摘要
目的:本文通过研究microRNA172a(miR172a)对成花途径中关键靶基因APETALA2类(AP2-like)的调控作用及其机理分析,探索通过操纵miRNA表达改变观赏植物花期的基因工程分子育种新策略。创新点:本研究基于植物miR172序列和功能的高度保守性,通过转基因的方法操纵大岩桐miR172的表达,进而影响AP2-like基因的表达,并起到调控花期的作用。方法:本研究借用拟南芥miR172a的已知序列构建组成型过表达载体35S:miR172a和抑制miR172表达的35S:MIM172载体。利用农杆菌介导法成功获得了35S:miR172a过表达株系以及抑制miR172作用的35S:MIM172株系,并利用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆得到了大岩桐AP2-likecDNA全序列,并通过实时荧光定量聚合酶链式反应技术(qPCR)检测转基因株系中AP2-like的表达变化。结论:短日照条件下,35S:miR172a过表达株系花期比野生型提前(47.00±2.16)天;35S:MIM172株系花期延迟(7.00±4.28)天。在35S:miR172a过表达株系中miR172的表达水平明显上升,其靶基因SsAP2-like的表达量明显下降;35S:MIM172株系中miR172的积累水平受到抑制,而SsAP2-like的表达量明显上升,表明miR172介导调控SsAP2的表达对大岩桐成花转变具有促进作用。通过改变miR172的表达调控关键靶基因进而改变花期的方法可以作为调节观赏植物开花时间的有效策略。
We investigated the microRNA172(miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA(cDNA) of gloxinia(Sinningia speciosa) APETALA2-like(SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up-or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172 a, whereas the expression pattern of miR172 a was negatively correlated with that of miR156 a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
基金
Project supported by the National Natural Science Foundation of China(Nos.31171615 and 31401913)
