小麦小分子RNA TaMIR1118介导植株抵御低钾营养胁迫的分子机理

Through regulation of the target genes at posttranscriptional and translational levels, microRNAs (miRNAs) play critical roles in mediating plant abiotic stress responses via modifying the target transcripts abundance. Our previous expression studies on wheat miRNA family members responding to potassium deprivation revealed that a miRNA member TaMIR1118 showed significantly upregulated expression upon low-K stress. In contrast, its target genes including camodulin encoding genes TaCaM2-1, TaCaM2-2, and TaCaM2-3 exhibited reverse expression patterns in response to potassium deficiency contrasting with TaMIR1118, suggesting that the TaMIR1118/TaCaM2-1~TaCaM2-3 module functions largely in mediating plant low-K stress tolerance. In this current project, by using RLM-RACE and PPM-RACE techniques, we will experimentally assay the cleaved characterization of the target genes TaCaM2-1, TaCaM2-2, and TaCaM2-3 under TaMIR1118. By adopting gene overexpression and RNAi gene knockdown approaches, we will elucidate the function of module members in mediating plant low-K stress tolerance. In addition, high throughput RNA-seq and proteomics techniques will be applied to identify the differential genes associated with plant potassium deprivation tolerance under the control of the miRNA member, by which to establish the corresponding molecular networks and biochemical metabolic pathways that contribute to the miRNA/target module-modulated low-K stress tolerance. This project will unravel the molecular mechanism as to how TaMIR1118 mediates the low-K stress tolerance of plants in wheat.

植物种属小分子RNA（miRNAs）通过转录后和翻译水平调节，在介导植株抵御非生物逆境过程中发挥着重要作用。申请人前期对小麦miRNA家族成员应答低钾胁迫的研究发现，TaMIR1118在低钾处理下表达水平显著上调，而其作用靶基因钙调素TaCaM2家族基因TaCaM2-1~TaCaM2-3应答低钾特征与该miRNA相反，表明TaMIR1118/TaCaM2模块在介导植株应答低钾胁迫中发挥重要功能。本项研究以该miRNA功能模块为基础，利用RLM-RACE 和PPM-RACE技术，鉴定供试miRNA靶基因TaCaM2-1~TaCaM2-3转录本剪切特征；采用基因过表达和RNAi干涉技术，系统研究上述模块成员介导植株抵御低钾逆境能力；采用转录组测序和蛋白谱检测技术，鉴定TaMIR1118调控的低钾响应基因、分子网络和生化代谢途径。通过本研究解析TaMIR1118介导植株抵御低钾胁迫的分子机理。

MicroRNAs（miRNAs）是一类在转录后调控中发挥重要作用的非编码小RNA，是植物抵御各种非生物逆境调控网络的重要组成部分。本课题以对低钾逆境应答的小麦TaMIR1118/TaCaM5-3A模块为基础，系统阐明了该功能模块介导植株抵御低钾逆境的生物学功能和分子机理。课题取得下述结果：.1.TaMIR1118/TaCaM5-3A模块应答低钾逆境的表达模式.TaMIR1118呈低钾诱导上调的表达模式，TaCaM5-3A与TaMIR1118呈相反的表达模式。TaMIR1118/TaCaM5-3A模块在介导小麦应答和抵御低钾逆境中发挥着重要作用。.2.TaMIR1118/TaCaM5-3A模块的靶向关系验证.利用降解组测序、5′ RACE、qRT-PCR和双荧光素酶报告系统，证明TaMIR1118可以精确地切割TaCaM5-3A的3′-UTR，从而抑制靶基因TaCaM5-3A的表达。.3.TaMIR1118/TaCaM5-3A模块介导植株钾素吸收和利用能力的特征.利用TaMIR1118/TaCaM5-3A模块组分遗传转化烟草和小麦株系，并对植株抵御低钾胁迫的生物学功能进行鉴定。研究表明TaMIR1118/TaCaM5-3A模块可通过调控植株根系对K+的吸收和积累，介导小麦植株的低钾耐受性。.4.TaMIR1118调控的转录谱特征研究.对低钾处理的过表达TaMIR1118的烟草株系和野生型根系转录组进行分析。TaMIR1118作为一个重要的调节因子，通过调节与细胞内稳态、次生代谢、植物激素信号转导和碳代谢等相关的调控网络，介导植物对低钾胁迫的耐受性。.5.TaCaM5-3A下游互作蛋白的筛选及功能解析.利用酵母双杂交筛选技术筛选TaCaM5-3A下游互作蛋白，进行一对一互作、BiFC和K+吸收缺陷型酵母功能互补等试验解析其生物学功能。表明该模块通过调控下游基因TaHAK1，参与植株对低钾逆境响应过程。.课题关键数据：获得了TaMIR1118/TaCaM5-3A模块转录、调控下游转录谱、下游互作蛋白以及相关低钾胁迫响应基因调控植株钾效率等方面关键数据。.课题科学意义：揭示了TaMIR1118/TaCaM5-3A功能模块介导植株抵御低钾逆境的生物学功能和分子机理。旨在深入阐明小麦应答低钾逆境的分子机制，为今后钾高效小麦的遗传改良和生产实践提供理论依据。