烟草microRNA827及其靶基因的鉴定与分析

[目的]本文旨在明确林烟草（Nicotiana sylvestris）microRNA827（miR827）及其靶基因的表达调控特征，揭示其靶基因在进化上的变异性及其功能，为全面解析植物磷信号途径提供新的证据。[方法]基于本课题组前期工作从普通烟草（Nicotiana tabacum）中克隆到的miR827基因序列，采用生物信息学手段结合RACE和RT-qPCR，以及本氏烟草叶片表皮细胞瞬时表达和酵母突变体回补试验，对林烟草miR827的靶基因进行克隆，对miR827及其靶基因的表达模式、靶基因的定位和功能进行研究。[结果]获得林烟草miR827两个靶基因Ns-SPX-MFS1/2的cDNA全长序列，发现Ns-miR827受缺磷诱导表达，而Ns-SPX-MFS1和Ns-SPX-MFS2响应缺磷信号分别发生下调和上调。Ns-SPX-MFS1/2均定位于液泡膜和细胞核。此外，不论Ns-SPX-MFS1/2的开放阅读框全长或截去SPX结构域的2个基因片段均能回补酵母磷吸收突变株。[结论]miR827及其靶基因调控模块在物种进化中存在复杂的变异性，Ns-SPX-MFS1/2具有转运磷的活性，其可能通过介导液泡和细胞质间磷的转运实现对植株磷稳态的调控。
[Objectives] This work is aimed at clarifying the expression pattern of microRNA827(miR827) and the target genes in Nicotiana sylverstris,elucidating the evolutionary divergence and function of Ns-miR827 target genes,as well as providing novel evidences for the comprehensive understanding of the phosphate signaling network in plants. [Methods] Based on the miR827 sequences obtained in our previous work,bioinformatic approach,RACE,RT-qPCR,transient expression in tobacco leaf epidermal cells,and complementation of a yeast mutant line defective in phosphate(Pi) transport were utilized to clone the miR827 target genes,and to investigate the expression pattern of miR827 and the target genes,as well as the subcellular localization and the function of miR827 target genes. [Results] The full-length cDNA sequences of the two Ns-miR827 target genes(Ns-SPX-MFS1/2) were acquired. The transcriptional expression of Ns-miR827s were up-regulated by Pi starvation stress,whereas Ns-SPX-MFS1 and Ns-SPX-MFS2 were repressed and induced,respectively,upon Pi starvation. Ns-SPX-MFS1/2 were both localized to the tonoplast and the nucleus. Furthermore,the growth defects of the yeast mutant line EY917 could be restored by complementation with both the intact open reading frames of Ns-SPX-MFS1/2 and their truncated versions lacking the SPX domains. [Conclusions] The miR827-target gene regulatory modules display complex evolutionary divergence. Ns-SPX-MFS1/2 both have Pi transport activities,which might modulate plant Pi homeostasis through mediating Pi transport across the tonoplast