Using combinatorial control to improve transgene expression specificity in plants

描述（由申请人提供）：据估计，目前世界上有15%的人口营养不良，每年有500万儿童死于营养不良。由于人口增长、气候变化、水资源短缺和对土地的竞争，未来养活世界的能力将更加困难。人们普遍认识到，要满足世界未来的营养需求，就必须在农业生物技术方面取得进展。在自然界中，基因表达在多个水平上调节，包括转录、可变剪接、转录物稳定性和翻译。相比之下，农业生物技术工业主要依赖于组成型转录控制来调节植物生长。 引入作物的性状的表达。这对于迄今为止已经商业化的相对简单的性状已经足够了，但是预期将需要满足未来食品需求的复杂性状需要增加的表达特异性。该项目的长期目标是组装一系列模块化表达元件，这些元件可以以组合方式部署，为农业生物技术行业提供可预测和可调的表达解决方案。该项目的I期部分的目标是证明异源miRNA结合元件和前mRNA剪接元件可用于增加模式植物拟南芥中转录启动子的表达特异性的概念验证。作为一个具体的产品概念，第一阶段的研究将集中在设计一个根中柱特异性表达盒，用于调节目标大豆胞囊线虫的性状。我们的具体目标是：1）验证增强中柱表达特异性的模块化转录后调控元件，和2）产生由转录启动子和转录后调控元件组合控制的中柱特异性表达盒。miRNA结合元件的选择将基于最近发表的数据，这些数据表明特定miRNA的细胞类型特异性表达模式与其各自靶标的表达模式呈负相关。选择性剪接事件将通过细胞类型特异性RNA-seq和微阵列表达数据的生物信息学分析来鉴定。鉴定的转录后调控元件将在组成型启动子的背景下进行验证，然后测试它们增加中柱富集启动子的表达特异性的能力。在展示了组合控制的概念验证之后，第二阶段的研究将把这种方法扩展到包括玉米等经济上重要的谷物在内的作物。 公共卫生相关性：据估计，目前世界上有15%的人口营养不良，每年有500万儿童死于营养不良。由于人口增长、气候变化、水资源短缺和对土地的竞争，未来养活世界的能力将更加困难。专家们普遍承认，需要通过生物技术提高农业生产力，以满足未来的粮食需求。该项目的目标是促进生物技术方法，从而在更少的土地上用更少的资源生产更多的作物。

DESCRIPTION (provided by applicant): It is currently estimated that 15% of the world's population is undernourished and 5 million childhood deaths a year are attributable to malnutrition. The ability to feed the world in the future will be even more difficult due to population growth, climate change, water scarcity, and competition for land. It is widely recognized that advances in agricultural biotechnology will be required to meet the world's future nutritional needs. In nature, gene expression is regulated at multiple levels including transcription, alternative splicing, transcript stability, and translation. In contrast, the agricutural biotechnology industry has relied primarily on constitutive transcriptional control to regulate the expression of traits introduced into crops. This has been sufficient for the relatively simple trais that have been commercialized to date, but the complex traits that will be needed to meet future food demands are expected to require increased expression specificity. The long term goal of this project is to assemble a portfolio of modular expression elements that can be deployed in a combinatorial fashion to provide predictable and tunable expression solutions to the agricultural biotechnology industry. The goal of the Phase I component of this project is to demonstrate proof-of-concept that heterologous miRNA binding elements and pre-mRNA splicing elements can be used to increase the expression specificity of transcriptional promoters in the model plant Arabidopsis. As a specific product concept, Phase I research will focus on designing a root stele-specific expression cassette useful for regulating traits that target soybean cyst nematode. Our specific aims are to: 1) validate a modular post-transcriptional regulatory element that enhances stele expression specificity, and 2) create stele-specific expression cassettes that are combinatorially controlled by transcriptional promoters and post-transcriptional regulatory elements. Selection of miRNA binding elements will be based on recently published data demonstrating cell type-specific expression patterns of specific miRNAs that are inversely correlated with the expression patterns of their respective targets. Alternative splicing events wil be identified through bioinformatic analysis of cell type-specific RNA-seq and microarray expression data. Identified posttranscriptional regulatory elements will be validated in the context of a constitutive promoter and then tested for their ability to increase the expression specificity of a stele-enriched promoter. After demonstrating proof-of-concept of combinatorial control, Phase II research will expand this approach to crops including economically important cereals like corn. PUBLIC HEALTH RELEVANCE: It is currently estimated that 15% of the world's population is undernourished and 5 million childhood deaths a year are attributable to malnutrition. The ability to feed the world in the future will be even more difficult due to population growth, climate change, water scarcity, and competition for land. It is widely acknowledged by experts that increased agronomic productivity through biotechnology will be needed to meet future food demands. The goal of this project is to facilitate biotechnology approaches that lead to the production of more crops on less land with fewer resources.