Function of Plant Protein Synthesis Initiation Factors: eIF4G and eIFiso4G

植物蛋白合成起始因子的功能：eIF4G 和 eIFiso4G

• 批准号: 1052530
• 负责人: Karen Browning
• 金额: $ 98万
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-15 至 2018-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1052530&HistoricalAwards=false
• 关键词: Function; Plant; Protein; Synthesis; Initiation

Intellectual Merit: The aim of the project is to understand the mechanism by which plant cellsstart the process of making proteins. The model plant, Arabidopsis, provides excellent genetic tools to dissect the role of various cellular components in this process during normal plant growth and development as well as under a variety of environmental stresses such as changes of temperature, drought, or infection by plant pathogens. Although the mechanism of protein synthesis in plants is similar to that of animals or insects, some aspects are unique to plants. Many of these differences may be related to the fact that plants must interact with the environment while remaining in a single place and must obtain their energy through photosynthesis. A variety of genetic and biochemical tools will be applied to determine the specialized nature of how plant cells synthesize proteins. Biochemical and phenotypic analysis of T-DNA mutants of initiation factors eIF4G, eIFiso4G1 and eIFiso4G2 will be carried out, as well as analysis using tagged proteins (regulated by both native and CaMV 35S promoters) in various plant backgrounds. Environmental stressors (light, heat, cold, hormones, redox, etc.) will be applied to determine if any of the mutant or transgenic plants display phenotypes associated with particular stresses. Changes in protein expression will be assessed in the various mutants and transgenic expression lines and any proteins with altered expression will be identified. Several methods will be utilized to further identify any mRNA targets that are reduced on polysomes in the T-DNA knockout mutants for eIFiso4G1, eIFiso4G2 or eIF4G or that associate directly with these proteins or complexes. Those mRNAs identified from DNA array analysis of polysomes from mutant plant lines will be validated using using in vitro translation assays dependent upon the presence of eIFiso4F and confirmation by qRT-PCR of reduced polysome loading of the mRNAs in the mutant plant lines. Further validation will be done using in vivo expression of tagged versions of the identified mRNA?s coding region in plants lacking one or more of the genes for the subunits eIFiso4F. The mRNAs will also be used as ?bait? to pull down and identify additional RNA binding proteins.Broader Impact. High school, community college, undergraduate, graduate and post-doctoral students will be involved in this research project. Undergraduates will enter through the Freshman Research Initiative (FRI), a unique program at the University of Texas at Austin for training students in science, technology, engineering and math (STEM) disciplines. Beginning students will be taught the necessary skills to engage productively in research and will learning science by participation. The students in the FRI will be responsible for construction of in vitro expression clones for the mRNAs, construction of plasmids for over-expression of proteins and tagged proteins in plants and will be involved in the phenotype analysis of mutant plant lines. In addition to training future research scientists, this project will lead to new information about how plant cells make proteins. This knowledge may be applied to improve plants to better resist environmental insults or virus infection and to provide better quality protein for food for humans and livestock.

智力价值：该项目的目的是了解植物细胞开始制造蛋白质过程的机制。模式植物拟南芥（Arabidopsis）提供了很好的遗传学工具来分析在正常植物生长发育过程中，以及在温度变化、干旱或植物病原体感染等各种环境胁迫下，各种细胞成分在这一过程中的作用。虽然植物的蛋白质合成机制与动物或昆虫相似，但有些方面是植物所特有的。这些差异中的许多可能与植物必须与环境相互作用而保持在一个地方并且必须通过光合作用获得能量这一事实有关。各种遗传和生化工具将被应用于确定植物细胞如何合成蛋白质的特殊性质。将对起始因子eIF4G、eIFiso4G1和eIFiso4G2的T-DNA突变体进行生化和表型分析，并在各种植物背景下使用标记蛋白（由天然和CaMV 35S启动子调控）进行分析。环境胁迫源（光、热、冷、激素、氧化还原等）将被应用于确定是否有任何突变或转基因植物显示与特定胁迫相关的表型。将评估各种突变体和转基因表达系中蛋白质表达的变化，并鉴定任何表达改变的蛋白质。将使用几种方法来进一步鉴定eIFiso4G1、eIFiso4G2或eIF4G T-DNA敲除突变体中多体上减少的mRNA靶标或直接与这些蛋白或复合物相关的mRNA靶标。从突变植物系的多体DNA阵列分析中鉴定出的mrna将使用体外翻译试验进行验证，该试验依赖于eIFiso4F的存在，并通过qRT-PCR确认突变植物系中mrna的减少多体负载。进一步的验证将使用已鉴定mRNA的标记版本在体内的表达。缺乏一个或多个eIFiso4F亚基基因的植物编码区。这些mrna也将被用作诱饵。拉下并识别额外的RNA结合蛋白。更广泛的影响。高中、社区学院、本科生、研究生和博士后均可参与本研究项目。本科生将通过新生研究计划（FRI）进入，这是德克萨斯大学奥斯汀分校的一个独特项目，旨在培养科学、技术、工程和数学（STEM）学科的学生。初学的学生将被教授必要的技能，以从事富有成效的研究，并将通过参与学习科学。FRI的学生将负责构建mrna的体外表达克隆，构建植物中蛋白质和标记蛋白质的过表达质粒，并将参与突变植物系的表型分析。除了训练未来的研究科学家，这个项目还将带来关于植物细胞如何制造蛋白质的新信息。这些知识可用于改良植物，使其更好地抵抗环境损害或病毒感染，并为人类和牲畜提供更优质的食物蛋白质。