小麦籽粒胚乳发育相关miRNA的鉴定及其调控机制研究

The development of endosperm directly affects the grain yield and processing quality of wheat. In our previous research, the expression patterns of miRNAs were profiled in developing wheat grains and 32 candidates were associated with the developmental processes of wheat grains. Based on the previous research, this project will systematically analyze the spatial and temporal expression patterns of these candidate miRNAs, especially the expression characters in developing endosperm and embryo tissues, in 4 wheat cultivars. According to the expression patterns, candidate miRNAs associated with the endosperm development will be identified. The regulation effects of endosperm development associated miRNAs will be validated via BSMV based miRNA over-expression or down-regulation techniques and those focused miRNA candidates will be used in further study. By using the iTRAQ (isobaric tags for relative and absolute quantitation) and the tandem mass spectrometry analysis, those candidate proteins involved in miRNA-mediated regulation during grain development will be isolated, their interaction characteristics and regulatory roles during the endosperm development will also be confirmed via BSMV based VIGS methods in wheat. Finally, the miRNA-dependent regulatory effects and gene networks during the development of endosperm will be comprehensively analyzed, which will provide important theoretic basis for further molecular breeding focused on high yield in wheat.

小麦籽粒胚乳的发育关系到小麦的产量和品质。课题组前期研究中，分析了小麦籽粒发育过程中的miRNA表达谱，筛选到32个籽粒发育相关的miRNA。在此基础上，本项目拟系统分析这些miRNA在4个小麦品种中的时空表达特性，依据它们在籽粒发育过程（特别是胚乳和胚组织）中的表达模式，确定胚乳发育相关候选miRNA；利用基于BSMV的miRNA过表达和抑制表达技术，鉴定候选miRNA对籽粒胚乳发育的调控功能，确定参与胚乳发育调控的重点miRNA；利用iTRAQ和串联质谱鉴定技术，鉴定小麦籽粒中受重点miRNA调控的候选蛋白，并利用BSMV-VIGS对候选蛋白编码基因的功能进一步验证；综合上述结果，确定miRNA对小麦籽粒胚乳发育的调控作用及基因网络，为小麦高产分子育种提供理论依据。

小麦籽粒（特别是胚乳）的发育调控直接影响小麦的产量和品质，miRNA是一类在植物生长发育调控过程中发挥重要功能的小分子非编码RNA，探讨小麦籽粒发育过程中依赖于miRNA的分子调控机制具有重要意义。在前期研究基础上，系统分析了候选miRNA在籽粒发育过程中的时空表达特性，筛选出与籽粒发育调控密切相关的重点miRNA。建立并优化了基于大麦花叶病毒（BSMV）的小麦miRNA短串联模拟靶标（STTM）瞬时抑制表达技术体系，并开展了重点miRNA在籽粒发育调控过程中的功能研究，初步确定了部分miRNA对小麦籽粒发育的调控作用。结果显示，小麦miR397a和Ta-miR154-5p对籽粒发育具有正调控作用，采用STTM法将miRNA抑制表达后千粒重降低了22-23%；Ta-miR167e对籽粒发育具有负调控作用，该miRNA抑制表达后千粒重提高了约10%。系统分析了重点miRNA调控的靶基因及籽粒发育过程中差异表达的蛋白，鉴定出一批参与籽粒发育调控的重点候选蛋白，通过对靶基因和差异表达蛋白谱的关联分析确定了miRNA调控的重点靶基因，并进行了初步的功能验证分析。对Ta-miR154-5p的候选靶基因TaNRX1的功能分析显示，该基因抑制表达后小麦粒长和粒宽显著增加，千粒重提高了5-15%。为了进一步明确候选miRNA及其靶基因在籽粒发育过程中的调控机制，开展了基于遗传转化的miRNA和靶基因过表达（或抑制表达）功能鉴定研究。研究结果为阐明依赖于miRNA的籽粒发育分子调控机制提供了参考信息，对于开展小麦高产分子育种具有重要意义。