甘蓝型油菜种皮色泽转录调控因子BnMYB47的功能分析

Brassica napus is widely grown across the world for the most vegetable oil or biofuel production. Compared with black-seeded phenotype, the yellow-seeded phenotype has decreased some toxic materials or antinutritives in the seeds and used as livestock feeding for high protein-reach after oil extraction. Using the composite interval mapping (CIM) method, a major QTL (BnSCA09) for seed coat colour has been identified in our lab. Further backcrossing and fine-mapping successfully delimited the BnSCA09 locus to a 60 kb region. Annotation reveald the BnMYB47 was in this region, belonging to the plant R2R3-MYB transcription factor, but the precise function and regulation mechanism still remains unclear. According to the results of cloning and transgenic evidence, we found that BnMYB47 involved in the diversification of seed coat colour in Brassica napus. In this project, transgenic materials of overexpression and RNAi will be used to reveal the function and molecular mechanism of BnMYB47, the major contents are as follows: 1) Phenotypic and cytological characterization of seed coat during development in transformed plants; 2) Determination of some physiological and biochemical for the content of flaconoids and the enzyme activities; 3) Subcellular localization of BnMYB47 and the molecular mechanism involving in the regulation of flavonoid biosynthesis; 4) Identifing the target genes of BnMYB47 transcription factor by RNA-seq and ChIP-seq, revealing the function and molecular mechanism. This project aimed to elucidate the role of BnMYB47 and understand the molecular mechanism about the formation of yellow-seeded B. napus. It will provide a substantial foundation for breeding the yellow-seeded B. napus.

甘蓝型黄籽油菜菜籽品质和含油量较高，榨取后饼粕饲用价值进一步提高。项目组已通过对色泽主效QTL（BnSCA09）的精细定位将QTL区间缩小为约60kb，通过全区间基因克隆和转基因结果分析，发现BnMYB47调控影响了油菜种皮色泽的变化，该基因属R2R3-MYB转录因子，但具体功能和分子机制尚不明确。因此，本项目拟以超表达和RNAi干扰的转基因后代为对象，主要工作包括：1）转基因植株表型鉴定与种皮发育的细胞学分析；2）种皮发育过程中类黄酮组分与关键酶活性等生理生化指标测定；3）BnMYB47亚细胞定位及参与类黄酮代谢途径的调控作用机制；4）采用RNA-seq和ChIP-seq等技术手段，获取BnMYB47的下游靶基因调控作用位点，揭示其功能与分子调控机制，进一步确定BnMYB47在甘蓝型黄籽形成中的作用，将有助于提高对甘蓝型黄籽性状形成的分子遗传机制的认识，为甘蓝型黄籽油菜育种实践奠定基础。

甘蓝型黄籽油菜菜籽品质和含油量较高，榨取后饼粕饲用价值进一步提高。前期研究结果表明BnMYB47可能参与调控影响了油菜种皮色泽的变化，但具体功能和分子机制尚不明确。因此，本项目主要工作包括：1）转基因植株表型鉴定与种皮发育的细胞学分析；2）种皮发育过程中类黄酮组分与关键酶活性等生理生化指标测定；3）鉴定BnMYB47生物学功能，进一步确定BnMYB47在甘蓝型黄籽形成中的作用。通过本项目的顺利实施，获得了稳定遗传的黄籽株系，与对照相比，RNAi株系转色延迟，含油量在RNAi株系中升高5%，在超表达株系中降低2.5%。石蜡切片分析结果表明，在RNAi转基因株系中色素和多酚类化合物也显著降低。利用LC-MS方法共检测到27种主要的类黄酮组份，主要包括表儿茶素、山奈酚、异鼠李素和花青素苷及其衍生物等次生代谢产物。其中8种物质存在显著差异，主要包括Procyanidin B2[DP2], Epicatechin, Km-3-O-glucoside-7-O-glucoside, [DP3], [DP3]-2, Is-O-glucoside-sulfate, [DP2]-2, Quercetin-3-O-glucoside，说明BnMYB47可能参与了这些代谢产物的合成。同时，我们测定了可溶性与不溶性原花色素，其结果表明不溶性原花色素含量可能是引起种皮颜色差异的主要原因。同时，在抑制BnMYB47表达后，还显著降低了PAL、PPO和POD活性，而超表达后，三种酶活性均明显升高。通过RNA-seq和qRT-PCR分析发现在抑制BnMYB47基因表达后，苯丙烷与类黄酮途径的关键基因PAL、TT4、TT6、TT7、TT3和TT18等关键基因在种皮发育中后期也显著受到抑制表达，说明基因BnMYB47直接或间接参与调控了甘蓝型油菜苯丙烷与类黄酮途径的代谢。因此，本项目的深入研究将有助于提高对甘蓝型黄籽性状形成的分子遗传机制的认识，为甘蓝型黄籽油菜育种实践奠定基础。通过本项目研究，共发表论文11篇，（其中SCI收录7篇），申请专利1项，在国际会议中做报告2次；培养研究生2名。