大麦盐胁迫相关miRNA的鉴定与功能分析

Soil salinity represents a growing threat to crop productivity. Barley is considered as one of the most notably salt tolerant crops. Clarify the molecular mechanism of salt tolerance of barely is the foundation for genetic improvement in salt resistance.MicroRNAs (miRNAs) are a class of negative regulators of gene expression in eukaryotic cells, which play an important role in affecting plants growth, development, signal transduction and also adaptation. In this study, we adopt high-throughput sequencing technology and specially designed microarray to analyze the miRNA profiles of barleys (salt-tolerant and salt-Sensitive) under normal conditions and under salt stress at genome-wide level. And then, the systemic identification and characterization of miRNA genes which involving salt stress response upon comparison of high-throughput sequencing with miRNA microarray profiling data. Northern blotting and real-time quantitative PCR was used to further validate the candidate miRNA molecules that respond to salt stress. The targets of those candidate miRNAs were verified by 5'RACE (Rapid Amplify cDNA Ends) of the spliced fragments combined with bioinformatics' prediction. The annotation of the targets will help to identify the role which miRNAs involve in the signal transduction pathway and mechanism of barley response to salt stress. Finally, the verified candidate miRNA will be transferred to Brachypodium to identify their biological function. This study emphasized on identification and characterization of more salt stress-response miNRAs from barley to clarify the regulation mechanism of miRNA in barley salt stress response, which could provide helpful information for revealing the molecular mechanisms of salt resistance and could be useful for genetic improvement to increase salt tolerance in barley.

盐害是限制作物产量提高的重要因素。大麦是最抗盐的作物之一，因此弄清大麦抗盐耐盐的分子机理是麦类作物抗盐性遗传改良的重要基础。MicroRNA (miRNA)是一类重要的真核细胞基因表达的负调控因子。本研究利用高通量测序技术和特制的miRNA芯片在全基因组水平研究不同抗盐性大麦在正常条件和盐胁迫下的miRNA表达谱，系统鉴定大麦响应盐胁迫的miRNA，然后采用Northern杂交结合实时定量PCR验证响应盐胁迫的候选miRNA分子，采用生物信息学手段结合5'RACE技术分析候选miRNA的靶基因和作用位点，初步明确miRNA在大麦盐胁迫响应方面的信号传导途径和作用机制，最后将验证后的候选miRNA基因转入模式植物中进一步鉴定其功能。本研究通过大麦盐胁迫相关miRNA的挖掘，阐明miRNA在大麦盐胁迫响应与抗性方面的调控机制，以期为揭示大麦抗盐分子机理奠定基础，为大麦抗盐耐盐育种提供基因资源。

MicroRNA是一类重要的真核细胞基因表达的负调控因子，其在植物感受逆境胁迫及适应性的过程中发挥重要作用。本研究以大麦为研究材料，取盐胁迫(150 mM)和对照处理后3h、8h和27h的样品，分别构建了6个小RNA文库并分别进行了高通量测序和降解组测序。根据miRNA的表达谱分析，鉴定出了134个miRNA家族，其中包含124个隶属于97个miRNA家族的保守miRNA和10个新鉴定的miRNA。我们发现与大麦盐胁迫响应过程相关的miRNA有97个，来自于73个miRNA家族。我们进一步采用生物信息学预测和降解组测序的数据，对这些候选miRNA的靶基因进行了分析，发现盐胁迫响应miRNA主要通过对基因渗透物质合成相关基因(如甜菜碱醛脱氢酶 BADH)、逆境相关特异表达蛋白(如热激蛋白Hsps)、过氧化物清除基因(如超氧化物歧化酶SOD，过氧化物酶)及一些胁迫相关转录因子SBP、NAC、bZIP等的调控，从而介导了大麦对盐胁迫的响应。本研究为大麦抗耐盐相关miRNA的鉴定和大麦抗盐研究提供了重要的基础数据，为揭示miRNA介导的大麦响应盐胁迫的分子机制奠定了基础。