拟南芥mRNA前体3’末端加工在生长素信号转导中的作用机理研究

Auxin is essential for plant growth and development; however, the molecular mechanisms of auxin signal transduction have not been fully understood. By using chemical genetics approach, we isolated two Arabidopsis sirtinol resistant mutants, which are auxin resistant as well, indicating these two loci are involved in auxin signaling. Map-based cloning and genomic DNA complementation indicated that the auxin resistance phenotypes of the two mutants were caused by mutations in symplekin (At5g01400) or CstF77(At1g17760). Preliminary genetic analysis suggested that these two genes play important roles in auxin signaling, which may not overlap with the TIR1/AFB mediated protein degradation auxin signal transduction pathway. Symplekin and CstF77 are two core components of the pre-mRNA 3'-end processing machinery, and involved in RNA silencing. We hypothesize that symplekin and CstF77 participate in mRNA 3'-end formation and RNA silencing of some auxin responsive genes, whose alternative polyadenylation or protein levels may be altered in the two mutants. We propose to dissect the mechanisms of symplekin and CstF77 in auxin signaling by using genetics, biochemistry, proteomics and PAS-Seq deep sequencing approaches.

生长素在植物的生长发育中必不可少，但其信号转导作用机理还不完全清楚。我们利用化学遗传学手段，分离到两个拟南芥sirtinol和生长素抗性突变体atsym1和atcstf77，说明它们参与生长素信号转导。图位克隆和基因组DNA互补实验表明symplekin(At5g01400)和CstF77(At1g17760) 基因突变导致突变体表型。通过初步的遗传分析推测它们可能不在TIR1/AFB信号通路中起作用。CstF77和symplekin是mRNA 3'末端形成复合体的重要成员，调控基因沉默。我们推测它们可能在某些生长素响应基因的mRNA前体3'末端加工和基因沉默过程中发挥作用，突变体中一些基因mRNA的选择性多聚腺苷酸化或者蛋白质可能会发生变化。我们将运用遗传学、生化、蛋白质组学和PAS-Seq深度测序技术等手段，深入解析CstF77和symplekin在生长素信号转导中的作用机制。

生长素是一类调控植物生长发育的重要激素，目前对于生长素信号转导机理的理解主要集中在它对基因表达的转录调控方面，而对转录后调控了解很少。为了进一步理解生长素调控植物发育的机理，我们通过化学遗传学的方法，分离得到两个新的对生长素敏感度减弱的拟南芥突变体，并克隆了相关基因AtSYM1 和AtCstF77，它们是mRNA前体3’末端多腺苷酸化加尾复合体的成员。本项目中，我们发现它们在植物的生长发育过程中起着重要作用，atsym1 和atcstf77 突变体对生长素的响应减弱并具有严重的发育缺陷。AtCstF77 和AtSYM1基因在植物体内具有广泛的表达，它们所编码的蛋白质定位于细胞核中，这与它们在mRNA前体加工中的功能是一致的。cstf77与其他生长素相关突变体具有遗传互作，进一步说明它参与生长素信号转导。我们通过免疫共沉淀和质谱的方法鉴定出可能与CstF77相互作用的蛋白，并通过转录组测序的方法揭示AtCstF77和AtSYM1调控的生长素响应基因及其对这些基因mRNA 前体3’末端加工的调控机制。这些结果揭示了mRNA前体加工这一转录后调控机制在生长素信号转导中的作用，加深了我们对于生长素调控植物生长发育作用机制的理解。