二系杂交小麦杂种优势相关microRNA分离与功能鉴定

Heterosis is a widespread phenomenon in plant and animal, but its genetic mechanism is not yet clear. MicroRNAs (miRNAs) are endogenous small RNA products of non-protein-coding genes and serve as sequence-specific, trans-acting regulators for controlling negatively eukaryotic gene expression at the post-transcription level. They have been predicted to regulate a variety of biological processes, such as plant morphogenesis, development and stress responses,and played important roles in gene expression and regulatory network. In this study, we will utilize genomics, high throughput sequencing, bioinformatic tools, and molecular techniques to study the involvement of miRNAs in heterosis of two-line hybrid wheat. Different organ types of wheat photo-thermo-sensitive genic male sterile line BS366, the restorer line TY 806, and their F1 hybrid will be sampled at the key period of growth and development in the field. The extracted low-molecular-weight RNA will be deeply sequenced by the Illumina High-seq2000 technology. Differentially expressed miRNAs among different wheat lines will be identified using our recently developed program miRDeep-P. Through the functional analysis of RNA in situ hybridization and transgenic wheat, we will aim to illustrate the function of key miRNAs and their target genes. Knowledge gained from the proposed research will be cirtical to understand the role of microRNA-mediated molecular events in the expression of heterosis and provide new insights into the molecular mechanisms of heterosis. It also will lay important theoretic foundation for further perfecting the two-line hybrid wheat system and broadening its application in China.

杂种优势是广泛存在的遗传学现象，但其遗传学机理尚未十分清楚。MicroRNA（miRNA）是近年来从真核生物发现的一类负调控因子，调节内源基因表达，广泛参与植物的生长发育与抗逆响应，在基因调控网络中扮演重要角色。本研究以小麦光温敏雄性不育系BS366、恢复系TY806及F1为实验材料，采集不同生长时期、不同组织部位的样品并提取RNA。通过应用High-Seq2000高通量测序技术和miRNADeep-P生物信息学方法，分析并分离特异组织的miRNA分子，进行靶基因验证，克隆全长miRNA基因。通过miRNA原位杂交、转基因功能验证，明确关键miRNA以及其靶基因与杂种优势的关系，初步构建小麦杂种优势分子调控网络模型，为揭示小麦杂种优势机理提供理论依据，并为促进我国原创性二系杂交小麦应用体系的进一步完善和杂交小麦的大面积应用奠定重要基础。

杂交小麦是世界性难题，其大面积应用将对保障我国粮食安全和种业安全具有重要意义。基于小麦光温敏核雄性不育种质的发现和应用创建的二系杂交小麦技术体系属我国原创性成果，但是关于二系小麦杂种优势分子机理研究仍然非常薄弱，已经成为限制二系杂交小麦进一步突破的技术瓶颈。本项目开展了二系杂交小麦杂种优势相关的miRNA特性、相关基因克隆等研究，完成了项目预期的任务指标，发表论文8篇，其中SCI论文5篇；申请专利2项。.本研究以小麦光温敏雄性不育系BS366、恢复系CP806及F1为实验材料，采集不同生长时期、不同组织部位的样品并提取RNA，采用高通量测序技术、生物信息学技术等分离和鉴定小麦杂种优势相关的miRNA，并对其功能进行研究，初步明确miR33、miR82、miR5384等参与杂种优势表达的组织特异性和表达模式，鉴定出产量和抗逆小麦杂种优势相关miRNA 14个，分离了其中5个miRNA及其靶基因，并进行了组织特异性和表达模式等研究；鉴定出bHLH、bZIP、C2H2、ERF等7个与花药细胞骨架发育相关的转录因子，构建了转录因子调控的花药细胞骨架发育的基因调控网络；克隆了4个相关基因，分别是TaSWEET6，TaPRF7，TaCu/Zn-SOD和TaAnnexin。本项目研究结果将对进一步揭示作物杂种优势现象，提高小麦杂种优势潜力和杂交小麦分子育种技术水平，促进我国二系杂交小麦应用技术体系的进一步完善和应用具有重要意义。