小麦miR5062靶基因的鉴定及功能研究

Identification and Functional Analysis of miR5062 Target Genes in Wheat(Triticum aestivum)

miRNA作为一种植物中重要的内源小分子,通过调控靶基因的表达来行使其功能。为了探索小麦(Triticum aestivum)中新发现的miRNA tae-miR5062的靶基因,利用生物信息学方法对tae-miR5062的靶基因进行预测和进一步分析,将属于小麦AGO2家族的TaAGO2确定为其靶基因;进一步构建重组表达载体pBI121-pre-miR5062和pBI121-TaAGO2,分别转入农杆菌GV3101感受态细胞,然后在烟草叶片上分区注射进行农杆菌介导的烟草瞬时共转化实验。结果表明,tae-miR5062与其靶基因TaAGO2互作降解,并降低了TaAGO2的表达。利用实时定量PCR方法检测TaAGO2在小麦不同组织中的转录水平,结果显示,TaAGO2在小麦根和叶中表达量不高,但在旗叶、茎、穗、籽粒和生长点中都有较高的表达。将C端连有GFP的TaAGO2融合表达载体转入水稻黄化苗叶鞘的原生质体后瞬时表达的亚细胞定位结果显示,TaAGO2蛋白定位于细胞核和细胞质中;与拟南芥过量表达TaAGO2的转基因植株相比,野生型对照并无明显的表型。

As an important endogenous small molecule in plants,miRNA performs its function by regulating the expression of target genes.In order to explore the newly discovered target genes of miRNA tae-miR5062 in wheat(Triticum aestivum),the target genes of tae-miR5062 were predicted and further analyzed by bioinformatics method in this study,and TaAGO2 belonging to the wheat AGO2 family was identified as its target gene.The recombinant expression vectors pBI121-pre-miR5062 and pBI121-TaAGO2 were further constructed,transformed into Agrobacterium GV3101 competent cells,respectively,and then injected into tobacco leaves in sections to carry out Agrobacterium-mediated transient co-transformation experiments.The results showed that tae-miR5062 interacts with TaAGO2 and reduces its expression.The transcription level of TaAGO2 in different tissues of wheat was detected by the real-time quantitative PCR method.The results showed that TaAGO2 was not highly expressed in roots and leaves,but was highly expressed in flag leaves,stems,spikes,grains and apical meristem.The transient expression of the TaAGO2 fusion expression vector with GFP linked to the C-terminus into the protoplast of the leaf sheath of rice etiolated seedlings showed that the TaAGO2 protein was localized in the nucleus and cytoplasm.The transgenic Arabidopsis overexpressing TaAGO2 had no obvious phenotype,compared to wild-type control.