3个小麦种子特异表达的全新microRNA的功能研究

Wheat seed development is the most important base of its yield and quality, uncovering the regulatory molecular mechanism of wheat seed growth and development will provide scientific theory basis for wheat quality and high yield breeding. MicroRNAs (miRNAs) are a class of small RNAs that are the products of endogenous non-protein-coding genes and serve as sequence-specific trans-acting regulators for controlling eukaryotic gene expression at the post-transcription levels. They have been found to regulate a various biological process, such as plant growth, development, differential, and stress responses, and play key roles in gene expression and regulatory network. Recently, we identified some seed-specific novel miRNAs, however, the biological functions of these miRNAs are unknown. In this research project, we will focus on functional identification of three wheat seed-specific novel miRNAs, tae-miR2026, tae-miR39 and tae-miR15, based on our previous study. RNA degradoms from wheat seedlings, flag leaves, and developing seeds will be deeply sequenced by high-throughtput, to determine the candidate miRNA target mRNAs. The pre-miRNA sequences and the miRNA target genes will be cloned, and the biological functions of these three miRNAs and their target gens will be identified by two approaches, i.e. VIGS of wheat seed-specific gene expression and wheat transgenic overexpression. The main purpose of this study is to unveil the functions of the 3 miRNAs and their regulatory network in wheat seed growth and development process. We also hope to fund miRNA or miRNA target having potential application in quality and high-yield wheat breeding.

籽粒生长发育是小麦产量和品质的重要基础，揭示其分子调控机制可为小麦优质高产育种提供重要理论依据。miRNA是近年来发现的一类调控因子，调节内源基因表达，广泛参与植物生长发育，在基因调控网络中起关键调控作用。最近,我们在小偃6号小麦中鉴定出籽粒特异表达miRNA，它们如何调控籽粒的生长发育及品质性状形成尚不清楚。在前期研究基础上，本项目重点研究3个小麦籽粒特异表达新miRNA(tae-miR2026, tae-miR39和tae-miR15)的功能。拟对幼苗、旗叶和不同发育时期籽粒的RNA降解组进行高通量测序，确定候选miRNA的靶基因；分离克隆miRNA及其靶基因序列；利用病毒诱导小麦籽粒内源基因沉默和转基因过表达2种策略进行候选miRNA及其靶基因的功能鉴定。旨在揭示候选miRNA对籽粒生长发育的调控作用及调控方式，并希望能鉴定出在小麦优质高产育种上具有应用价值的miRNA或靶标基因。

研究揭示小麦种子生长发育的调控机制可为优质高产小麦育种提供理论依据。项目旨在研究揭示了小麦种子特异表达的3个新miRNA（tae-miR9655，tae-miR9677和tae-miR9658）和种子高表达的2个已知miRNA（miR164和mi319）的在小麦中调控作用。小麦种子特异表达的3个新目标miRNA和种子高水平表达的2个已知miRNA的调控表基因预测和实验验证，结果表明tae-miR9655的调控靶基因是TaRZ1A是一种RNA结合蛋白； tae-miR9677 的靶基因是在小麦基因组中被注释为长链非编码RNA lncRNA(Tm77)； tae-miR9658的靶基因是木糖转移酶基因TaXYLT； miR164的调控靶基因是TaNAC14； miR319调控靶基因是TaMYB34。克隆到5个miRN的前体序列及其各自的靶基因全长cDNA序列。用小麦不同器官/发育时期样品RNA-seq和qRT-PCR方法分析揭示了这些miRNA及其各自调控靶基因在小麦中的时空表达模式。研究建立了小麦内源靶标miRNA过表达和沉默的技术体系（研究成果已申请国家发明专利，专利申请号201710046709.9）。利用该技术快速鉴定了2个已知miRNA（miR164和miR319）及其靶基因TaNAC14和TaMYB34，和2个新miRNA（tae-miR9655 和tae-miR9677）和其调控靶基因TaRZ1A 和lncRNA(Tm77)的功能；结果发现在小麦发育期的tae-miR319正调控种子大小，其靶基因TaMYB34 负调控种子大小。miR164通过调控靶基因TaNAC4表达水平对籽粒中淀粉形成和籽粒饱满程度有一定影响。开展miRNA（tae-miR9655，tae-miR9658，tae-miR9677，tae-miR164和tae-miR319）及其各自靶基因转基因过表达小麦研究，已经获得了目标基因基因稳定表达的T2代转基因株系。研究结果发现，与受体小麦品种相比，tae-miR164靶基因TaNAC14的转基因T2过表达株系（OE—TaNAC14）的种子粒重和收获系数显著增大；表明tae-miR164通过控调TaNAC14表达水平调控种子粒重和产量；其在小麦优质高产育种上可能具有潜在的应用价值。其它转基因株系小麦的农艺性状和产量性状测定正在进行中。