【作者】 李娜；

【导师】 陈集双； 廖乾生；

【作者基本信息】 浙江理工大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 黄瓜花叶病毒卫星RNA(Cucumber mosaic virus satellite RNA CMV-satRNA)是一类大小在307-405nt之间,线型、单链,非编码的RNA分子。目前已经报道了100多条CMV-satRNA的序列,分别寄生在65种以上的亚组I和II的CMV上。CMV-satRNA完全依赖于辅助病毒(helper virus)进行复制,包被并传播。CMV-satRNA大多集中在332-342nt,有一些特大satRNA,大约为386-405nt[1]。在CMV-sat RNA的5’端近80个碱基和3’端近45个碱基相当保守。与小片段的CMV-satRNA相比,大片段的卫星序列增加了一些中间插入片段[39]。CMV-satRNA对辅助病毒在寄主植物上的症状为减轻、加重或无影响,对辅助病毒症状的影响与卫星RNA、辅助病毒和寄主植物三者都相关。本论文旨在研究sat405的活性,及其以CMV-Fny为辅助病毒在寄主植物上的症状。sat405序列由NCBI数据库下载得到,并人工合成在PUC51质粒上。sat405的cDNA经体外转录与CMV-Fny的基因组RNA按一定比例混合接种于心叶烟,观察并检测其存活特性及其对CMV-Fny致病症状的调节。本文进一步分析了sat405的序列及其二级结构。对CMV-Fny携带卫星前后在本生烟上引起的光合作用的差异进行了研究。分别观察了CMV-Fny携带卫星前后在心叶烟(Nicotiana glutinosa),本生烟(Nicotiana benthamiana),番茄合作903(Solanum lycopersicum hezuo 903),普通烟(Nicotiana)等植物上的症状,研究卫星、病毒、与植株之间的互作情况。主要研究结果:sat405以CMV-Fny为辅助病毒在寄主植物上经过反复转接传代后仍保持序列的稳定性。sat405的cDNA的体外转录产物与被CMV-Fny+sat405侵染的寄主植物叶子的汁液一样都具有侵染活性。经过双链RNA的分析检测发现sat405以CMV-Fny为辅助病毒在寄主植物上高含量积累。荧光定量PCR显示了sat405与CMV-Fny在番茄合作903上假重组后,sat405对CMV-Fny病毒基因组表达量起到上调作用。本生烟被病毒侵染前后的光合作用检测结果为:被四种毒源侵染后的本生烟相对于健康植物光合作用减小。CMV-Fny侵染后减小本生烟的光合作用,携带satT1和sat405后的CMV-Fny侵染本生烟后进一步减小光合作用,减小的程度大于CMV-Fny ,而CMV-Fny+satYN12相对于CMV-Fny增大本生烟的光合作用。序列分析结果显示sat405相对于satT1和satYN12在186位至213位插入一段序列。利用DNA Star软件,将sa405与satT1和satYN12的序列比对,结果显示:satT1相对于sat405和satYN12在81位至107位缺失一段序列,在114,115位和155—157位缺失碱基。satYN12相对于sat405的序列在186位至213位缺失一段序列,satT1相对于sat405的序列在181位至213位缺失一段序列。satYN12相对于sat405的序列在177位至182位缺失6个碱基。三个卫星在3’端和5’端基本保守。研究表明其致病区域的定位不能单纯以sat405与其他CMV-satRNA的序列比对而得出。而应该利用sat405序列的突变(替换、插入或缺失)具体定位致病碱基,并研究具体的突变对其二级结构的影响。更多还原

【Abstract】 Cucumber Mosaic Virus Satellite RNA (CMV-satRNA) are small, linear, single-stranded and non-coding RNA that depend on the helper viruses for their replication, encapsidation, and transmission. The sequences of more than 100 CMV–satRNA variants, found associated with over 65 CMV isolates in both subgroups I and II of CMV, have been reported. Most variants found all over the world contain 332–342 nucleotides. Some are considerably larger, with 386–405 nt. The 80nt at 5’end and 45nt at 3’end of CMV satelliteRNA were highly concervative.This paper aims to study the property of sat405 and identify the sypmptoms induced by sat405 with its helper virus CMV-Fny. It was doubted that CMV can contain satellite RNA larger than 400nt. The sequence of sat405 was obtained from NCBI and synthesized into PUC51 vector. The in vitro transcriptional product of sat405 was inoculated onto Nicotiana glutinosa after being mixed with CMV-Fny genomes. CMV-Fny+sat405 was then inoculated onto Nicotiana benthamiana, Nicotiana glutinosa, Solanum lycopersicum hezuo 903 and Nicotiana tabacum. The interaction between satellite RNA,CMV-Fny and host plant was analyzed.The study results showed that the sequence of sat405 remained consisitent after several passages. Both in vitro transcriptional products and leaf sap of CMV-Fny+ sat405 are all infectious. It showed that sat405 accumulation was very high in host plant with CMV-Fny.The existence of sat405 and its interaction with CMV was confirmed. The real time PCR results showed that CMV genome accumulation was upregulated by sat405. The photosynthesis analyzation showed that CMV-Fny decreased the photosynthetic rate of N. benthamiana. CMV-Fny+sat405 and CMV-Fny+satT1 decreased the photosynthetic rate of N. benthamiana compared to CMV-Fny. CMV-Fny+satYN12 increased the photosynthetic rate of N. benthamiana compared to CMV-Fny. It was discovered that sat405 aggravated symptoms induced by CMV-Fny and sat405 increased the accumulation of CMV genomes. The alignment between sat405, satT1 and satYN12 was done by DNASTAR. Comparisons of the sequences reveal that the 5’region and 3’region were homologous. SatT1 lost the middle segment (position 81-107, 114,155—157) compared to sat405 and satYN12. Compared to sat405, satYN12 lost the middle segment position 177-182). Sat405 contained insertion sequences (position 186-213) compared to satT1 and satYN12. Our study showed that the necrogenic region of satRNA can not be determined by alignment of the known satellite RNA only. It should be determined by creating insertion or deletion mutants of satellite RNA and analyze its secondary structure. It showed that sat405 aggravated the symptom of CMV-Fny on host plants.更多还原