Function of Plant Protein Synthesis Initiation Factors: eIF4G and eIFiso4G

智力价值：该项目的目的是了解植物细胞启动蛋白质制造过程的机制。模式植物拟南芥提供了很好的遗传工具来剖析各种细胞成分在植物正常生长发育过程中的作用，以及在各种环境胁迫下的作用，如温度变化、干旱或植物病原体的感染。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独有的。这些差异中的许多可能与这样一个事实有关，即植物必须与环境相互作用，同时保持在一个单一的地方，并且必须通过光合作用获得能量。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专门性。将对启动因子eIF4G、eIFiso4G1和eIFiso4G2的T-DNA突变体进行生化和表型分析，并在不同的植物背景下使用标记蛋白(受天然启动子和CaMV 35S启动子调节)进行分析。环境应激源(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表现出与特定胁迫相关的表型。蛋白质表达的变化将在不同的突变体和转基因表达系中进行评估，并将识别任何表达变化的蛋白质。将利用几种方法进一步确定在eIFso4G1、eIFso4G2或eIF4G的T-DNA敲除突变体中减少的多聚体上的任何mRNA靶标，或与这些蛋白质或复合体直接相关的任何信使核糖核酸靶标。从突变植物系多聚体的DNA阵列分析中鉴定出的那些mRNAs将使用体外翻译分析来验证，这取决于eIFiso4F的存在，并通过qRT-PCR确认突变植物系中mRNAs的多聚体负载量减少。进一步的验证将使用已识别的S编码区的标记版本在缺少一个或多个eIFso4F亚单位基因的植物中的体内表达。MRNAs也将被用作诱饵？下拉并鉴定更多的RNA结合蛋白。高中、社区大学、本科生、研究生和博士后将参与这一研究项目。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，旨在培训学生的科学、技术、工程和数学(STEM)学科。初学者将被教授必要的技能，以有效地参与研究，并将通过参与学习科学。FRI的学生将负责构建mRNAs的体外表达克隆，构建用于在植物中过度表达蛋白质和标记蛋白质的质粒，并将参与突变植物品系的表型分析。除了培训未来的研究科学家外，该项目还将带来有关植物细胞如何制造蛋白质的新信息。这些知识可能被应用于改良植物，以更好地抵御环境侮辱或病毒感染，并为人类和牲畜提供更高质量的食物蛋白质。

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.