Abstract
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 miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
概 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-like cDNA 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。
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Project supported by the National Natural Science Foundation of China (Nos. 31171615 and 31401913)
Electronic supplementary materials: The online version of this article (https://doi.org/10.1631/jzus.B1800003) contains supplementary materials, which are available to authorized users
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Li, Xy., Guo, F., Ma, Sy. et al. Regulation of flowering time via miR172-mediated APETALA2-like expression in ornamental gloxinia (Sinningia speciosa). J. Zhejiang Univ. Sci. B 20, 322–331 (2019). https://doi.org/10.1631/jzus.B1800003
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DOI: https://doi.org/10.1631/jzus.B1800003