甘蓝型油菜种皮色素合成相关miRNA的鉴定及机理研究

黄籽甘蓝型油菜与褐籽相比具有含油量高、色素少、饼粕蛋白质含量高、纤维素和木质素含量低等优点，已成为当前油菜育种的重要目标之一。目前关于油菜黄籽性状形成机理的研究主要集中在类黄酮合成路径相关的基因，油菜种皮色素形成和积累的转录水平调控机制仍不清楚，miRNA是如何调控油菜种皮色素的积累还未见报道。本项目以细胞工程创制的黄籽株系W82和褐籽亲本为材料，通过高通量测序挖掘调控油菜种皮色素合成相关基因/转录因子的miRNA，通过qRT-PCR及Northern blot对黄/褐籽油菜种子发育过程中的差异表达miRNA进行验证；借助生物信息学预测差异表达miRNA的靶基因，通过RACE克隆分析其对靶基因的切割特性；分别构建 miRNA和候选靶基因的过表达载体，分析转基因株系种子表型和种皮色素成分含量的变化，探讨miRNA调控油菜种皮色素形成的机制，为阐明油菜黄籽性状形成的分子机理提供理论依据。

Yellow-seeded Brassica napus possesses several quality advantages over its black-seeded counterpart, including higher oil content, less seed coat pigment, and higher protein content, lower fiber and lignin content in seed meal. Yellow-seeded rapeseed has been elevated as one of the important aims of rapeseed breeding. Hitherto, molecular analysis of yellow seed character in B. napus is mostly focused on genes participated in flavonoid biosynthesis pathway. Transcriptional regulation mechanisms of pigment synthesis and accumulation in seed coat are in need to be uncovered, and papers on miRNA regulating seed coat pigmentation of B. napus have never been reported yet. In this project, using the yellow seed line W82, which was created using cell engineering technique, and brown-seeded parent as material, we aim to figure out miRNAs regulating genes or transcription factors functioned in seed coat pigmentation, applying high-throughput sequencing. Expression of differential miRNAs between developing seeds of yellow and brown-seeded B. napus will be verified by qRT-PCR and Northern blot. The target genes of differential expressed miRNAs will be predicted using bioinformatics analysis, and the splicing sites of miRNAs on candidate genes will be analyzed using RACE cloning. Based on the construction of over-expressing vectors, rapeseed lines over-expressing miRNA and candidate target genes will be created respectively to further into their molecular functions in regulating seed coat pigmentation, supporting with the phenotypic analysis and variation of seed coat pigments in transgenic rapeseed lines. This work will facilitate the dissection of molecular mechanisms on yellow seed character of B. napus.