含有WD-40重复基元的蛋白在拟南芥miRNA通路中机理的研究

microRNAs (miRNAs) are a class of 20-24 nucleotide (nt) non-coding RNAs, which negatively regulate gene expression through cleaving their target RNA or repressing translation. We have chosen a genetic screening for Arabidopsis miRNA biogenesis pathway mutants, utilizing vein beaching phenotype caused by phloem specific expression of an artificial miRNA (amiR-SUL) targeting CH42 gene as a visible selection marker, and isolated a suppressive mutant named ab57. It shows that the mutation gene encodes a WD-40 repeat protein (At5g64730) by genome re-sequencing and experimental validation. Until now, the function of the protein (At5g64730) is unknown. The WD-40 proteins are a class of proteins that contain WD-40 repeat domains and are evolutionarily conserved in eukaryotes. Both exogenous amiR-SUL and the endogenous miRNAs levels are reduced in ab57 seedlings, along with the decrease of their primary transcripts. All the available data show that this new protein may affect the miRNA pathway. We will comprehensively focus on the biological function of the new protein in miRNA biogenesis by genetic and molecular biological methods as well as bioinformatics methods. This will provide theoretical basis and novel strategies for the class of WD-40 proteins in future.

miRNA是一类长度在20-24nt的非编码RNA，通过对其靶基因进行特异切割或者抑制蛋白翻译进行负调控。我们采用一个正向遗传筛选系统筛来寻找miRNA通路的突变体。该系统通过构建人工miRNA特异降解叶绿素合成基因SUL，并在叶片韧皮部伴胞细胞特异启动子SUC2驱动下使转基因植物呈现叶斑。通过叶脉叶斑大小作为筛选标记，我们筛选到一个参与miRNA通路的突变体（ab57），在一个编码含有WD-40 重复基元的蛋白发生点突变。该类蛋白是一类包含WD40重复基元的蛋白。和对照相比该突变体叶脉叶斑明显减小，且内源miRNA和外源的amiR-SUL水平都显著下降。此外pri-miRNAs水平在突变体里面也显著下降。本项目将从采用遗传学和分子生物学手段，并结合生物信息学手段系统的研究该蛋白如何影响miRNA的通路，为具有WD-40重复原件的这类蛋白在miRNA通路的作用提供理论依据和实验基础。