油菜显性黄籽基因的克隆及其功能研究

Seed-coat color is one of the most important quality traits in Brassica napus. Yellow seeds have several advantages over blasck seeds with the same genetic background such as thinner seed-coat, higher oil content, higher protein and lower fiber content. Seed-coat color in B. napus is controlled by multiple genes and its genetic basis is very complex. Black seed-coat is generally dominant over yellow seed-coat. Only when all these loci are recessive homozygous, the seed-coat will be in yellow color. Thus, development of high yielding, high quality and yellow-seeded rapeseed is difficult. No2127-17 is an artificially syntheszed pure yellow-seeded inbred line. Its yellow-seeded trait is controlled by a single semi-dominant gene, which is obviously different from the genetic models of all yellow seed-coat color in B. napus.In previous studies, we fine mapped the yellow seed-coat gene and constructed a BAC library of No2127-17, and identified two BAC clones covering the genomic region of the yellow seed-coat gene.In this study, we are planning to sequence the BAC clones and identify the gene by comparing the genomic sequences from the BAC, B. carinata, B. oleracea and B. napus, and to characterize its function through genetic transformation, gene expression analysis, RNA sequencing, seed-coat pigment staining and metobolites analysis. The goals are to reveal its roles in the biosynthesis of seed-coat pigment and its regulation, and finally reveal the molecular mechanisms of the dominant yellow-seeded trait. We expect that the results of this study could faciliate yellow-seeded rapeseed breeding.

油菜籽粒种皮颜色是重要的品质性状。黄籽具有种皮薄、油分和蛋白质含量高、色素和纤维素含量低等优点。油菜的种皮颜色受多基因控制，黑籽一般为显性，多个位点都为隐性纯合时才表现为黄籽，所以选育高产优质的黄籽甘蓝型油菜品种非常困难。人工合成的甘蓝型油菜品系No2127-17的籽粒纯黄，其黄籽性状受一个不完全显性基因控制，这种遗传模式明显不同于油菜中的其它黄籽材料。前期的研究中，我们完成了显性黄籽基因的精细定位，构建了No2127-17的BAC文库，并筛选到覆盖黄籽基因区段的BAC克隆。本研究中，我们拟对BAC克隆进行测序，通过与埃塞俄比亚芥、甘蓝和甘蓝型油菜基因组序列相比较，鉴定黄籽候选基因，进而通过转基因、基因表达分析、转录组分析、种皮色素染色和不同发育时期种皮色素成分分析，确定基因的功能及组织表达特异性，阐明该基因控制的代谢途径及其调控规律，揭示显性黄籽性状形成的分子机理，为黄籽育种提供服务。

在前期研究中，我们利用黄籽品种No.2127和黑籽品种中油821构建的分离群体将黄籽基因定位在Ys37和Ys45之间共58 kb的染色体区段。本项目中，我们利用紧密连锁的分子标记筛选到16个BAC克隆，构建了覆盖黄籽基因区段的BAC重叠群。根据BAC测序及与参考基因组Darmor比对结果，我们发现筛选到的BAC克隆位于A9染色体，而不是以前定位的C8染色体，表明人工合成的黄籽品种No.2127的A9和C8染色体之间发生了部分同源染色体交换事件（HE）。为验证此染色体结构变异，我们对包括黄籽亲本No.2127在内的8个品种进行了全基因组de-novo测序和组装，获得了8个高质量参考基因组。全基因组范围内鉴定到大量变异，并在目标区段鉴定出由HE事件导致的基因组结构变异。结合基因组注释、转录组分析及RT-PCR验证，鉴定到2个候选基因TFXF和ASK11,分别编码物质的跨膜运输相关蛋白和泛素化降解相关蛋白。转录组分析及RT-PCR分析发现，TFXF和ASK11的表达量在黄籽和黑籽材料之间存在显著差异，另外类黄酮合成过程的多个基因在黄、黑籽品种之间也存在显著差异。分别构建了TFXF和ASK11的超表达载体和基因编辑载体，分别转化黄籽和黑籽品种，都已获得相应的转基因阳性植株，等待鉴定转基因后代植株基因型和观察表型。本项目的研究结果为最终克隆显性黄籽基因，揭示黄籽形成的分子机理及品种选育奠定了良好基础。组装的8个油菜品种高质量参考基因组序列为油菜基因组研究、重要农艺性状基因定位和克隆、油菜基因组育种提供了重要资源。