杨树5个木质部特异表达miRNAs的功能鉴定及调控机制研究

microRNA (miRNA) is a negative regulator of gene expression in eukaryotes，which can regulate various biological processes of an organism through down-regulation of the target genes. Our previous studies had identified quite a few miRNAs and predicted their target genes during the regeneration process of secondary vascular system in P. tomentosa，and 5 of which were specifically expressed in xylem thus might be involved in the regulation of the differentiation and formation of secondary vascular tissues. This proposal intends to systematically study the biological functions of these 5 miRNAs and their regulatory role on wood formation. We will construct the transient expression vectors with miRNA and its target gene(s) respectively to verify the effect of miRNA on its putative target gene(s) by co-transformation of them into poplar protoplasts. In addition, we will create the transgenic plants with over-expression and inhibition of miRNA and its target gene, by analyzing which we could reveal the regulation mechanism of miRNA and its target genes on wood formation by comparison of gene expression, phenotype and vascular tissue structure among these plants. The results obtained from this study will lay the foundation for the molecular breeding of wood properties of trees.

microRNA（miRNA）是真核生物基因表达中的一类负调控因子，它能够通过负调控靶基因的表达来实现对整个生物体生命活动的调控。我们前期对毛白杨次生维管组织再生过程中的miRNAs及其靶基因进行了预测和鉴定，筛选出5个在木质部特异表达的miRNAs可能参与调控次生维管组织的形成和分化。本项目拟系统地研究这5个miRNAs的生物学功能以及其对木材形成的调控作用。通过构建miRNA与其靶基因的瞬时表达载体并共转化杨树原生质体，验证并确定各个miRNA对其靶基因的作用；创制miRNA过表达转基因植株、miRNA作用抑制的STTM转基因植株、靶基因的过表达转基因植株和靶基因定点敲除突变体植株，并对转基因植株进行表达量、表型和维管组织结构分析，揭示miRNA与其靶基因对木材形成的调控机制，为林木材性的分子育种奠定基础。

microRNAs（miRNAs）是一类负调控因子，主要通过介导靶基因mRNA的切割或抑制翻译来调节植物基因的表达。目前，对物种特有的miRNAs特别是林木miRNAs功能研究的报道还比较少，因此，对木材形成相关miRNAs的挖掘和功能研究尤为重要。本项目拟对在杨树木质部特异表达的3个新miRNAs（pto-miR005、pto-miR037和pto-miR056）和2个已知miRNAs（ptc-miR160和ptc-miR393）进行生物学功能和调控机制的研究。通过对miRNAs前体序列的基因组分析、组织表达分析和克隆测序比对，选择合适的miRNA家族成员构建过表达以及STTM抑制表达载体并转化84K杨。通过表型分析，过表达pto-miR005、pto-miR037和pto-miR056了抑制植株的生长以及木质部的分化和发育。过表达ptc-miR160h促进杨树植株的生长，而过表达ptc-miR160b抑制了植株节间的分化，但两者都促进了木质部的分化。相反，抑制了ptc-miR160b和ptc-miR160h的表达作用，则会促进植株节间的分生生长，抑制了木质部的次生发育，同时叶片和根系发育也受到抑制。此外，过表达ptc-miR393减弱了植株的生长和木质部的发育，抑制ptc-miR393的表达则促进植株生长和茎增粗，但不影响木质部的发育。通过对上述5个维管发育相关miRNAs调控机制的初步研究和探讨，可以为培育速生优质的转基因品种以及研究木材形成的分子机理奠定基础。