拟南芥尿苷酸转移酶URT1参与成花转换调控的分子机制

Floral transition or flowering refers to the transition from vegetative to reproductive growth in plants and is controlled by multiple genes in the six major flowering pathways along with several integrator genes. Gene expression regulation is closely related to mRNA stability and translatability. Results from our previous research and published articles suggest that plant RNA uridylyltransferases uridylate microRNAs and messenger RNAs, thereby affecting the stability of these targets and regulating gene expression at the post-transcriptional level. Our recent study found that Arabidopsis uridylyltransferase urt1 mutants exhibited an early flowering phenotype, which coincided with altered expression levels of FLC and AP1, which are key flowering time genes. To elucidate the molecular mechanism of URT1-regulated floral transition, we propose to adopt genetic, molecular and bio-informatics strategies to identify the flowering pathways through which URT1 regulates floral transition and to identify its direct target genes. We will also determine the modes of action of URT1 in regulating the stability and translatability of its target mRNAs. The results of this study should improve our understanding of the multilayer network of gene expression regulation and the mechanisms regulating floral transition; in addition, this study should provide theoretical guidance for manipulating flowering time in crops and horticultural plants to improve their agricultural and ornamental value.

成花转换，即植物由营养生长向生殖生长的转换，受多条遗传途径中众多基因的调控。基因的表达与mRNA的稳定性和翻译能力密切相关。本课题组的前期研究和已有的文献报道显示：植物尿苷酸转移酶可对miRNA和mRNA尿苷化加尾并影响其稳定性，在转录后水平参与基因表达的调控；而且课题组最近发现：拟南芥尿苷酸转移酶urt1突变体有早花表型，且开花调控关键基因FLC和AP1表达量的变化与早花表型相吻合，显示URT1参与拟南芥成花转换的调控。为了阐明该调控的分子机制，本课题拟利用遗传学、分子生物学和生物信息学等手段，明确URT1所参与的开花调控遗传途径，并鉴定其直接靶基因，还将探究URT1靶基因mRNA的尿苷化对其稳定性和翻译能力的影响模式。研究结果将有助于完善基因表达的多层次调控网络，也将加深我们对调控植物成花转变分子机制的认识，还将为改良农作物或园艺作物的开花时间以提高其农业价值或观赏价值提供理论指导。

尿苷化是广泛存在的RNA3’末端非模板修饰，该过程由RNA末端尿苷转移酶催化。RNA 尿苷化发挥多种不同的作用，然而，在植物中尿苷化和RNA稳定性之间的关系尚未报道。拟南芥中尿苷酸转移酶主要有HESO1和URT1，本项目利用遗传学、分子生物学、和生物信息学方法探究URT1介导的尿苷化影响基因表达的分子机制以及对成花转换的调控机制，研究结果如下：.1）高温（28℃）胁迫下AtURT1影响拟南芥植株开花时间。在高温（28℃）长日照下urt1突变体有明显早花表型，而URT1过表达株系有明显晚花表型，URT1回补株系可以恢复突变体的早花表型。促进开花的关键基因CO和FT等在urt1中有显著上调，而在URT1过表达株系中有显著下调。.2）AtUTR1、 OsNTP4 和OsNTP10不影响miRNA加尾及丰度。我们对10个NTPs单突变体和Col-0进行小RNA测序，发现10个NTPs的单突变体中miRNA的丰度并没有显著变化；我们体外重组表达及纯化了与拟南芥AtURT1同源性最高的水稻OsNTP4 和OsNTP10蛋白，以miRNA156作为底物，没有鉴定到OsNTP4、OsNTP10对sRNA的加尾活性。表明AtURT1、OsNTP4和OsNTP10对sRNA没有加尾活性。.3）确定URT1尿苷化加尾的靶标RNA为mRNA。我们创建了mRNA 3’末端测序文库构建流程，比较野生型和urt1突变体中mRNA加尾、mRNA丰度，发现高温（28℃）胁迫条件下Col-0和urt1-1中mRNA 3’末端加尾比例分别约为6.00%和2.05%。对加尾基因的表达量进一步分析发现加尾使mRNA更稳定,说明URT1尿苷化加尾的靶标RNA为mRNA。.4）尿苷化加尾影响mRNA稳定性和翻译效率。mRNA 半衰期检测结果发现urt1突变体中mRNA半衰期显著缩短，Ribosome profiling的实验，发现高温（28℃）胁迫条件下urt1-1突变体monosome和polysome中的加尾基因mRNA显著下调，表明URT1可能影响mRNA的稳定性和翻译效率。对URT1的靶标基因进行GO分析，发现URT1的靶标基因中包括抑制开花的基因，urt1突变体中抑制开花基因表达量明显降低，显示URT1可能通过对开花抑制基因mRNA加尾，提高其稳定性，促进其对开花的抑制, 具有延迟开花的功能。.