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

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我的主要研究项目集中在芜菁皱纹病毒（TCV）与其宿主植物的相互作用。 我们正在分析 TCV 和模式植物拟南芥的遗传相互作用。 目前，我们正在使用两种 TCV 毒株，一种是野生型毒株，另一种是由内布拉斯加州大学莫里斯实验室开发的毒株，其病毒外壳蛋白 R6a 中的一个氨基酸发生了改变。 野生型 TCV 外壳蛋白与拟南芥中属于 NAC 转录因子家族的转录激活因子结合。 这种相互作用会导致受感染植物中的 RNA 沉默。 突变的 R6a TCV 不表现出这种相互作用，并会导致更严重且症状不同的疾病。 我们通过分别用正常和突变 TCV 接种野生型拟南芥以及不接种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 小时后收获了所有三个植物群体的叶子。 我们从所有六种组织中一式三份地提取了总 RNA。 这些 RNA 很快将用于拟南芥全基因组 cDNA 阵列的微阵列分析。 在不久的将来，将使用实时 PCR 进一步研究这些阵列中被确定为最一致上调或下调的转录本，以验证微阵列结果。 我还正在进行其他几个项目，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。 与博士。 Sarath 和 Tobias，我一直在研究一项基因组学计划，该计划旨在描述生物燃料植物柳枝稷（Panicum virgatum）的遗传学特征。 我和我的学生迄今为止已经从柳枝稷愈伤组织、茎、叶、冠和幼苗中构建了 cDNA 文库。 我们与 Tobias 博士一起对来自叶、愈伤组织、茎和冠文库的 15,272 个 cDNA 克隆的 5' 端进行了测序和分析。 所有这些序列很快就会被存入 GenBank，上面有我和我的学生的名字以及相应的 INBRe 确认。 从这些序列中，我们收集了许多在各种代谢过程中重要的基因，以供进一步表征。 其中最主要的是参与木质素生物合成的基因，这将很快成为我们关于该材料的第一份出版物的基础。 我目前正在与约翰·沃格尔博士一起开始一项类似的基因组学计划，研究另一种名为Bradypodium distachyon的生物燃料草。 该项目还处于起步阶段，但我们目前正在从该植物的不同组织构建四个 cDNA 文库。 我还与 Sarath 博士合作，对一种名为鹧鸪豌豆 (Chamaecrista fasciculata) 的草原豆科植物进行基因组学研究。 该植物是重要的草原物种，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构建了 cDNA 文库，并开始表征各种序列的 5' 端。 我们分离出了一种称为豆血红蛋白的共生蛋白克隆，我们选择该克隆进行进一步表征。 我们目前正集中精力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。 我们打算分析豆血红蛋白的氧结合行为和结构。 这项工作很快就会产生第一份手稿。 我的最后一个项目涉及一种名为玉米百合（Veratrum californicum）的植物。 这种植物之所以引起人们的关注，是因为它能产生一种叫做环杷明的致畸和致癌的甾体生物碱。 这种化合物主要集中在玉米百合的根部，但怀孕母羊食用其叶子后，会产生一些可怕的后代，它们只有一只眼睛或两只眼睛合并到一个眼窝中。 为了研究这种有趣的植物，我们构建了根和叶的 cDNA 文库。 我们还使用 cDNA-AFLP 分析分析了这些组织转录组的差异。 我们已经分离并测序了超过 100 个单独的 AFLP 片段，这些片段对于一种组织或另一种组织来说是独特的。 我们目前正在分析这些序列，希望能够确定一些有助于环杷明合成的遗传因素。 我们希望尽快出版这项工作。

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.