Arabidopsis 2010: Functional Genomics of Arabidopsis P450s

获奖摘要：该项目旨在从功能上定义拟南芥细胞色素P450单加氧酶（P450）基因家族的表达模式和底物反应性，该基因家族约占该植物基因组的0.6%。 由这些基因编码的蛋白质在植物生长所必需的广泛的生物合成和解毒途径中介导氧化转化。 由于它们在代谢过程的广泛多样性中的作用以及它们相对缺乏翻译后修饰，它们作为下游报告基因直接激活响应化学、发育和环境线索的许多不同生化途径。 催化位点和非催化位点残基的广泛分歧导致了这个非常多样化的P450基因超家族的一级结构变异程度很高，在将功能分配给单个基因组DNA序列时呈现出无与伦比的复杂性。 我们将进一步的拟南芥P450的功能定义，通过创建微阵列的mRNA表达模式在不同发育阶段的组织分析。 成像个别P450基因相对于各种内部和外部化学线索以及不断变化的环境条件（紫外线损伤，病原体攻击，昆虫攻击，冷应激等）的反应。这些反应模式与植物生化途径的整理将为这些酶的预期功能提供基础。 在酵母和杆状病毒蛋白生产系统中过表达这些P450的一个子集，并将其掺入适合于高通量筛选底物反应性的膜支架蛋白复合物中，将为这些酶的确定功能提供基础。 这些结果有助于阐明该模式植物对各种胁迫条件的生化反应，并提供评估不同P450基因家族以及其他膜蛋白家族所需的基因组技术工具。 关于273个P450基因的组织、可用EST的比对、通过微阵列分析定义的mRNA表达模式和通过功能蛋白表达定义的P450底物偏好的数据将在http://arabidopsis-P450.biotec.uiuc.edu上提供。

Award Abstract: This project is aimed at functionally defining the expression patterns and substrate reactivities of the Arabidopsis thaliana cytochrome P450 monooxygenase (P450) gene family that represents approximately 0.6% of this plant's genome. The proteins encoded by these genes mediate oxidative transformations in a wide array of biosynthetic and detoxicative pathways essential for plant growth. Because of their roles in this wide diversity of metabolic processes and their relative lack of post-translational modification, they serve as downstream reporters for the direct activation of many different biochemical pathways responding to chemical, developmental and environmental cues. Extensive divergence of catalytic site as well as noncatalytic site residues has resulted in a high degree of primary structure variation in this very diverse P450 gene superfamily that presents unsurpassed complexity in assigning function to individual genomic DNA sequences. We will further the functional definition of Arabidopsis P450s by creating microarrays for analysis of the mRNA expression patterns in tissues at different developmental stages. Imaging of the responses of individual P450 genes with respect to a variety of internal and external chemical cues as well as changing environmental conditions (UV damage, pathogen attack, insect attack, cold stress, etc.) and collations of these response patterns with respect to plant biochemical pathways will provide the basis for assigning prospective functions to these enzymes. Overexpression of a subset of these P450s in yeast and baculovirus protein production systems and incorporation into membrane-scaffolding protein complexes suitable for high-throughput screening of substrate reactivities will provide the basis for assigning definitive function(s) to these enzymes. These results serve to elucidate this model plant's biochemical responses to a variety of stress conditions and provide genomic technology tools needed for assessment of the diverse P450 gene family as well as other membrane protein families. Data on the organization of the 273 P450 genes, alignments of available ESTs, patterns of mRNA expression defined by microarray analysis and P450 substrate preferences defined by functional protein expression will be made available at http://arabidopsis-P450.biotec.uiuc.edu as collected.