拟南芥AGO3及其结合小RNA调控根系盐胁迫响应的机理研究

Salt Stress is one of the major abiotic stresses and becomes a growing threaten to the agricultural production worldwide. It is pivotal to unravel the mechanism of the response to salt stress in root for engineering more salt-tolerant crop and improving the crop yield. Recent evidence suggests that epigenetic mechanism, such as RNA-directed DNA methylation (RdDM), also plays a significant role in gene regulation in response to the environmental stresses. In Arabidopsis argonaute (AtAGO) family, AGO4 clade proteins are well known as its key role in RdDM process. We previously identified that AGO3, which is the least characterized member in AtAGO family, predominantly binds 24-nucleotide (nt) small RNAs, and its expression is specifically and highly induced under salt stress. Further results indicate that AGO3, similar to AGO4, is a dark-horse component in the epigenetic pathway， although AGO3 does not belong to AGO4 clade. In this proposal, we are going to investigate the detailed molecular mechanism of AGO3 and its associated 24-nt small RNAs in response to salt stress by the methods of genetics, bioinformatics, molecular biology and biochemistry. Our study will provide new insight into the mechanism of epigenetic regulation in response to salt stress and be beneficial to the application in the genetic modification of salt-tolerant crop.

研究根系在盐胁迫过程中的响应机理，对改良农作物耐盐性状、维持和提高盐碱地作物产量有重要的理论和应用意义。RNA介导的DNA甲基化（RdDM）相关的表观遗传调控在植物进行环境胁迫响应过程中发挥着重要作用。拟南芥Argonaute（AGO）家族中AGO4亚支蛋白是RdDM效应复合物的核心元件。我们前期研究发现目前AGO家族了解最少的AGO3，尽管不属于AGO4亚支，却在功能上与AGO4相似，通过结合24nt小RNA参与植物DNA甲基化调控。AGO3在根部特异性受到盐胁迫诱导表达，因此推测AGO3参与根系盐胁迫响应中的表观遗传调控。本项目拟运用遗传学、生物信息学、分子生物学以及生物化学等方法研究AGO3及其结合24nt小RNA在植物根系盐胁迫响应过程中的分子机理，探明AGO3及其结合小RNA的下游靶位点和调控机理，最终为耐盐农作物遗传改良提供理论依据。

研究根系在盐胁迫过程中的响应机理，对改良农作物耐盐性状、维持和提高盐碱地作物产量有重要的理论和应用意义。RNA介导的DNA甲基化（RdDM）相关的表观遗传调控在植物进行环境胁迫响应过程中发挥着重要作用。我们前期研究发现目前AGO家族了解最少的AGO3可以通过结合24nt小RNA参与植物DNA甲基化调控。AGO3在根部特异性受到盐胁迫诱导表达，因此推测AGO3参与根系盐胁迫响应中的表观遗传调控。本项目运用转录组、甲基化组和小RNA测序相结合研究AGO3突变体及野生型在植物根系盐胁迫响应过程中的分子机理。转录组数据显示拟南芥野生型在高盐胁迫处理前后的差异表达基因与ago3突变体在相应条件下的差异表达基因（DEG）存在明显差异，其中种子萌发调节、脂肪酸代谢、脱水反应、离子转运等过程相关基因在野生型盐处理过程中特异性上调，而糖类、氧化还原水平、茉莉酸、乙烯的反应以及光合系统捕光复合体等过程相关基因则特异性下调，而这些基因在ago3盐处理条件下变化不显著，可能受到了AGO3的特异性调控。甲基化组测序显示大部分DEGs表达模式与相应DNA甲基化差异区域未见明显关联关系，少数基因表达差异与其启动子区的DNA甲基化差异有关。小RNA测序显示，野生型和ago3突变体中小RNA在DNA/MuDR、LTR/Gypsy和RC/Helitron等不同类型TE中的分布存在差异，推测AGO3可能通过结合的小RNA影响不同类型转座子的转录活性进而影响下游编码基因的表达。这对于深入解析AGO3及小RNA在植物高盐胁迫响应过程中的影响有着重要意义。