Hvu-miR6194 调控大麦抗盐性的分子机制研究

Soil salinity represents a growing threat to crop productivity.MicroRNAs (miRNAs) are a class of negative regulators of gene expression in eukaryotic cells, which play an important role in plants growth, development, signal transduction and adaptation. Barley is one of the most notably salt tolerant crops. Clarify the molecular mechanism of salt tolerance of barely is the foundation for salt resistance improvement in crops. Our previous study has found that Hvu-miRNA6194 was one of barley-specfic miRNA involving in response to salt stress. In this study, we firstly ascertained its extract target based on bioinformatic prediction and splicer 5’RACE methods. And then, northern blotting and real-time quantitative PCR were used to study the spatial and temporal expression patterns of Hvu-miRNA6194. The promoter and upstream cis-elements of its precursor, together with the genetic variation of target were futher analyzed to reveal the regulationary effect of Hvu-miRNA6194. Futhermore, the VIGS and CRISPR/Cas9 vector of Hvu-miR6194 precursor were constructed and transformed into barley, to validate its biological function through gene silencing and targeted gene editing. Our study will provide important informaton for revealing the on the molecular mechanism of salt tolerance in barley, and also shed light on making use of small RNA to improve the salt tolerance in barley and beyond.

盐害是限制作物生长及产量提高的重要因素。MicroRNA (miRNA)是一类重要的基因表达调控因子，在植物逆境响应和抗性方面起着重要作用。大麦是最抗盐的作物之一，弄清miRNA在大麦盐胁迫响应中的作用机制对揭示大麦乃至其他麦类作物的耐盐机理具有重要意义。本研究在前期研究基础上，以盐胁迫下特异表达的大麦Hvu-miR6194为研究对象，首先通过生物信息学预测结合剪切产物的5’RACE鉴定获得其准确的靶位点，然后采用Northern杂交和荧光定量方法明确其时空表达特性，结合前体启动子、顺式作用元件及靶基因的遗传变异等分析，初步阐明其产生、表达及调控途径；进一步利用VIGS和CRISPR/Cas9基因打靶技术，深入研究并揭示其生物学功能，以期阐明Hvu-miR6194参与大麦盐胁迫响应与抗性的作用机理，揭示大麦耐盐的分子机制，为利用小RNA改良大麦乃至其他麦类作物的抗盐性提供新的思路。

本研究以大麦特异的盐胁迫响应关键miRNA Hvu-miR6194为对象，对其参与调控大麦盐胁迫响应的分子机制进行了初步研究。首先，根据参考基因组序列设计引物，从大麦品种Morex的克隆的得到了Hvu-miR6194的前体序列，并对前体上游2kb的序列也进行了克隆，并其顺式元件进行了分析发现其中含有较多的逆境响应相关顺式元件；结合生信预测、降解组测序以及接头PCR验证，发现Hvu-miR6194作用的靶基因是锌指蛋白基因HORVU3Hr1G021400；然后对Hvu-miR6194及其靶基因在不同品种、不同时间节点、不同组织中的表达特性进行了分析，发现Hvu-miR6194在耐盐品系中高表达，而在盐敏感品系显著低表达；进一步，利用扩增子测序，对Hvu-miR6194前体及其靶基因在148份不同大麦材料的遗传多样性进行了初步分析，发现前体序列比较保守，而靶基因序列差异较大；最后，综合利用VIGS和过表达技术，获得了沉默Hvu-miR6194的大麦株系以及过表达Hvu-miR6194的转基因拟南芥株系，并对转基因后代的抗盐性进行了初步分析，初步解析了Hvu-miR6194的生物学功能。