gma-miR4359b介导DNA甲基化在大豆根盐胁迫应答中的功能及分子机制研究

MiRNAs as a gene expression regulator plays an important role in the process of plant response to salt stress. 24-nt non-conserved miRNAs are able to regulate target gene expression at the transcriptional level by methylated DNA. Previous work found that 24-nt non-conserved miRNA miR4359b was significantly down-regulated in response to salt stress in soybean roots. Yet, little has been known about the role of 24-nt non-conserved miRNAs-mediated DNA methylation in plant response to salt stress. This project intends to identify the salt-tolerance of the transgenic hairy roots of miR4359b and reveal its function in response to salt stress in soybean; Identification of miR4359b regulated target mRNA; To further detect the dynamic changes of miR4359b-mediated DNA methylation of target genes under salt stress, and from the perspective of DNA methylation to explore the molecular mechanism of miR4359b regulating response to salt stress. To analyze the downstream genes and metabolic pathways regulated by miR4359b and its target mRNA and to explore the molecular mechanism of miR4359b-mediated regulation of target mRNAs response to salt stress at the transcriptional level. To provide a theoretical basis for revealing the function and mechanism of 24-nt non-conserved miRNA-mediated regulate of target gene DNA methylation in response to salt stress in soybean roots.

miRNA参与调控植物对盐胁迫的应答。24nt非保守miRNA可通过甲基化DNA在转录水平调控靶基因表达。前期工作发现大豆的24nt非保守miRNA miR4359b在根中受盐胁迫诱导显著下调表达。目前，24nt非保守miRNA是否可以通过介导甲基化参与盐胁迫应答还鲜有报道。本项目拟对转miR4359b的大豆毛状根进行耐盐性鉴定，揭示其在大豆盐胁迫应答中的功能；鉴定miR4359b调控的靶mRNA；进一步检测盐胁迫下miR4359b介导靶基因DNA甲基化的动态变化，从DNA甲基化角度探讨miR4359b调控大豆应答盐胁迫的分子机制；分析miR4359b及其靶mRNA调控的下游基因及代谢途径，在转录水平探讨miR4359b介导调控靶mRNA应答盐胁迫的分子机制，为揭示24nt非保守miRNA介导调控靶基因DNA甲基化在大豆根盐胁迫应答中的功能及作用机制提供理论依据。

MicroRNAs（miRNAs）在植物盐胁迫应答中具有重要的功能。然而，非保守MiRNA在大豆盐胁迫适应性中的功能仍不清楚。在本研究中，大豆非保守MiRNA gma-miR4359b及其靶mRNA F-box基因GmFBX193在盐胁迫应答中功能被鉴定。在大豆毛状根、茎和叶中gma-miR4359b应答盐胁迫。在盐胁迫下，相比K599（对照）大豆毛状根，过表达gma-miR4359b的大豆毛状根（MIR4359b）的根长比率，根表面积比率，根体积比率，根尖数，存活率和叶绿素含量均提高，而相对电导率和丙二醛含量下降；相反，模拟靶标（Target mimicry，MIM4359b）和短串联靶标模拟（Short tandem target mimic，STTM4359b）抑制gma-miR4359b的大豆毛状根表现出盐敏感表型。在MIR4359b大豆毛状根中GmFBX193的转录水平降低，而在MIM4359b和STTM4359b大豆毛状根中GmFBX193的转录水平增加。GmFBX193在大豆根中应答盐胁迫。GmFBX193是一个核定位蛋白。在盐胁迫下，过表达GmFBX193的大豆毛状根与MIM4359b和STTM4359b大豆毛状根的表型类似。相比在K599（对照）大豆毛状根中，在MIR4359b大豆毛状根中有172个差异表达基因；在MIM4359b大豆毛状根中有个2129差异表达基因。这些差异表达基因中包括一些已知的与逆境胁迫相关的转录因子。与K599（对照）相比，MIR4359b大豆毛状根中gma-miR4359b前体序列的CHH类型甲基程度较高。正常条件下在GmFBX193的靶位点处第17位胞嘧啶有77.7%发生甲基化，盐胁迫后第17位胞嘧啶未被甲基化。这可能表明gma-miR4359b通过较高CHH甲基化程度和介导GmFBX193甲基化影响转基因大豆毛状根的耐盐性。总之，这些结果表明gma-miR4359b通过负调控GmFBX193在大豆盐胁迫应答中发挥正向作用。本研究解析了gma-miR4359b/GmFBX193分子模块在大豆盐胁迫应答中的功能及作用的分子机制，为大豆耐盐生物育种提供功能明确的分子模块。