甘蓝型油菜籽粒大小与千粒重主效QTL区间候选基因的高效筛选与功能分析

Rapeseed is one of the most important oil crops in China and worldwide, improving the seed oil production was considered to be the most focused issues in the past decades. Exploring the key genes relative to controlling the size of seed will be helpful to the research of improving oil production. We had acquired two consensus QTLs cqTSW-C1-1 and cqTSW-A7-4 contributing over 10% relative to seed size and thousand seed weight traits in KN DH population and 41 candidate genes were found in the two QTL intervals. In this study, we trying to use the FOX hunting system to screen 41 candidate genes we had acquired previously and select the target genes that obviously affecting seed size for further functional analysis in rapeseed. Overexpression, knockdown and knockout lines of target genes in rapeseed will be constructed by RNAi and CRISPR/Cas9 technology. The morphology of seeds and gene expression profiles relative to seed size in transgenic lines will be detected in order to discover the relationship between the seed size and target gene related metabolic networks. This project will lay the foundation for rapid identification pivotal genes controlling seed size derived from QTL candidate intervals.

油菜是中国乃至全球最重要油料作物之一，提高油产量是油菜研究的热点。发掘控制种子籽粒大小相关基因将对提高油菜油产量有重要意义。课题组前期利用KN DH群体，将种子大小和千粒重的QTL区间共定位获得了贡献率超过10%的整合QTL位点cqTSW-C1-1和cqTSW-A7-4并初步筛选了41个候选基因。本课题拟将41个候选基因导入高通量筛选基因系统—FOX hunting system 进行初步筛选并将明显引起种子大小改变的候选基因在油菜中进行进一步功能分析。利用CRISPR/Cas9构建目标基因的敲除载体以及构建超表达载体和RNA干扰载体并转化甘蓝型油菜。对目标基因转基因株系后代种子进行形态学鉴定分析和种子发育相关代谢网络的变化进行解析，找到目标基因引起种子大小改变内在规律。本研究为快速鉴定QTL候选区间内控制种子大小的关键基因奠定基础。

籽粒重是影响作物产量的重要因素，籽粒重很大程度上由种子大小决定，阐明控制籽粒大小与千粒重的分子机理对提高油菜产量具有重要意义。本课题将FOX hunting技术应用于油菜种子大小QTL定位区间候选基因的筛选成功筛选到了影响种子大小的基因。具体内容如下： .利用KN群体，将种子大小和千粒重的QTL区间共定位，将QTL位点cqTSW-C1-1和cqTSW-A7-4中的候选基因初步筛选后构建了基于FOX-hunting的筛选库，最终筛选到了一个有稳定表型的候选基因SCL；通过将SCL基因分别在拟南芥和甘蓝型油菜中过表达显著提高了种子的大小和千粒重；为进一步提高油菜产量提供有价值的遗传位点，具有重要生产价值。SCL是定位在细胞核的GRAS家族转录因子，在KN群体亲本间存在显著的表达差异；细胞学切片表明，过表达SCL通过使细胞增大并最终实现种子变大；SCL是如何在基因和分子水平上提高细胞大小仍有待进一步研究。.种子大小的改变通常主要由细胞数目和细胞大小改变引起；本研究发现，亚麻酸超量积累会引起油料种子大小发生改变，即脂肪酸代谢也会显著影响油料种子大小。这一发现为油料种子中的脂肪酸改良和产油量研究提供借鉴。.本研究证明了将FOX-hunting技术应用于油菜种子大小QTL定位区间候选基因的方法是可行且有效的，为加快筛选QTL定位候选基因提供方法借鉴。.