新型油料作物亚麻荠CsbZIP转录因子调控ω-3脂肪酸生物合成机制的研究

ω-3 fatty acid is one kind of high-value healthy fatty acids with large market demand. Common oil crops such as soybean and rape seed only contain small amount of ω-3 fatty acid while Camelina sativa, a new-type oilseed with “low input, stress tolerance and high output” features, could accumulate high up to 45% of the unusual fatty acid. For understanding the unique molecular mechanism underlying high biosynthesis and accumulation of ω-3 fatty acid in camelina seeds, transcription fatty CsbZIP16 and fatty acid desaturase CsFAD3-2 are selected as the target gene in the present study. To investigate their functions responsible for seed oil metabolism and other phenotypes in camelina, we will develop the transgenic seed-specifically expressing the two targets, respectively, and also the CRISPR/case9-mediated gene-knockout mutants by an effective genetic transformation method established in our lab. ELISA-based DNA binding assay and chromatin immunoprecipitation will be employed to test if CsbZIP16 can transactivate CsFAD3-2 expression in vivo and in vitro. Yeast-1-hybrid and yeast-2-hybrid will be used to identify proteins interacted with CsbZIP16. Finally, we will develop a model to elucidate CsbZIP16-mediated regulation network controlling biosynthesis and high accumulation of ω-3 fatty acid in camelina seeds. The present study will uncover new knowledge, enriching theory and practice of plant oil biosynthesis and metabolic engineering, and promoting large-scale green production and sustainable utilization of this high-value unusual fatty acid.

ω-3脂肪酸是一种市场需求量大的高值健康型脂肪酸，在大豆和油菜等普通油料作物种子中含量较低，而在“低投入、耐逆境、高产出”的新型油料作物亚麻荠种子中高达45%。为解析亚麻荠种子高水平合成积累ω-3脂肪酸独特分子机制，本项目以转录因子CsbZIP16和脂肪酸脱氢酶CsFAD3-2基因为靶标，用我们优化的亚麻荠高效遗传转化技术，培育CsbZIP16和CsFAD3-2超表达株系和CRISPR/case9介导基因敲出突变体，分析靶基因对种子油脂合成等表型的影响。通过酶联免疫和染色质免疫沉淀法检测CsbZIP16是否直接激活CsFAD3-2基因表达。用酵母单、双杂交检测与CsbZIP16互作蛋白。建立CsbZIP16介导ω-3脂肪酸生物合成及高水平积累的调控网络。研究将获得植物油脂合成调控新知识，拓展油脂代谢工程的理论和策略，推动这一高值珍稀脂肪酸的规模化绿色生产和可持续利用。

本项目以种子富含ω-3脂肪酸的亚麻荠这一特色油料作物种质为试材，采用分子生物学和组学等技术，深入探讨ω-3油脂生物合成及高水平积累的分子机理。主要研究内容包括亚麻荠转录因子CsbZIP16和脂肪酸去包和酶CsFAD3-2基因的分子克隆，CsbZIP16和CsFAD3-2基因超表达及敲除突变体创制及表征， CsbZIP16激活CsFAD3-2基因表达的活体和离体检测，以及CsbZIP16转录因子上游调控因子的鉴定及功能分析等。研究发现在亚麻荠发育种子油脂合成积累过程中CsbZIP16和CsFAD3-2基因呈共上调表达特征。分别过表达CsbZIP16和CsFAD3-2基因导致亚麻荠种子总油脂和ω-3 脂肪酸含量分别提高9.8%（干重）和29.4%（%总脂肪酸）。相反，这两个基因敲除突变体种子总油脂和ω-3 脂肪酸含量分别减低19.8%（干重）和38.7%（%总脂肪酸）。活体和离体测试显示转录因子CsbZIP16能与CsFAD3-2基因启动子区结合激活CsFAD3-2基因表达。鉴定获得了CsbZIP16互作蛋白CsFUS3以及可调控CsbZIP16表达的CsWRKY42。整合研究发现提出了CsbZIP16转录因子介导亚麻荠种子ω-3油脂生物合成及调控的分子机制模型，以及可用于在亚麻荠及普通油料作物种子中组装富集ω-3脂肪酸途径的代谢工程策略。本研究为全面解析ω-3 脂肪酸等特异高值脂肪酸生物合成及其调控网络的提供了新见解，亦为进一步应用转基因技术及代谢工程培育种子富集ω-3 脂肪酸的亚麻荠及其他普通油料作物新品系提供新的基因元件和技术体系，丰富有关植物油脂生物合成调控及遗传改良的理论和实践，进而加速实现ω-3脂肪酸等高值珍稀植物油脂的规模化绿色生产和广泛工业应用，提高油料作物生产效益和拓展植物油脂加工利用产业链，促进植物油脂相关产业的可持续发展。