早熟禾亚科特异microRNA2032调控植物开花时间的机制研究

Flowering time is crucial for plants to adapt a wide range of growing environments and is one of the most targeted traits for crop breeding. Photoperiod and vernalization are two major factors affecting flowering time in temperate grasses such as Brachypoidum and wheat. Using high-throughput sequencing of small RNAs, we identified a microRNA (miR2032) in Brachypoidum that was predicted to target the putative Arabidopsis FLOWER LOCUS T (FT) orthologue genes. Preliminary analysis showed that the expression of miR2032 was affected by several flowering pathways such as day length and vernalization. Bioimformatic and Northern blot analysis showed that miR2032 was Pooideae-specific, suggesting that miR2032 may represent a novel molecular mechanism for flowering time regulation which may not exist in other model plants such as Arabidopsis and rice. Based on these previous results, in this programme, we propose: (1) to provide evidence that miR2032 regulates targets through cleavage or translation repression (2) to study the effects of other known flowering factors (e.g. VRN1, VRN2, and PPD) on miR2032 accumulation; (3) to identify transcription factors that regulates miR2032 expression; (4) to establish the relationship between flowering time and miR2032 abundance in different wheat germplasms; (5) to compare the evolution events between miR2032 and target loci in Pooideae plants using both bioinformatics and experimental approaches. This work will,elucidate a novel, Pooideae-specific, microRNA-mediated mechanism for flowering time regulation that may provide great insights for wheat heading date breeding and genetic improvement.

适时开花是作物适应广大种植地域和气候环境的重要育种目标。春化和光周期是影响小麦等重要农作物开花期的重要因素。我们通过短柄草小分子RNA高通量测序发现一个受光周期影响的microRNA (miR2032)，预测其靶基因是FLOWER LOCUS T (FT)家族同源基因。初步研究表明miR2032表达受多条开花路径影响，并为短柄草、小麦等早熟禾亚科植物所特有，暗示它代表一个不同于拟南芥和水稻的全新开花调控机制。本项目拟在前期发现的基础上，将明确miR2032对FT的作用方式；研究其他重要开花影响因子对miR2032的调节作用；寻找miR2032上游调控转录因子；阐述不同小麦开花种质与miR2032的表达关系；探究miR2032前体和靶基因在早熟禾亚科植物中的协同进化规律。本研究将首次阐明一种由miRNA介导的早熟禾亚科特有开花调控分子机制，为麦类作物广适性分子育种和遗传改良提供重要依据。

植物从营养生长时期到生殖生长时期的转变过程对于其生长和繁殖至关重要。植物的多条开花路径几乎均整合于对成花素（florigen）蛋白的调控，FT 是成花素蛋白的编码基因。MicroRNA （miRNA）是真核生物中对基因表达调控具有重要作用的一类小分子RNA。我们在短柄草、大麦和小麦等早熟禾亚科植物中发现了可以调控FT表达的miR5200 （先前命名为miR2032），并对该miRNA的表达、功能以及作用方式进行了深入研究，取得的主要研究结果包括：.1.miR5200在植物叶片中特异表达，以切割方式调控靶标FT基因；过量表达miR5200的转基因植物严重延迟植物开花时间。.2.miR5200受到光周期条件的严格控制，表现为短日照诱导表达，长日照抑制表达；miR5200受到植物生长时期的影响，前期表达较低，后期表达较高；不同光周期条件下miR5200前体基因受不同组蛋白的修饰，H3K27me3修饰在短日照条件下降低，在长日照条件下增加，而H3K4me3修饰在长日照条件下降低，在短日照条件下增加。.3.利用Target mimicry方法人工抑制miR5200的活性可以特异影响短柄草短日照条件下的开花时间，而对长日照条件下的开花并无影响。.4.miR5200在不同早熟禾亚科植物光周期介导的开花路径中具有一定的保守作用。. 本项目对miR5200的深入研究，首次阐明了一种由miRNA 介导的对植物成花素基因表达调控的特异分子机制，其意义不仅深化了我们对整个植物开花路径的认识，而且也为将来利用基因工程手段开展麦类作物的广适性分子育种和遗传改良提供了一定的理论依据。