胡杨ptc-miR213及其靶基因参与盐碱胁迫应答的机制研究

Salinity is the most important environmental factor that affects the productivity of forest tree. It's a large area of salinity land that need improvement and utilization in China, and it's important to breed salt-tolerance tree viateries during coastal shelter forest construction and salinity land improvement. Slow progress had been make up by conventional breeding. During the long-term evolutionary, different kinds of stress tolerance mechanisms had produced by high-salt-tolerant species. So it's important for salinity land phytoremediation for identifying salt tolerance molecular mechanism, cloning salt tolerance key genes and analyzing the expression model response to salt stress. MicroRNAs (miRNAs), the regulators of gene transcription levels play an important role in plant stress response. Populus euphratica was selected for studying ptc-miR213 function under salt stress in the project, The target genes MYB transcription factor, pp2c related protein and protein kinase putative will be verified, to explore its specific biological function, tissue-specific expression, and its relationship with negative regulation of target genes by transgenic technology. The regulation mechanism of ptc-miR213 and target genes response to salt stress will be revealed following the completion the project, theoretical basis and new salt tolerance relation gene may be used for new high salt tolerance varieties breeding.

我国有大面积的盐碱化土地亟待于改良和利用，沿海防护林建设急需培育高耐盐树种，但传统育种进展缓慢。因此查清耐盐树种的分子机制，发掘耐盐关键基因对于高抗树种培育和盐碱地开发利用具有重要意义。研究表明MicroRNA（miRNA）对基因转录后水平的调节在植物逆境反应中发挥重要作用。本项目以胡杨为材料,以前期研究分离出的耐盐相关microRNA ptc-miR213为研究对象，实验验证它的靶基因：MYB4、PP2C和蛋白激酶，分离ptc-miR213和靶基因及其相应的启动子，明确ptc-miR213及其靶基因的组织时序的特异性表达及启动子区的胁迫诱导元件，研究ptc-miR213对其靶基因的负调控作用及它们在胡杨盐碱胁迫应答中的调控机制，为揭示胡杨高耐盐的分子机制提供理论依据，为林木高耐盐新品种培育提供有效手段和物质基础。

项目通过miRNA及其降解组测序，分析发现39个旱柳基因组中新的可能特异性响应盐胁迫的miRNA，其中miRNA160, miRNA171, miRNA172和miRNA399家族中的大部分成员表达上调。在186个差异表达的miRNA中，有29个miRNA参与到了与硫酸盐运输相关的进程中，其中有14个仅受盐胁迫后特异表达的。在37个所调控的靶基因中，有14个表达量在胁迫前后发生了变化。利用PCR技术分离了ptcmiR-213及其靶基因Cal B和EAP30，研究发现ptcmiR-213及其靶基因Cal B和EAP30在盐胁迫前后其表达量基本相反。构建了ptcmiR-213及其靶基因Cal B和EAP30的植物表达载体并转化拟南芥，对T3代转基因拟南芥进行盐胁迫实验发现拟南芥植株叶片中脂膜过氧化产物MDA含量升高，WT的MDA含量升高到胁迫前的2.8倍以上，而转基因植株MDA含量为胁迫前的1.1-1.9倍，NaCl胁迫后，相对电导率升高，WT相对电导率为胁迫前3倍，而转基因植株相对电导率为胁迫前1.2-2.3倍，表明转基因植株细胞膜受损害程度相对较低。NaCl 胁迫后，WT植株的总叶绿素含量下降了29%左右，转基因植株叶绿素含量受胁迫略有下降，但差异没有达到显著水平。转ptcmiR-213转基因拟南芥盐胁迫转录组测序发现上调表达的基因有参与脂质分解代谢的GDSL类脂肪酶（AT4G18790和AT1G29660）、与植物角质合成相关的脂质转运蛋白（AT3G22142）和磷酸酶GPAT8（AT4G00400）、与衰老调节相关的蛋白磷酸酶（AT5G02760）和生长素响应蛋白（AT4G32280）、胞质转化酶（AT4G09510）、参与果胶分解代谢的果胶裂解酶（AT3G07010）。