INTERACTION OF TURNIP CRINKLE VIRUS (TCV) AND ITS HOST PLANTS

芜菁皱缩病毒 (TCV) 与其寄主植物的相互作用

• 批准号: 7381531
• 负责人: PAUL TWIGG
• 金额: $ 5.52万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381531
• 关键词: INTERACTION; TURNIP; CRINKLE; VIRUS; TCV

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. My main research project is focused on the interaction of Turnip Crinkle Virus (TCV) and its host plants. We are analyzing the genetic interactions of TCV and the model plant Arabidopsis. Currently, we are using two strains of TCV, a wild type strain and a strain developed by the Morris lab at UNL with one amino acid altered in the viral coat protein called R6a. Wild type TCV coat protein shows a binding with a transcriptional activator in Arabidopsis belonging to the NAC family of transcription factors. This interaction causes RNA silencing in the infected plants. The mutated R6a TCV does not show this interaction and causes a more severe and symptomatically different disease. We are analyzing this interaction by inoculating wild type Arabidopsis with normal and mutant TCV individually, as well as a mock inoculation with no virus. We then harvested leaves after 24 and 48 hrs form all three plant populations. We have extracted total RNA from all six tissues in triplicate. These RNAs will soon be used in microarray analysis of Arabidopsis whole genome cDNA arrays. Transcripts identified as being the most consistently up or down regulated from these arrays will be further studied in the near future using real-time PCR to verify the microarray results. I also have several other projects underway mostly in collaboration with Dr. Gautam Sarath, but also with Dr. Christian Tobias and Dr. John Vogel all of the USDA. With Drs. Sarath and Tobias, I have been working on a genomics inititative characterizing the genetics of the biofuel plant Switchgrass (Panicum virgatum). My students and I have to date constructed cDNA libraries from Switchgrass callus tissue, stems, leaves, crowns, and seedlings. Together with Dr. Tobias, we have sequenced and analyzed the 5' end of 15,272 cDNA clones from the leaf, callus, stem ,and crown libraries. All of these sequences are soon to be deposited in GenBank bearing my and my students names as well as the appropriate INBRe acknowledgement. From these sequences, we have gleaned many genes important in a variety of metabolic processes for further characterization. Chief amongst these are the genes involved in lignin biosynthesis which will soon be the basis of our first publications on this material. I am currently beginning a similar genomics initiative with Dr. John Vogel studying another biofuel grass called Bradypodium distachyon. This project is in its infancy, but we are currently constructing four cDNA libraries from various tissues from this plant. I am also working with Dr. Sarath on a genomics study of a prairie legume called Partridge Pea (Chamaecrista fasciculata). This plant is an important prairie species that participates in a symbiotic nitrogen-fixing symbiosis with Rhizobium forming root nodules. We have constructed a cDNA library from the nodule tissue of this plant, and have begun to characterize the 5' end of various sequences. We have isolated a clone for a symbiotic protein called leghemoglobin which we have selected for further characterization. We are currently concentrating on subcloning the open-reading frame into an appropriate expression vector for the production of recombinant protein. We intend to analyze the oxygen-binding behavior and structure of the leghemoglobin protein. This work will shortly produce its first manuscripts. My last project involves a plant called corn lily (Veratrum californicum). This plant is of interest due to its production of the teratogenic and carcinogenic steroidal alkaloid called cyclopamine. This compound is largely concentrated in the roots of the corn lily, but consumption of its leaves by pregnant ewes can produce somewhat gruesome offspring with only one eye or both eyes merged into one socket. To study this interesting plant, we have constructed cDNA libraries from the roots and leaves. We have also analyzed the differences in the transcriptomes of these tissues using cDNA-AFLP analysis. We have isolated and sequenced in excess of 100 individual AFLP fragments which are unique to one tissue or the other. We are currently analyzing these sequences with the hope of identifying some of the genetic factors contributing to cyclopamine synthesis. We hope to publish this work shortly.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我的主要研究项目是芜菁皱纹病毒(TCV)与其寄主植物的相互作用。我们正在分析TCV和模式植物拟南芥的遗传互作。目前，我们正在使用两种TCV菌株，一种是野生型菌株，另一种是由UNL的莫里斯实验室开发的菌株，其外壳蛋白中的一种氨基酸发生了变化，称为R6a。野生型TCV外壳蛋白与拟南芥中的转录激活子结合，属于NAC转录因子家族。这种相互作用导致受感染植物中的RNA沉默。突变的R6a TCV不显示这种相互作用，并导致更严重和症状不同的疾病。我们正在通过分别用正常和突变的TCV接种野生型拟南芥以及没有病毒的模拟接种来分析这种相互作用。然后，我们在24小时和48小时后从所有三个植物种群中收获叶子。我们已经从所有六个组织中提取了总RNA一式三份。这些RNA将很快用于拟南芥全基因组基因阵列的微阵列分析。在不久的将来，将使用实时聚合酶链式反应进一步研究从这些阵列中被确定为最一致上调或下调的转录本，以验证微阵列的结果。我还有其他几个项目正在进行中，主要是与Gautam Sarath博士合作，但也与美国农业部的Christian Tobias博士和John Vogel博士合作。与Sarath博士和Tobias博士一起，我一直在进行基因组学研究，初步确定了生物燃料植物柳枝菊(Panicum Virgatum)的遗传学特征。到目前为止，我和我的学生们必须从柳枝花愈伤组织、茎、叶、树冠和幼苗中构建cdna文库。我们和托拜厄斯博士一起，对叶、愈伤组织、茎和树冠文库中的15,272个克隆的5‘端进行了测序和分析。所有这些序列都将很快存储在GenBank中，带有我和我的学生的名字以及适当的INBRE认可。从这些序列中，我们已经收集了许多在各种代谢过程中重要的基因，以供进一步鉴定。其中最主要的是涉及木质素生物合成的基因，这将很快成为我们关于这种材料的第一批出版物的基础。我目前正在与约翰·沃格尔博士一起开始一个类似的基因组学计划，研究另一种名为Bradypodium disachyon的生物燃料草。这个项目还处于初级阶段，但我们目前正在从这种植物的不同组织中构建四个cdna文库。我还在与萨拉特博士合作，对一种草原豆类植物进行基因组学研究，这种豆类植物被称为山豆(Chamaecrista Fasculata)。该植物是一种重要的草原植物，参与与根瘤菌形成根瘤的共生固氮共生。我们已经从该植物的根瘤组织中构建了一个cDNA文库，并开始对各种序列的5‘端进行特征分析。我们已经分离了一种名为豆状血红蛋白的共生蛋白的克隆，我们已经选择了这种蛋白进行进一步的鉴定。我们目前正致力于将开放阅读框亚克隆到合适的表达载体中，以生产重组蛋白。我们打算分析豆状血红蛋白的氧结合行为和结构。这项工作将很快产生它的第一个手稿。我的最后一个项目涉及一种名为玉米百合(Veratrum CalforNicum)的植物。这种植物之所以令人感兴趣，是因为它产生了一种名为环多巴胺的致畸和致癌类固醇生物碱。这种化合物主要集中在玉米百合的根部，但怀孕的母羊食用它的叶子会产生一些可怕的后代，只有一只眼睛或两只眼睛合并到一个眼窝里。为了研究这种有趣的植物，我们从根和叶中构建了cDNA文库。我们还利用cDNA-AFLP分析了这些组织转录本的差异。我们已经分离和测序了超过100个单独的AFLP片段，这些片段是一个或另一个组织所独有的。我们目前正在分析这些序列，希望确定一些有助于环多巴胺合成的遗传因素。我们希望不久就能出版这部作品。