRSV-USA)、墨西哥(PRSV-Y)核苷酸序列同源性均低于90%。     

侵染百合的黄瓜花叶病毒亚组Ⅱ分离物cp基因克隆和序列分析

 从云南大理的东方型百合上得到黄瓜花叶病毒分离物(CMV-DL), ELISA检测初步确定为CMV亚组Ⅱ分离物, 设计并合成CMV亚组Ⅱ的特异引物, RT-PCR扩增得到1条约800 nt的特异片段, 经克隆及序列测定, 该片段长828 nt, 包含的外壳蛋白(CP)基因由657 nt组成。将该分离物的cp基因与其它14个CMV分离物进行同源性比较, 在核苷酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为76.8%~78.1%和98.6%~99.2%;在氨基酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为82.0%~84.3%和95.9%~100.0%。结果表明CMV-DL为CMV亚组Ⅱ成员。     

北京地区地黄花叶病病原的分子鉴定

为明确北京地区发生的地黄花叶病的病原,在进行生物学接种分离纯化的基础上利用RT-PCR方法对其进行了分子鉴定。通过接种指示植物和进行单斑分离,获得了病毒的纯分离物。经RT-PCR和序列测定及分析,有明显花叶症状的北京地黄样品受黄瓜花叶病毒Cucumber mosaic virus(CMV)和蚕豆萎蔫病毒2Broad bean wilt virus2(BBWV2)复合侵染。为进一步明确BBWV2地黄分离物(BBWV2-Rg)的分类地位,克隆了BBWV2-Rg RNA2多聚蛋白基因,并进行了序列测定和分析,结果表明该多聚蛋白基因由3 195个核苷酸组成,编码1 064个氨基酸。经序列比对分析,BBWV2-Rg编码的外壳蛋白大亚基LCP基因和小亚基SCP基因核苷酸序列与已发表的BBWV2其它株系相应基因核苷酸序列的同源性分别为78.69%~89.30%和76.99%~90.52%,氨基酸序列同源性分别为91.29%~97.51%和87.82%~96.45%。     

西瓜花叶病毒2号新疆昌吉分离物外壳蛋白基因核苷酸序列分析

 利用RT-PCR从新疆昌吉地区表现花叶、疱斑、扭曲等症状的南瓜病株上检测到西瓜花叶病毒2号新疆昌吉分离物(简称WMV-2-XJ-CJ),并测定了该分离物外壳蛋白(CP)基因序列。序列分析表明,新疆昌吉分离物CP基因全长850个核苷酸,编码197个氨基酸。与国内外报道的12个WMV-2CP基因相比,其核苷酸序列同源性为92.6%~98.3%,由此推导的氨基酸序列同源性为94.7%~99.3%。新疆昌吉分离物在CP N'端可变区明显不同于国内外报道的核苷酸序列。WMV-2新疆昌吉分离物与日本和郑州分离物较其它国家和地区的分离物多出6个核苷酸,但其核苷酸及其推导的氨基酸序列差异较大。新疆昌吉分离物外壳蛋白有2个氨基酸残基明显不同于其它分离物,其中蚜传株系的特征结构域DAG突变为DAE。     

大麦黄矮病毒PAV中国分离物外壳蛋白基因的克隆及同源性分析

 大麦黄矮病毒PAV株系由麦长管蚜和禾谷缢管蚜传毒。本研究通过RT-PCR、克隆和序列测定后,确认所得到的我国小麦PAV分离物的外壳蛋白基因片段由600个核苷酸组成,编码199个氨基酸。序列同源性比较结果显示,与BYDV的其它株系典型分离物的外壳蛋白基因同源性最高为74.5%,而与国外发表的PAV 8个分离物的CP基因核苷酸同源性为81%左右,且同源性比较的分值也较其它株系高。氨基酸序列的比较中,仅在46到60位氨基酸差别较大。     

来源于桃和苹果的苹果褪绿叶斑病毒的部分分子生物学特性和cp基因的原核表达

 从桃和苹果上分离得到苹果褪绿叶斑病毒ACLSV-HBP和ACLSV-C2个分离物,采用RT-PCR法进行扩增,所获扩增片段经序列测定,其全长分别为1768nt(ACLSV-HBP)和1751nt(ACLSV-C)。这2个分离物扩增片段全长的同源性为83%,mp基因片段核苷酸和推导编码氨基酸序列同源性分别为82.6%和87.1%;cp基因均由582nt组成,其核苷酸和推导编码氨基酸序列同源性分别为87.8%和95.9%。将2个分离物的cp基因与已报道ACLSV分离物进行序列同源性比较,结果显示ACLSV-HBP与SX/2的cp基因核苷酸序列及推导编码氨基酸序列同源性最高,分别为94.0%和96.4%。将ACLSV-HBP分离物的cp基因克隆到原核表达载体pGEX-KG,在大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析表明,融合蛋白大小约为46kDa。Western-blot分析表明,该基因在大肠杆菌内得到高效表达,融合蛋白具有抗原性。     

李属坏死环斑病毒新疆分离物运动蛋白基因片段的克隆与序列分析

为进一步揭示李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)新疆巴旦木分离物的分子特征、遗传变异及其与宿主之间的相互关系,采用RT-PCR方法扩增并克隆了4个PNRSV新疆巴旦木分离物运动蛋白(move protein,MP)基因片段,并进行了测序及序列同源性分析。结果表明,4个新疆巴旦木PNRSV分离物MP基因片段分别为259、258、254、260 bp;其核苷酸和氨基酸序列与已报道的PNRSV分离物的同源性分别为72.7%~91.7%和75.6%~92.9%,表现出明显差异,其中与美国分离物CH9同源性最高,分别达88.8%~91.7%和82.6%~92.9%;而与同属03亚组的苹果花叶病毒(Apple mosaic virus,Ap MV)同源性较低,仅为51.2%~58.1%和52.3%~61.9%;新疆PNRSV各分离株之间MP基因核苷酸序列同源性较高。系统发育树显示,4个新疆巴旦木PNRSV分离物与Ⅰ组代表毒株PV32的核苷酸序列同源性达88.4%~91.3%,并与Ⅰ组分离物聚集成簇,表明PNRSV新疆巴旦木分离物属于引起严重症状的Ⅰ组株系,且Ⅰ组中各分离物之间表现出一定的寄主相关性,而Ⅱ组和Ⅲ组中各分离物之间未表现出明显的寄主相关性。     

灰飞虱来源的水稻条纹病毒外壳蛋白基因遗传多样性

 采用单雌产卵法获得来自江苏、云南、山东、河北等地灰飞虱（Laodelphax striatellus Fallén,SBPH）来源的21个水稻条纹病毒（Rice stripe virus,RSV）分离物,提取灰飞虱总RNA,经RT-PCR扩增,获得21个RSV分离物的包含外壳蛋白（cp）基因在内的约1 000 bp左右的DNA片段。测序结果显示,参试分离物的cp由969个核苷酸组成,编码322个氨基酸。采用DNASTAR软件进行分析,21个灰飞虱来源的RSV-cp核苷酸序列和推导出的编码蛋白的氨基酸序列同源性分别为95.7%~100%和96.0%~100%。与已报道水稻来源的32个RSV分离物一起进行序列同源性比较和系统进化树分析结果表明,总体而言RSV-cp较为保守,其遗传多样性首先与地缘相关,从地理位置上可以分成中国云南、中国沿海和日本3个地理种群;其次与寄主相关,在同一地理种群中可以划分为灰飞虱和水稻2个寄主种群。     

侵染白附子的芋花叶病毒分子变异研究

为明确侵染白附子的芋花叶病毒(dasheen mosaic virus, DsMV)的分子变异情况,对51个DsMV白附子分离物(DsMV-BF)的外壳蛋白(Coat Protein,CP)基因和3个分离物的近全长基因组序列进行了克隆和测定,DsMV-BF的CP基因大小有855个和942个核苷酸两种类型,51个白附子分离物之间CP基因的核苷酸和氨基酸一致率分别为88.3%～100%和91.9%～100%,BF8、BF30和BF38分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为82.9%～95.9%和90.7%～95.9%,与GenBank中其他分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为76.9%～99.4%和85.6%～99.0%;P1基因的分子变异较大,P1基因大小有987个和990个核苷酸两种类型;CP基因核苷酸序列系统进化树分析结果表明,侵染白附子的DsMV分离物可分为两个亚组;重组分析结果表明BF8和BF30分离物各检测到1个重组事件,BF38检测到2个重组事件。     

Biological, serological, and molecular variabilities of clover yellow vein virus

ABSTRACT A comparative study was made on the host reactions, serological properties, and nucleotide sequences of the coat protein (CP) gene of 10 clover yellow vein virus (C1YVV) isolates and one bean yellow mosaic virus (BYMV) isolate collected from different host plant species and locations in Japan. Two strains of C1YVV isolates, grouped on the basis of host reactions on Chenopodium amaranticolor, C. quinoa, Nicotianaclevelandii, N. benthamiana, Vicia faba, and Trifolium repens, corresponded to two serotypes determined by double-antibody sandwich- and triple-antibody sandwich-enzyme-linked immunosorbent assay using three polyclonal and nine monoclonal antibodies. These results were also confirmed by nucleotide sequence analysis of the CP gene. The CP gene of C1YVV isolates of strain 1, including the Australian isolate C1YVV-B, had 93 to 98% nucleotide identities and 97 to 99.6% amino acid identities. The CP of C1YVV isolates of strain 2, including the New Zealand isolate C1YVV-NZ, had 92 to 98% nucleotide identities and 95 to 98% amino acid identities. The nucleotide identities and the amino acid identities between the two C1YVV strains were 82 to 84%, and 90 to 94%, respectively. When compared with the CP sequences of 12 C1YVV isolates, the CP sequence of the BYMV isolate had 71 to 73% nucleotide identity and 73 to 77% amino acid identity. Amino acid sequence differences among C1YVV isolates from strains 1 and 2 were located mostly at the N-terminal regions of the CP. Our results indicated that the C1YVV isolates studied could be separated into two strains on the basis of host reactions, serology, and the nucleotide sequence of the CP gene.     

云南美人蕉病毒病病原鉴定

In this study,the causal agents were identified from Canna indica viral diseased plants in Yunnan Province.The diseased C.indica plants mainly exhibited the symptoms like veinal chlorosis and yellowing,streak mosaic or interveinal chlorosis,while older leaves always showed veinal necrosis as well as chlorosis.Viral pathogens were detected by RT-PCR/PCR in 24 diseased C.indica samples collected from Kunming and Yuxi City in Yunnan Province.The results indicated that the main C.indica-infecting viruses were canna yellow mottle virus (CaYMV),bean yellow mosaic virus (BYMV),sugarcane mosaic virus (SCMV).CaYMV showed the highest detection rate of 87.5 %,whereas,the BYMV had the lowest rate of 16.7% in the 24 samples.Co-infections of CaYMV+SCMV,CaYMV+BYMV and CaYMV+SCMV+BYMV were also detected in the diseased samples.However,cucumber mosaic virus (CMV),tobamovirus,luteovirus,orthotospovirus,begomovirus and umbravirus were not detected in these samples.This is the first report of CaYMV and SCMV infecting C.indica in Yunnan province.     

云南美人蕉病毒病病原鉴定

In this study,the causal agents were identified from Canna indica viral diseased plants in Yunnan Province.The diseased C.indica plants mainly exhibited the symptoms like veinal chlorosis and yellowing,streak mosaic or interveinal chlorosis,while older leaves always showed veinal necrosis as well as chlorosis.Viral pathogens were detected by RT-PCR/PCR in 24 diseased C.indica samples collected from Kunming and Yuxi City in Yunnan Province.The results indicated that the main C.indica-infecting viruses were canna yellow mottle virus (CaYMV),bean yellow mosaic virus (BYMV),sugarcane mosaic virus (SCMV).CaYMV showed the highest detection rate of 87.5 %,whereas,the BYMV had the lowest rate of 16.7% in the 24 samples.Co-infections of CaYMV+SCMV,CaYMV+BYMV and CaYMV+SCMV+BYMV were also detected in the diseased samples.However,cucumber mosaic virus (CMV),tobamovirus,luteovirus,orthotospovirus,begomovirus and umbravirus were not detected in these samples.This is the first report of CaYMV and SCMV infecting C.indica in Yunnan province.     

百合斑驳病毒云南分离物 全基因组序列分析及CP结构预测

 对云南嵩明百合上发生的百合斑驳病毒(Lily mottle virus, LMoV)进行全基因组序列测定及分析,并对LMoV嵩明分离物(LMoV-SMi1、LMoV-SMi2)和玉溪分离物(LMoV-YXi1、LMoV-YXi2)外壳蛋白(coat protein,CP)基因进行序列比较,发现云南的LMoV分为2个类群,玉溪分离物属于种群I,嵩明分离物属于种群II。2个类群间的核苷酸和氨基酸同源性分别为86.7%~89.5%、90.1%~92.7%,玉溪分离物和嵩明分离物相比,cp基因发生了3个核苷酸的缺失。对国内外LMoV所有分离物的cp基因氨基酸序列进行系统进化分析,结果表明所有LMoV分离物可划分为2个种群,种群I分离物较种群II分离物几乎均存在1个苏氨酸缺失的差异。此外,对LMoV-SMi2的CP相关特性和空间结构进行了初步预测,认为该蛋白为球状,具有较强的表面可能性,不存在跨膜区域,大多数区域能够形成主要的抗原决定簇,主要集中在aa12-22、aa31-42、aa83-99、aa179-191、aa215-223、aa249-259区段,可作为制备抗血清选择抗原的参考。LMoV-SMi2和LMoV-YXi1在二级结构和三级结构上存在一定的差异,但总体空间结构差异不大。     

Comparison of Pathogenicity and Nucleotide Sequences of 3′-terminal Regions of Bean yellow mosaic virus Isolates from Gladiolus

Sixty-four isolates of Bean yellow mosaic virus (BYMV) from cultivated and naturalized gladioli were divided into two pathogenic groups, necrotic spot (NS) and chlorotic spot (CS) groups on Chenopodium quinoa. NS-type isolates (S-22N and E-24N), CS-type isolates (S-22C and E-92C), and broad bean isolates (Sb-50C and Sb-12C) differed in their pathogenicity on Antirrhinum majus, Nicotiana benthamiana, Phaseolus vulgaris, Spinacia oleracea and Vigna unguiculata. The four gladiolus isolates were different from BYMV-B, -P, -O and C1YVV-N in their pathogenicity on these plants, while the two broad bean isolates were similar to BYMV-B, originally from broad bean. The nucleotide (nt) sequences of the 3′-terminal region of the BYMV RNA genome of the two NS-type isolates, the two CS-type isolates, the two broad bean isolates and BYMV-B, -P and -O were determined. In a phylogenetic tree based on the CP amino acid (aa) sequences, the two NS-type isolates clustered together (identity 98.4% and 98.2% at the nt and aa level, respectively). The two CS-type isolates clustered with BYMV-O (93.2 to 99.3% nt identity and 95.6 to 98.5% aa identity). The two broad bean isolates clustered with BYMV-B (99.0 to 99.5% nt identity and 98.9 to 99.6% aa identity). BYMV-P clustered with BYMV-CS (identity 97.7% and 99.3% at the nt and aa level, respectively). The obtained sequences were compared with those of the 3′-terminal regions of seven published BYMV isolates. In a phylogenetic tree based on deduced aa sequences, BYMV isolates were divided into four clusters. Received 1 July 1999/ Accepted in revised form 22 May 2000     

蚕豆和豌豆锈病病原菌的分子鉴定

为明确我国热带和亚热带地区蚕豆Vicia faba和豌豆Pisum sativum锈病的病原菌种类,通过致病性测定和ITS序列系统发育分析对来自我国云南省玉溪市的4份豌豆锈菌分离物及云南、广西、重庆和四川省(区、市)的5份蚕豆锈菌分离物进行系统鉴定。结果显示,分离自豌豆的锈菌WX1分离物对蚕豆和豌豆均具有高致病性,在侵染叶片上产生大量锈子器;分离自蚕豆的锈菌CX3分离物仅对蚕豆具有高致病性,能在叶片上产生大量夏孢子,而对豌豆的致病性相对较低,仅产生少量的夏孢子堆;分离物WX1和CX3对小扁豆和鹰嘴豆不具有致病性。基于ITS序列系统发育分析表明,所有不同寄主来源的蚕豆单胞锈菌分离物均聚类于一个系统发育组,但分离自蚕豆和豌豆的分离物分别聚类在不同的亚组。表明分离自云南省玉溪市豌豆上的蚕豆单胞锈菌Uromyces viciae-fabae应为豌豆专化型,定名为U. viciae-fabae ex P. sativaum,而来源于云南、广西、重庆和四川省(区、市)的蚕豆锈病病原菌为蚕豆专化型U. viciae-fabae ex V. faba。     

太子参蚕豆萎蔫病毒2号CP基因的遗传多样性及分子进化分析

为了解太子参蚕豆萎蔫病毒2(Broad bean wilt virus 2,BBWV2)的遗传多样性及分子进化关系,本研究对5个不同地理来源的6份太子参病叶样品进行了BBWV2外壳蛋白(coat protein,CP)基因的RT-PCR扩增和克隆。序列分析结果表明,所得13条太子参BBWV2 CP基因序列均为1 345 bp,其核苷酸序列同一性在81.0%~99.0%之间,氨基酸序列同一性在93.5%~99.3%之间;核苷酸多样性为0.084 00;存在280个核苷酸多态性位点,其中232个为地域特异性位点;总变异数为278个,其中同义突变29个,异义突变249个。不同地理来源的太子参BBWV2分离物之间遗传分化程度较高,基因交流不频繁,遗传漂变可能导致分离物间明显的遗传分化。在系统进化树中,太子参分离物的聚类呈现出明显的地域特异性,江苏、贵州、湖南、福建分离物均聚类在I-a亚组,而山东分离物聚类在II-c亚组,遗传距离较远。研究表明,太子参BBW V2存在很高的遗传变异,基因突变是其产生遗传多样性的主要驱动力,而地理因素与其具有较高的核苷酸多样性密切相关。