Ath-miR169d介导的拟南芥叶片发育的分子调控机制

doi:10.3864/j.issn.0578-1752.2017.16.001

摘要/Abstract

摘要： 【目的】探究ath-miR169d在拟南芥叶片发育过程中的作用，明确其调控的分子机制，为增强叶菜类植物的光合效率以及增加生物量和提高作物产量提供理论依据。【方法】选取ath-miR169d过表达和降低表达的拟南芥转基因株系，以及野生型拟南芥，在22℃、相对湿度60%、16 h/8 h光周期条件下培养，观察不同生长阶段叶片发育表型的差异；同时构建pmiR169d::GUS表达载体，转化农杆菌EHA105，并利用蘸花法转化野生型拟南芥（Columbia，Col-0），获得转基因拟南芥，利用GUS染色对ath-miR169d在拟南芥不同组织器官中的表达模式进行研究；选取8周龄的过表达材料和野生型对照株系，对其叶片表皮细胞形态进行扫描电镜分析；对4周龄过表达材料和野生型对照株系的幼苗顶端分生组织和叶片中总IAA进行定量分析，并进行基因芯片表达谱分析，筛选差异表达基因；通过实时荧光定量PCR对生长素信号途径部分差异表达关键基因的表达情况进行分析。【结果】Ath-miR169d过表达拟南芥株系莲座叶数目较野生型少，叶片小，而ath-miR169d降低表达的拟南芥株系的莲座叶较野生型多，叶片大，且在抽薹和种子成熟之后还持续长出新叶，而野生型莲座叶则在种子成熟之后逐渐衰老干枯；扫描电镜观察到ath-miR169d过表达拟南芥株系叶片中表皮细胞小于野生型；表达模式分析表明，ath-miR169d主要在顶端分生组织（shoot apical meristem，SAM）和叶片维管系统中表达，且新叶中表达强。SAM是产生生长素的部位，IAA测定结果表明ath-miR169d过表达拟南芥株系中IAA含量显著降低，为野生型植株的38.6%，同时基因芯片表达谱分析也验证了ath-miR169d参与调控植物体内激素信号转导途径。进一步研究发现，ath-miR169d过表达株系中生长素合成关键基因YUC2和转运体蛋白基因PIN1均显著下调表达，而具有转录抑制功能的生长素响应因子1和2基因（ARF1和ARF2）表达上调；STTM miR169d低表达株系中这4个基因的表达则为相反趋势，该结果与观察到的表型相一致。【结论】Ath-miR169d通过介导生长素信号途径参与了拟南芥叶片发育的调控，ath-miR169d表达量发生变化会影响叶片的数量和大小，最终影响整个植株的生物量。

关键词: 拟南芥, miR169, 叶片, 发育, 生长素信号途径

Abstract: 【Objective】The aims of this study are 1) to analyze the role of ath-miR169d in the progress of leaf development in Arabidopsis, 2) to illuminate the mechanisms of molecular regulation mediated by ath-miR169d, and 3) to provide a new insight into improving the photosynthetic performance and the genetic engineering of vegetables biomass and crop productivity.【Method】Ath-miR169d over-expression transgenic plants, STTM ath-miR169d knockdown transgenic plants, and WT plants were grown in a controlled culture room at 22℃ with a relative humidity of 60% and 16 h/8 h photoperiod. Number and size of rosette leaf were measured; pmiR169d::GUS vector were constructed and transformed into Agrobacterium tumefaciens EHA105, and then infected the buds of wildtype Arabidopsis (Columbia, Col-0), the pmiR169d::GUS transgenic Arabidopsis plants were used to observe the expression profile of ath-miR169d in different organs and tissues by GUS staining method. Using 8-week-old seedlings of ath-miR169d over-expressing transgenic Arabidopsis thaliana and wild type plants as materials, morphology of leaf epidemic cells were observed by scanning electron microscope (SEM). Whole shoots were harvested from 4-week-old seedlings of ath-miR169d over-expressing transgenic A. thaliana and wild type plants, subsequently the total endogenous IAA were detected and quantified as methyl esters by gas chromatography-mass spectroscopy (GC-MS). The mRNA expression profiles were detected by microarray analysis to screen differentially expressed genes. The expression of key genes in plant auxin signal pathway were verified by real-time fluorescent quantitative PCR (RT-qPCR). 【Result】In contrast to WT, ath-miR169d overexpression plants exhibited fewer and smaller rosettes, STTM ath-miR169d knockdown plants showed more and larger rosettes. Moreover, STTM ath-miR169d plants could generate new rosette leaves incessantly even after bolting and seeds harvest, whereas the leaves of ath-miR169d overexpression and WT plants generally decayed after harvest. The epidermal cells in the leaves of ath-miR169d over-expression plants were smaller than that in the WT leaves by SEM detection. Ath-miR169d was highly expressed in SAM and leaf vasculature, and expression profile was stronger in new leaves than in old ones. Endogenous IAA content reduced by 38.6% in ath-miR169d over-expressing plants compared to wild-type plants, showing Ath-miR169d was potentially involved in auxin signal pathway by microarray analysis. The auxin biosynthesis gene YUC2 and transporter gene PIN1 were downregulated in ath-miR169d over-expressing plants whereas, auxin response factors 1 and 2 (ARF1 and ARF2) genes were upregulated. Expression profile of YUC2, PIN1, ARF1 and ARF2 in STTM miR169d plantswere opposite to over-expressing plants. 【Conclusion】Results of the experiment showed that the leaf development was regulated by ath-miR169d through the auxin-signaling pathway. Number and size of rosette could be alerted by ath-miR169d expression level, and further affected biomass of the whole plant.

Key words: Arabidopsis, miR169, leaf, development, auxin signaling pathway

张敏，朱明，李文宗，马洁，刘悦萍，江海洋，王磊，徐妙云. Ath-miR169d介导的拟南芥叶片发育的分子调控机制[J]. 中国农业科学, 2017, 50(16): 3063-3070.

ZHANG Min, ZHU Ming, LI WenZong, MA Jie, LIU YuePing, JIANG HaiYang, WANG Lei, XU MiaoYun. Molecular Regulation Mechanism of Leaf Development Mediated by Ath-miR169d in Arabidopsis thaliana[J]. Scientia Agricultura Sinica, 2017, 50(16): 3063-3070.

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