甘蓝型油菜LPATs基因克隆及其调控含油量及品质的分子机理

Rapeseed is one of the most important oil crops in China and worldwide, improving the seed oil contents and quality was considered to be the most focused issues in the past decades. Exploring the key genes relative to the synthesis and accumulation of fatty acid will be helpful to the research of improving seed oil content and quality. In this study, lysophosphatidic acid acyltransferase genes as one of the candidate genes relative to oil content QTL location in Brassica napus will be cloned and analyzed. The gene sequence of LPATs will be found by matching the genetic linkage map with the physic map in B. napus. Seed specific over-expression vectors, and knockout vectors by using CRISPR/Cas9 will be applied to knockout LPAT genes in Arabidopsis and B. napus in order to investigate the effect of LPAT on oil contents and fatty acid composition in seeds. RNA-seq will be performed to characterize the expression profiles in the overexpression and defective lines. The expression levels of key genes in oil synthesis and fatty acid accumulation metabolic pathways both in overexpression and defective lines will be quantified and evaluated in order to explore the mechanism of oil synthesis and fatty acid accumulation.

油菜是中国乃至全球最重要油料作物之一，提高油菜种子含油量和改善品质是油菜研究的热点。发掘脂肪酸合成与积累方面的关键基因将有助于提高种子含油量和品质的研究。本课题依据前期含油量QTL定位结果，拟对QTL区间内候选基因溶血磷脂酸酰基转移酶基因（lysophosphatidic acid acyltransferase, LPAT）进行克隆及功能分析。根据甘蓝型油菜基因组物理图谱进行对应分析找到LPAT的基因并进行克隆；通过将LPAT在拟南芥和甘蓝型油菜超表达、以及通过CRISPR/Cas9在拟南芥或油菜LPAT基因进行敲除来研究LPAT对甘蓝型种子含油量及脂肪酸组分的影响；同时，对LPAT超表达株系和敲除株系进行转录组分析并对油脂代谢及其他物质代谢的关键基因进行表达差异分析，以探讨LPAT对油脂合成及脂肪酸积累的代谢通路影响，为解析LPAT影响油脂合成机理奠定基础。

提高含油量是当前油菜遗传育种的重要方向之一，项目执行期间，克隆得到了甘蓝型油菜含油量主效QTL区间的溶血磷脂酸酰基转移酶（Lysophosphatidic acid acyltransferase, LPAT）家族的LPAT2/5，并通过基因超表达和CRISPR/Cas9基因编辑获得了LPAT2/5转基因株系。结果显示超表达LPAT2和LPAT5均能提高含油量，但LPAT2超表达转基因株系提高的更高，其中BnLPAT2-A7超表达株系提高了19.7%；而CRISPR/Cas9敲除的Bnlpat2和Bnlpat5突变株系中种子含油量则下降明显，分别下降了32%和29%。对超表达株系和基因编辑株系的发育过程中种子进行了转录组分析，揭示了LPAT基因影响油脂合成和积累的分子机理。已发表了9篇学术论文，超额完成项目预定任务。