Cloning of miR164b precursor from Phyllostachys edulis and analysis of its function in leaf morphogenesis
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摘要: microRNA（miRNA）参与植物多种生理代谢过程，在调控植物形态建成中发挥着重要作用。miR164作为植物特有的miRNA，其主要的靶基因是NAC转录因子，参与调控植物茎、叶顶端分生组织的建立、器官的分化和植株衰老等过程。本研究以毛竹（Phyllostachys edulis（Carr.） Lehaie）为材料，从中分离出miR164b的前体序列（82 bp），二级结构分析结果发现该前体序列能够形成稳定的茎环结构，其成熟序列（21 bp）产生于茎环结构5'端的臂上，且碱基具有较高的保守性。本研究还构建了由CaMV 35S启动，包含毛竹miR164b前体序列的植物表达载体，并转化野生型拟南芥（Arabidopsis thaliana（L.） Heynh），获得了转基因植株。结果表明，转基因植株生长瘦弱，莲座叶数量明显减少，叶片变小且叶片边缘锯齿减少，更加光滑。实时定量PCR分析结果显示，转基因拟南芥中毛竹miR164b的表达量极显著上升，而拟南芥内源靶基因CUC1与CUC2的表达量极显著下降。表明毛竹miR164b通过调节CUC1和CUC2的表达来参与植物叶形态建成过程。研究结果可为利用miRNA开展竹子分子育种提供参考。Abstract: MicroRNA (miRNA) plays a crucial role in the regulation of plant morphogenesis by participating in a variety of physiological metabolic processes.The miR164 family is a group of plant-specific miRNAs,whose target genes are mainly the NAC transcription factors involved in the differentiation and development of apical meristems in stems and leaves,as well as in plant senesce.In this study,the precursor sequence of miR164b isolated from Phyllostachys edulis was 82 bp,which could form a stable stem-loop structure with the generated mature sequence on the 5'end of the arm.The mature sequences of the miR164 family were highly conserved.The expression vector with the target fragment harboring the miR164b precursor driven by CaMV 35S was constructed and transformed into Arabidopsis(Col-0).The phenotypes demonstrated that the transgenic Arabidopsis plants grew smaller and weaker,with fewer leaves,later flowering,and smoother leaf margins than those of Col-0.The real-time PCR results showed that the expression of miR164b was significantly increased (P < 0.01) and the expressions of CUC1 and CUC2 were significantly down-regulated (P < 0.01) in transgenic plants compared with Col-0.These results indicate that miR164b plays an important role in leaf morphogenesis by regulating the expression of CUC1 and CUC2.This study provides a useful reference for bamboo molecular breeding using miRNA strategies.
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Keywords:
- Phyllostachys edulis /
- MiR164b /
- Precursor /
- Leaf morphogenesis
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