miR164和miR167调控玉米胚乳发育的信号通路解析

miR164 and miR167 played important roles in plant morphogenesis, growth and development, hormones and signal transduction, and environmental stress responses.Our previous study showed that the expression level of two miRNAs (miR164 and miR167) were significantly difference and were involved in the regulation of maize endosperm development. We sequenced small RNA libraries, Transcriptome libraries, degradome libraries from developing maize endosperm to investigate expression pattern of miRNA and mRNA and identify the target genes of miRNA.The result showed that miR164 target the mRNA of NAC113 and miR167 target the mRNA of auxin response factors 18(ARF18) .Moreover, we generated the transgenic plants for the overexpression and silence of miR164 and miR167. In the present study,The target genes(NAC113,ARF18) of miR164 and miR167 were validated by transient expression technology. In addition, the monoclonalantibodies against NAC113 and ARF18, respectively, will be prepared for ChIP-Seqanalysis to identify their target genes.A variety of experimental methods, such as yeast one-hybrid assay, EMSA, transient expression analysis, Microarray， and so on, will be carried out to reveal the regulatory mechanism of miR164 and miR167 involved inmaize endosperm development. This study can shed light on the molecular mechanism involved in the interaction of miR164 and miR167 with other important biological molecules in maize endosperm.

miR164和miR167对植物的形态建成、生长发育，激素分泌等都具有重要的作用。在前期研究中，我们发现miR164和miR167在玉米胚乳发育过程中表达量差异显著，它们可能参与了胚乳发育的调节及信号通路。通过对胚乳发育7个不同时期的小RNA测序、降解组测序和RNA-seq，我们初步鉴定了其靶基因为转录因子NAC113和ARF18。同时，我们创制了玉米miR164和miR167的过表达及沉默转基因植株。在此基础上，本项目首先通过瞬时表达对靶基因NAC113和ARF18进行验证，然后通过制备NAC113和ARF18的单克隆抗体，利用ChIP-Seq技术鉴定转录因子NAC113和ARF18的下游调控基因。通过酵母单杂交、凝胶阻滞电泳、基因瞬时表达、基因芯片分析、生理指标测定等研究技术，在体内外分别验证miR164和miR167调控玉米胚乳发育的信号通路，为进一步解析其功能及调控机制奠定基础。

miR164和miR167对植物的形态建成、生长发育，激素分泌等都具有重要的作用。在前期研究中，我们发现miR164和miR167在玉米胚乳发育过程中表达量差异显著，表明它们可能参与了胚乳发育的调节及相关信号通路。在此基础上，本项目开展miR164和miR167对玉米胚乳发育的影响与调控机制研究。为探究miR164和miR167在玉米胚乳发育过程中的表达模式，我们进行了玉米胚乳小分子RNA测序以及qPCR检测。结果表明，miR164和miR167分别在玉米胚乳中呈下调和上调表达趋势。为鉴定miR164和miR167的靶基因，我们进行了降解组测序和瞬时表达验证，确定了NAC32和NAC40为miR164的靶基因，ARF9和ARF18为miR167的靶基因。为深入探究miR164的生物学功能，我们创制了过表达材料。与野生型相比，miR164转基因株系胚乳变小，粒长、粒宽和百粒重降低，表明miR164可能与种子膨大有关。为进一步分析miR164-NAC32/NAC40调控路径，本研究通过RNA-Seq以及共表达分析挖掘过表达玉米籽粒中的差异基因，发现两个扩张蛋白基因EXPB14和EXPB15可能作为NAC32和NAC40下游作用基因参与玉米种子的发育调控。为探究miR167-ARF9/ARF18调控路径，我们通过基因瞬时表达的方法获得ARF18过表达胚乳。RNA-Seq以及GO功能富集分析显示，miR167-ARF9/ARF18可能通过调控激素响应、细胞壁疏松和水运输基因参与玉米胚乳发育调控。本研究深入探究了miR164和miR167对胚乳发育的影响与调控机制，揭示玉米种子大小调控的新机制，为完善和补充玉米胚乳发育的调控机制奠定了基础。