油菜黄籽花粉直感基因的克隆与核不育表型标签的构建

Seed coat colour is determined by the type and content of pigment deposited in the seed coat cells. It is related with many important agronomic traits of seeds, such as seed dormancy, longevity, oil content, protein content and fibre content. Conventional genetic research shows that genetic control of seed coat colour is composed of maternal effect and pollen effect (xenia effect). Because phenotype of genes with pollen effects are exhibited one growing season ealier than the phenotype of genes with maternal effects, this kind of genes have specific useness in the breeding. For example, we can link the yellow seed coat gene with pollen effect together with male sertrile genes to construct yellow seed coat indicated male sterile lines for hybrid producing. .In this research, we isolated a mutation of yellow seeded Brassica napus line controlled by single mendel locus with pollen effect. Using the Brassica 60K Infinium BeadChip SNP genotyping array, we mapped the yellow seed coat gene on C4 genetic linkage map. The linked makers flank the gene within about 3.2M physical region.Using RNAseq technology, we accquired 50K unigenes transcripted in the seeds and 53 genes differently transcripted between brown wild type and yellow mutation. Only one of the 53 genes located on the 3.2M gene-mapped region. Another 81 transcripted genes accquired by RNAseq were loacted in the region with SNP polymorphism. Based on these work, this project is intergating fine-mapping and bioinfomatics to isolate the candidate genes, then verifys the gene function by genetic transformaiton. In order to intergate the breeding research, we are linking some male sterile gene to the candidate gene to construct yellow seed coat-male strile linked breeding lines.

种皮颜色由种皮色素类型和含量决定，是种子的重要表型性状之一，与种子休眠、种子寿命、含油量、蛋白含量、纤维素含量等重要农艺性状有关。经典遗传研究表明种皮颜色调控方式主要分为母体效应和花粉效应（花粉直感，种胚直感）。遗传育种中花粉直感效应基因表型比母体效应基因表型早一个种植世代，具有特殊的应用价值。本研究在筛选获得花粉效应基因调控油菜黄籽性状的单基因突变体的基础上，结合经典遗传分析、转录组测序和60K油菜SNP芯片作图，将该基因定位于C4染色体3.2M的区段，转录组数据表明该区域只有一个基因表达有差异，另外尚有81个无表达差异的基因被转录组测序检测到。本研究结合精细定位和候选基因多态性分析确定候选基因，通过转基因的方法进行基因功能验证。为与应用研究相结合，本研究将雄性不育基因和候选基因构建在同一转化载体上，尝试构建黄籽-不育连锁体系，开拓杂种优势利用的新途径。

种皮颜色由种皮色素类型和含量决定，是种子的重要表型性状之一，与种子休眠、种 子寿命、含油量、蛋白含量、纤维素含量等重要农艺性状有关。经典遗传研究表明种皮颜 色调控方式主要分为母体效应和花粉效应（花粉直感，种胚直感）。本研究在筛选获得花粉效应基因调控油菜黄籽性状的单基因突变体的基础上，结合经典遗传分析、转录 组测序和60K油菜SNP芯片作图，将该基因定位于C4染色体3.2M的区段。选取初定位染色体区域的SSR标记和新开发的indel标记，对黄籽基因精细了精细定位，定位目标区域大约200kb。通过转录组测序数据和近等基因系基因组重测序相结合，确定黄籽是由于一小段DNA插入突变引起。插入造成的效应是一个正负链双向表达的基因断裂，正链部分基因不完全转录，生成负链基因的反义基因，造成负链基因功能失活。将候选基因片段采用35S启动子构建载体验证该基因功能。根据油菜和拟南芥小孢子发育调控模型，分别在油菜中敲除了5个雄性不育基因，单由于油菜基因组的复杂性，在拟南芥中有功能的基因在油菜中敲除后都没有表型，于是重新设计了TA29-RNase的过期专利基因，与黄籽基因一起构建连锁表达载体。黄籽基因-不育基因构建的连锁表达载体转化油菜，获得了48棵油菜转化苗，2020年3月表型显示全部转化株都是雄性不育，是否黄籽由于作物生长季节关系尚处于评价中。取得的最主要进展就是确定了黄籽候选基因，设计了黄籽-雄性不育连锁的转基因油菜，转化获得的转基因油菜黄籽指示具有雄性不育表型的不育株，黑籽指示可育的非转基因株。杂交制种时选用黄籽，用于生产时选用黑籽，实现用转基因株系生产非转基因杂交种。