Analysis of mRNA splicing in plant hormone signaling

描述(由申请人提供)： 生物信息学驱动的分子遗传学方法将被用来研究选择性剪接作为基因调控机制在植物激素信号转导中的作用，以模式植物拟南芥为例。高密度寡核苷酸芯片将被用来检测外源生长素、油菜素内酯和赤霉酸处理对(I)编码假定的RNA结合蛋白剪接因子的拟南芥基因的表达的影响；以及(2)前mRNA剪接的影响。T-DNA突变插入系将确定编码激素调节的假定RNA结合蛋白的基因，并分析激素相关的表型变化。使用Affymetrix阵列检测到的可能受激素调控的选择性剪接变体将使用RT-PCR、变性高效液相色谱(DHPLC)进一步分析，并通过测序最终确认。被证实具有激素调节的选择性剪接的基因将被选择用于开发替代剪接报告基因系统，该系统将在转基因植物中进行实验测试。

DESCRIPTION (provided by applicant): A bioinformatics-driven molecular genetics approach will be used to study the role of alternative splicing as a gene regulation mechanism in plant hormone signaling using the model plant Arabidopsis thaliana. High-density oligonucleotide microarrays will be used to examine the effects of exogenous auxin, brassinolide, and gibberellic acid treatments on (i) the expression of Arabidopsis genes that encode putative RNA-binding protein splicing factors; and (2) pre-mRNA splicing. T-DNA mutant insertion lines will be identified for the genes encoding hormone-regulated putative RNA-binding proteins and assayed for hormone associated phenotypic changes. Putative hormone-regulated alternative splice variants detected using the Affymetrix arrays will be further analyzed using RT-PCR, denaturing high performance liquid chromatography (DHPLC) and final confirmation by sequencing. Genes confirmed to exhibit hormone-regulated alternative splicing will be selected for use in developing an alternative splicing reporter gene system that will be tested experimentally in transgenic plants.