BnFUSCA3调控油菜籽油脂合成的分子网络

The oil accumulation in seed is regulated by a complex network of gene expression that is controlled by some master transcription factor. Evidences indicated that AtFUSCA3 (AtFUS3) was one of key transcription factor controlling the seed development and oil synthesis in Arabidopsis. Mutation of AtFUS3 caused the seed abnormality and the oil content in seeds decreased significantly. Moreover, overexpression of AtFUS3 rendered the seed death of transgenic plants, and thus resulted in no available seeds of transgenic plants used to explore the network of gene and metabolism of AtFUS3 regulating oil synthesis in seeds. Brassica napus, one of the most important oilseed crops, is an allotetraploid formed by hybridization across Brassica rapa and Brassica oleracea. Recently, genome sequencing showed that there were four homologous/members in B. napus genome. Heretofore, the biological function of each BnFUS3 member and the molecular network of BnFUS3 regulating the oil synthesis in B. napus seed were unclear. In this project, we will create loss-of-function mutants of BnFUS3 members by CRISPR-cas9 site-directed mutagenesis system to investigate their biology functions. And then, using D6 promoter, a seed-specific and low-level -expression promoter constructed by our previous research, to drive BnFUS3 members expression in B. napus seed, from which distinguish the key member regulating oil synthesis in B. napus seed among these four members. Lastly, we will employ the seeds of mutant and transgenic plants of the key member to reveal the gene network of BnFUS3 regulating lipid synthesis in B. napus seeds by molecular biology, genetics, RNA-seq and metabolome. The present study would not only provide new insights into basic understanding of gene regulatory networks of oil synthesis in B. napus seed, but also help improve oil yield and quality of rapeseed varieties through molecular breeding and biotechnological tools.

种子油脂积累是受掌控种子成熟的转录因子，通过操纵其下游复杂的基因表达网络所控制。利用拟南芥研究表明AtFUSCA3（AtFUS3）是调控种子油脂合成的重要转录因子，其突变导致种子畸形，含油量降低，过表达则使种子死亡，而不能获得转基因植株，导致难以阐明AtFUS3调控种子油脂合成的机理。在前期工作中，我们已克隆并证明油菜BnFUS3具有调控油脂合成的功能。本项目在此基础上，拟用已开发的D6启动子驱动BnFUS3成员，获得发育正常的转基因油菜，以及采用CRISPR-cas9技术创制BnFUS3成员的功能缺失性突变体，研究BnFUS3成员调控油菜籽油脂合成的生物学功能。最后利用BnFUS3关键成员的突变体和转基因油菜，通过分子、遗传、转录组测序和高通量代谢组学等分析手段，揭示BnFUS3调控油菜籽发育和油脂合成的基因网络和代谢网络，为分子育种培育高含油量油菜新品种提供理论指导以及有效靶基因。

在拟南芥（Arabidopsis thaliana）中，AtFUSCA3是胚胎发育的重要调控因子。AtFUS3的突变导致种子储备减少，AtFUS3的过度表达导致转基因拟南芥植株脂肪酸水平升高，发育异常严重。迄今为止，AtFUS3调控种子油生物合成的机制还不完全清楚。在本项目中，我们在甘蓝型油菜中鉴定到了4个BnFUS3成员。这些BnFUS3成员在基因结构和蛋白质序列上都非常保守。它们都主要在甘蓝型油菜种子胚的晚期表达，但它们的各自表达方式不尽相同。这些成员蛋白的亚细胞定位都在细胞核中，表明它们均能在细胞核中调控下游基因的表达。通过XVE系统诱导BnFUS3-1在拟南芥幼苗中过表达，增加了拟南芥幼苗脂肪酸的含量，但转基因拟南芥幼苗发育迟缓，有的甚至死亡。在种子特异启动子D6的驱动下，转基因BnFUS3特异在油菜籽中表达，D6启动子驱动的BnFUS3成员能够在甘蓝型油菜种子中维持低水平表达，从而使得转基因BnFUS3种子含油量增加了6.21%～21.52%，而转基因BnFUS3植株的农艺性状值与非转基因野生型无明显变化。在转基因油菜种子中，大量与光合作用、脂肪酸合成和糖酵解相关的基因表达上调。转基因油菜种子发育过程中淀粉积累增加，蔗糖和葡萄糖积累反而减少，表明转基因BnFUS3油菜籽中脂肪酸合成途径的碳通量增加，从而提高油菜籽含油量。总之，我们的研究结果表明BnFUS3成员共同调控油菜籽成熟过程中的油脂合成，是遗传改良油菜品质的可靠靶点。我们的研究结果也可以用于其它油料作物的油脂品质改良。