Sun Yan, Liu Baoling, Xue Jinai, Wang Xiaodan, Cui Hongli, Li Runzhi, Jia Xiaoyun
Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Taigu, Jinzhong, China.
Biotechnol Biofuels Bioprod. 2022 Feb 25;15(1):21. doi: 10.1186/s13068-022-02120-2.
Vernonia galamensis native to Africa is an annual oleaginous plant of Asteraceae family. As a newly established industrial oil crop, this plant produces high level (> 70%) of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid), which is an unusual epoxy fatty acid (EFA) with multiple industrial applications. Here, transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation, aiming to identify the target genes that could be used in the biotechnological production of high-value oils.
Based on oil accumulation dynamics of V. galamensis seeds, we harvested seed samples from three stages (17, 38, and 45 days after pollination, DAP) representing the initial, fast and final EFA accumulation phases, and one mixed sample from different tissues for RNA-sequencing, with three biological replicates for each sample. Using Illumina platform, we have generated a total of 265 million raw cDNA reads. After filtering process, de novo assembly of clean reads yielded 67,114 unigenes with an N50 length of 1316 nt. Functional annotation resulted in the identification of almost all genes involved in diverse lipid-metabolic pathways, including the novel fatty acid desaturase/epoxygenase, diacylglycerol acyltransferases, and phospholipid:diacylglycerol acyltransferases. Expression profiling revealed that various genes associated with acyl editing, fatty acid β-oxidation, triacylglycerol assembly and oil-body formation had greater expression levels at middle developmental stage (38 DAP), which were consistent with the fast accumulation of EFA in V. galamensis developing seed, these genes were detected to play fundamental roles in EFA production. In addition, we isolated some transcription factors (such as WRI1, FUS3 and ABI4), which putatively regulated the production of V. galamensis seed oils. The transient expression of the selected genes resulted in a synergistic increase of EFA-enriched TAG accumulation in tobacco leaves. Transcriptome data were further confirmed by quantitative real-time PCR for twelve key genes in EFA biosynthesis. Finally, a comprehensive network for high EFA accumulation in V. galamensis seed was established.
Our findings provide new insights into molecular mechanisms underlying the natural epoxy oil production in V. galamensis. A set of genes identified here could be used as the targets to develop other oilseeds highly accumulating valued epoxy oils for commercial production.
原产于非洲的加拉姆斑鸠菊是菊科一年生油料植物。作为一种新确立的工业油料作物，这种植物能产生高水平（>70%）的斑鸠菊酸（顺式-12-环氧十八碳-顺式-9-烯酸），这是一种具有多种工业用途的特殊环氧脂肪酸（EFA）。在此，对加拉姆斑鸠菊发育种子进行转录组分析和脂肪酸谱分析，以揭示负责高EFA积累的关键代谢途径，旨在鉴定可用于高价值油脂生物技术生产的靶基因。
基于加拉姆斑鸠菊种子的油脂积累动态，我们采集了代表EFA积累初始、快速和最终阶段的三个阶段（授粉后17、38和45天，DAP）的种子样本，以及一个来自不同组织的混合样本用于RNA测序，每个样本有三个生物学重复。使用Illumina平台，我们共生成了2.65亿条原始cDNA读数。经过过滤过程，对clean reads进行从头组装得到67114个单基因，N50长度为1316 nt。功能注释鉴定出了几乎所有参与多种脂质代谢途径的基因，包括新型脂肪酸去饱和酶/环氧化酶、二酰甘油酰基转移酶和磷脂：二酰甘油酰基转移酶。表达谱分析表明，与酰基编辑、脂肪酸β-氧化、三酰甘油组装和油体形成相关的各种基因在发育中期（38 DAP）表达水平更高，这与加拉姆斑鸠菊发育种子中EFA的快速积累一致，这些基因在EFA产生中起重要作用。此外，我们分离了一些转录因子（如WRI1、FUS3和ABI4），它们可能调控加拉姆斑鸠菊种子油的产生。所选基因的瞬时表达导致烟草叶片中富含EFA的TAG积累协同增加。通过对EFA生物合成中的12个关键基因进行定量实时PCR进一步证实了转录组数据。最后，建立了加拉姆斑鸠菊种子中高EFA积累的综合网络。
我们的研究结果为加拉姆斑鸠菊天然环氧油生产的分子机制提供了新的见解。这里鉴定出的一组基因可作为开发其他高积累有价值环氧油的油料种子用于商业生产的靶标。
