盐胁迫下白榆种子萌发成苗过程中MicroRNA调控机制研究

Ulmus pumila，which has strong salt tolerance，is an ecologically and economically important species in saline-alkali land. Whereas, the recent survey found that its natural regeneration in saline-alkali land was difficult with very few seedlings. Previous studies have shown that the inhibiting effect of salt stress on Ulmus pumila seed germination and seedling establishment was a limiting factor of its natural regeneration in saline-alkali land. MicroRNA (miRNA) played a crucial regulatory role in seed germination and seedling establishment under salt stress, however, research on miRNA of Ulmus pumila has not been found in literature. We have already completed the study on influence of salt stress on Ulmus pumila seed germination and seedling establishment, which revealed its growth characteristics and we aslo got its salt-tolerant threshold. Moreover, we obtained its transcriptome by high-throughput sequencing. This project aims to conduct further research on physiological and biochemical characteristics in the process of Ulmus pumila seed germination and seedling establishment under salt stress, then build sequence library and create the expression profile of miRNA and their target genes in Ulmus pumila by utilizing the method of small RNA sequencing, bioinformatics analysis, and experimental verification (qPCR, Northern Blotting and 5′RACE). In addition, the study will screen out key salt-resistant miRNAs and then validate their function. Thus we can get insight into miRNA regulation mechanism during seed germination and seedling establishment of Ulmus pumila under salt stress, in order to providing basis for solving its difficult natural regeneration problem in saline-alkali land.

白榆（Ulmus pumila）耐盐性强，是盐碱地上的重要生态经济树种，近年发现盐碱地白榆天然更新困难，幼苗极少。研究表明盐胁迫抑制白榆种子萌发成苗是盐碱地白榆天然更新的限制因子，而MicroRNA在种子萌发成苗适应盐胁迫中发挥重要调控作用，但白榆miRNA研究尚未见报道。目前，申请人已完成盐胁迫对白榆种子萌发成苗生长影响的研究，揭示了其生长特性，确定了耐盐阈值，并测序获得了白榆转录组。本项目拟在进一步研究盐胁迫下白榆种子萌发成苗过程中生理生化特性的基础上，利用small RNA测序结合生物信息学分析及qPCR、Northern Blotting和5′RACE等实验方法，构建盐胁迫下白榆种子萌发成苗过程中miRNAs及其靶基因的序列数据库和表达谱，筛选耐盐关键miRNAs，进行功能验证分析，以揭示盐胁迫下白榆种子萌发成苗过程中miRNA调控机制，为解决盐碱地白榆天然更新困难奠定理论基础。

白榆（Ulmus pumila）耐盐性强，是盐碱地上的重要生态经济树种，近年发现盐碱地白榆天然更新困难，幼苗极少。研究表明盐胁迫抑制白榆种子萌发成苗是盐碱地白榆天然更新的限制因子，而MicroRNA（miRNA）在种子萌发成苗适应盐胁迫中发挥重要调控作用，但盐胁迫下白榆萌发期miRNA研究尚未见报道。本项目完成了盐碱胁迫对35个白榆优良家系种子萌发影响的研究及萌发期耐盐碱性评价，筛选出中高耐盐碱家系8个，不耐盐碱家系3个；完成了盐胁迫下白榆种子萌发成苗过程中miRNAs鉴定与表达谱构建，筛选出64个关键耐盐miRNAs，其中盐胁迫下30个miRNAs（具有负调控关系的靶基因50个）下调表达，34个miRNAs（具有负调控关系的靶基因63个）上调表达，并对测序数据进行了qRT-PCR验证。红砂（Reaumuria soongorica）是典型的木本盐生植物，在盐碱荒漠区广泛分布，能在盐碱逆境下渡过萌发期。因此，项目以红砂作为白榆种子萌发期研究的对比物种，在原来的基础上增加了盐胁迫下红砂种子萌发成苗过程中生长生理特性研究、miRNAs鉴定与表达谱构建和关键耐盐miRNAs筛选等工作。通过对白榆（非盐生植物）与红砂（盐生植物）萌发期适应盐胁迫机制的比较，发现白榆萌发期主要通过改变miRNAs表达量调控与生长和胁迫响应相关的功能基因以适应盐胁迫，但能适应的盐浓度较低，当盐浓度较高时，种子多能萌发，但其萌发后生长缓慢不能成活；红砂萌发期可能主要通过改变miRNAs表达量，调控GA、生长素、ABA形成途径，进而调节种子萌发能力适应盐胁迫，高盐下降低生长素和赤霉素水平，提高ABA浓度，抑制种子萌发保持存活，并在低盐下萌发，以提高萌发成活率。相关研究结果可为木本非盐生植物（白榆）种子萌发期耐盐定向分子育种和新品种培育提供新的基因资源和理论依据。最后，受项目研究工作的启发，本项目组发明了一种用于盐碱逆境的种子萌发装置和方法，可为攻克盐碱地植物种子萌发成苗受阻难题，实现盐碱地低成本规模化的生物修复与治理提供切实有效的技术支持。