甘蓝型油菜种皮色素合成相关miRNA的鉴定及机理研究

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.

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。

黄籽甘蓝型油菜较相同遗传背景的褐籽油菜具有明显的品质优势，因而成为油菜育种关注的性状之一。该性状的形成与类黄酮等次生代谢物的含量变化有关，目前关于植物色素合成机制的研究主要集中于类黄酮合成代谢路径，其中对MYB-bHLH-WD40（MBW）转录复合体的研究报道最多。而关于miRNA调控油菜种皮色素积累的研究尚未见报道。本研究利用sRNA-seq对黄/褐籽甘蓝型油菜不同发育时期种子（3-6 WAF和成熟期）的miRNA表达水平进行比较，发现大量材料特异和时期特异的miRNA。这些差异miRNA包括miR156、miR168、miR171、miR172等，且对差异miRNA的靶基因分析发现候选靶基因主要为一些转录因子，如mi156可调控转录因子SPL、miR172可调控转录因子AP2的表达。差异表达miRNA及其靶基因的qPCR验证结果与sRNA-seq结果一致。结合油菜降解组测序的现有数据，我们对miR156及其靶基因SPL3/9/10、miR172及其靶基因AP2、TOE1/2进行了功能验证，经PCR阳性鉴定和qPCR表达量鉴定后，获得preMIR156a/b/d和preMIR172a/b/d过表达植株、miR156和miR172的target mimicry植株，以及SPL3、SPL10、AP2、TOE1、TOE2的过表达植株。miR156、miR172均可观察到明显的发育相关的表型变化（如分枝数增加），且Pro35S:preMIR172b可观察到种皮色泽变异。亚细胞定位分析表明SPL3/9/10、AP2、TOE1/2均为核定位。通过酵母双杂交筛库发现SPL3可与MYB4、WD40、AP2互作，TOE2可与PAP18、NAC、DFR、SEP3、TPR互作。靶基因与上述互作蛋白及预测互作蛋白的点对点验证、BiFC等尚在重复中，现有初步结果表明TOE2与DFR确有互作。后续拟对夏繁获得的转基因纯系进行表型鉴定、类黄酮物质的含量分析、类黄酮合成相关基因的表达量分析，结合上述靶基因互作蛋白的鉴定，了解miRNA及其靶基因影响种皮色素的调控路径。本研究同时基于黄/褐籽甘蓝型油菜不同发育时期种子RNA-seq结果，对候选差异表达mRNA进行了功能验证，其中BnTTG1的RNAi株系获得花色变异的表型，且其与BnSPL3、BnTTG2、BnTT8、BnTT2存在明显的互作。