【作者】 陈洁云；

【导师】 陈集双；

【作者基本信息】 浙江大学 ， 微生物学， 2003， 硕士

【副题名】Ⅰ：主要病毒鉴定与基因组分析　Ⅱ：病毒生态学和致病性研究

【摘要】 葫芦科作物在浙江省和周边地区的栽培面积不断扩大，其栽培模式也呈现多样化趋势。凡是种植瓜类作物的地区都有病毒病的发生，但是，我国在该科作物的病毒病研究相对滞后。本研究对我国部分地区的葫芦科作物进行了病毒分离和鉴定，确定小西葫芦黄化花叶病毒(Zucchini yellow mosaic virus，ZYMV)和黄瓜花叶病毒(Cucumber mosaic virus，CMV)是侵染葫芦科植物的2种主要病毒病原，系统检测了浙江地区该科作物上的ZYMV和CMV的季节和寄主分布情况，并在分子水平上进行了病毒基因组变异规律和变异趋势的摸索和病毒致病性研究，研究结果如下。 在浙江地区和全国部分地区采集自然发病的葫芦科作物田间样品，通过病毒分离和寄主反应测定、病毒dsRNA分析、形态学观察等方法，获得了13个CMV分离物、16个ZYMV分离物，1个TMV分离物，4个未知病毒分离物。自然发病的田间植株通常表现重花叶、畸形，植株矮化，果实表面瘤状增生等病毒病症状。田间作物受2种病毒或多种病毒复合侵染时，表现的症状更为严重。中国大陆获得的ZYMV分离物，在藜科、豆科等植物上产生局部坏死斑，系统侵染葫芦科植物，不侵染茄科植物。病毒粒子呈弯曲线形，粒体长为750nm，CP蛋白分子量约为36.0kDa；ZYMV侵染的西葫芦叶肉细胞内观察到典型的柱状内含体。获得的CMV分离物在藜科、豆科等植物上产生局部症状，系统侵染茄科、葫芦科等植物，病毒粒体大小约为29nm，CP分子量约为24.2kDa。CMV接种侵染的心叶烟薄壁细胞中，大量球形病毒散布于细胞质中，病毒粒子结晶体呈菱形、多边形或不规则形等。 通过RT-PCR、T载体连接、转化和重组质粒测序，研究了来自中国大陆4种作物上9个ZYMV分离物的CP基因和3’-UTR区的核苷酸序列，并与其它地区报道的16个分离物进行了同源性比较。9个来自我国大陆地区ZYMV分离物的CP基因均为837个核苷酸(nt)，分别编码279个氨基酸，其3’-UTR区的序列(不包括PolyA尾)为210～211nt。ZYMV CP基因核苷酸序列同源性具有一定的寄主和地域相关性，但是，总体上其关联程度不甚明确。其氨基酸序列同源性的寄主相关性程度高于地域相关性程度。25个ZYMV分离物的CP氨基酸序列根据其变异程度分为2个区：N端约41个氨基酸为高度变异区、“CP核心区”和C端氨基酸序列为保守区。根据CP氨基酸保守区可以将ZYMV分为5个基因型，基因型内氨基酸同源性高于95％。保守区CP氨基酸同源性的寄主相关性程度高于地域相关性程度。3’-UTR区序列相对保守，不形成明显的基因型分组。但是，个别分离物UTR区明显高于其它分离物(达245nt)。以上研究结果初步揭示了ZYMV作为单链RNA病毒通过快速变异适应与寄主相互作用的趋势：ZYMV作为单链RNA病毒通过与寄主相互作用而表现寄主适应性变异的趋势。摘要 对来自杭州南瓜、番茄上的CMV分离物进行了CP基因核昔酸和CP氨基酸序列 分析，并与国内外已发表的不同地区、寄主来源的CMV分离物进行了比较。系统进 化树分析表明:CMV分离物明显分为2个亚组，本研究获得的2个分离物都属于亚组 I。4个葫芦科来源的CMV分离物均属于亚组I。不同来源的CMV分离物没有表现 出寄主和地区适应性。 通过在浙江地区分别于2002年夏季6一8月、秋季10一11月定点采集葫芦科作物田 间样品，以32P标记的ZYMv和CMV基因组cDNA作为探针进行杂交检测。检测结 果表明:ZYMV和CMV在葫芦科作物上的发生表现出显著的季节性差异。夏季采集 的22个样品中有21个感染CMV，发生率为95.5%;只有5个样品感染ZYMV，发生 率为22.7%，在秋季ZYMV明显成为优势病毒，其自然发生率为59.1%，所有秋季样 品中均没有检测到CMV。ZYMV的发生在不同作物、地区间具有一定的差异性:在夏季检测的7种作物中，南瓜上ZYMV的发生率为66.7%，2个甜瓜样品中有l个感染ZYMV，其余作物均没有检测到该病毒。秋季葫芦科栽培品种较少，主要为南瓜与丝瓜，其中南瓜上ZYMV的发生率为80.0%，丝瓜上为40.0%，2个葫芦样品中1个检测到ZYMV。不同作物和地区之间CMV的发生没有表现明显的差异性。 通过在3种葫芦科作物品种上单独和混合接种CMV和ZYMV，对各处理的症状和病毒负荷量进行了定量分析。ZYMV和CMV对3种寄主的致病性测定结果表明:丝瓜供试品种对CMV的侵染相对敏感，而西葫芦供试品种对ZYMV的侵染更敏感，2种病毒在黄瓜供试品种上均未表现出明显的致病性;ZYMv与CMV的复合侵染使丝瓜和西葫芦的发病症状明显加重，表现强协生作用。接种病毒后29d各处理的杂交检测结果显示:在病症表现最严重的西葫芦植株体内基本检测不到CMV或ZYMV;丝瓜上CMV接种处理和混合接种处理的植株体内检测到高含量存在的CMV，ZYMV接种处理中检测到高浓度的病毒，但混合接种丝瓜中未检测到ZYMV。尽管外观症状不明显，黄瓜3个接种处理中均可检测到高负荷量的CMV或ZYMV。显示了该黄瓜品种对病毒具备耐受抗性，与丝瓜和葫芦比较，黄瓜具有推迟发病的抗病性。接种29d各处理CMv含量的ELISA检测结果与杂交检测结果基本一致。 选择16个葫芦科作物常见品种进行田间接种试验，分别以 ZYMV、CMv单独接种更多还原

【Abstract】 The production of cucurbitaceous crops is increasing recently in Zhejiang Province and other parts of south-east China, where the agricultural economy is relatively well developed, and the crop system is become more flexible. Viral diseases occur in all the area where cucurbits are planted. But the research work on viruses infecting this family of plant remain behind in compare with that for other crops. In this report, viral pathogens naturally occurred on cucurbitaceous crops were isolated and identified systemically. We found that Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV) were both principal viruses infecting cucurbits in Zhejiang Province and many other parts of China’s mainland. Based on this, we studied the genomic diversity of the above two viruses and their seasonal occurrence. The pathogenicity of ZYMV and CMV was also investigated and crop resistance, for different varieties was also compared for against these viruses.Based on host reaction,"double-stranded RNA analysis, viral particle purification and morphological observation, thirteen virus isolates were identified as CMV, sixteen as ZYMV. one as Tobacco mosaic virus (TMV) and other four as unidentified. Infected cucurbit crops exhibited severe mosaic, distortion, stunt, and lump formation on fruits. The symptom usually become more severe when infected with more than one virus.ZYMV isolates obtained from China’s mainland, were found to produce local infection to Chenopodiaceae and Fabaceae species tested, and produced systemic infection to plants of Cucurbitaceae but no infection to Solanaceae. Their virus particles are of filamentous shape of about 750 nm in length, and the coat proteins (CP) are about 36.0 kDa when tested with SDS-PAGE. Cucurbits infecting CMV were found to produce local infection to Chenopodiaceae and Fabaceae species tested, and produced systemic infection to plants of both Cucurbitaceae and Solanaceae. Their virus particles are of spherical morphorlogy of about 29 nm in diameter, and the coat proteins (CP) are about 24.0 kDa.For understanding the genomic variation, geological distribution and host adoption of ZYMV the 3’ end sequence of its genome including the coat protein (CP) gene and 3’ endun-translated region (UTR) were compared, after RT-PCR and cloning of the part genome. Nine Mainland isolates originally isolated from naturally infected Cucurbita moschata , Cucumis melo, Benincasa hispida and B. hispida var. Chieh-qua, were collected and identified from Zhejiang Province, Hainan Province, Shangdong Province, Shanxi Province and Beijing, respectively. The coat protein genes of all the Mainland-China isolates consisted of 837 nucleotides (nt) coding for 279 amino aids. Their 3’ end UTR region composed of 210 to 211 nt not including the poly (A) tail. The above sequence data obtained in our study was then compared with previously reported sequences of 16 other ZYMV isolates obtained from other part of the word. In certain degree, similarity of nucleic acid sequence for CP gene was found being related with the host and geological origination, but not very obvious. Similarity of the CP amino acid sequences deduced from nucleic acid of the 25 ZYMV isolates reached a higher sequence similarity and a clearer relationship to the host and geological origination. The relationship extent of sequence similarity to host origin is higher than its extent to geological origin. According to sequence similarity, the amino acid sequence of ZYMV CP was divided into two parts-"the high variable region", containing about 41 amino acids at its N end, and "the conservative region", containing coat protein core-region and C terminal amino acids. The 25 ZYMV isolates could be classifiedinto five genotypes according to "the conservative region", with a 95% sequence similaritywithin each genotype. The 3’ end UTR of ZYMV was relatively conservative but some isolates were relatively longer. It was hard to differ ZYMV isolates into genotypes according to the UTR. The above results showed the trend of ZYMV variation更多还原