异源表达栅藻苹果酸酶基因SqME对烟叶光合固碳和油脂积累的影响

Effect of heterologous expression of Scenedesmus quadricauda malic enzyme gene SqME on photosynthetic carbon fixation and lipid accumulation in tobacco leaves

摘要:

微藻光合效率高、适应性强和生物量大，是一种易规模化养殖的优良生物种质。苹果酸酶(malic enzyme,ME)是一种生物体内广泛存在的代谢酶，催化苹果酸脱羧生成丙酮酸和CO₂，同时产生NAD (P) H。ME在能量、光合、呼吸和生物合成等代谢过程中发挥重要作用。本研究旨在鉴定栅藻(Scenedesmus quadricauda)苹果酸酶基因SqME并解析其生物学功能，为高等植物遗传改良提供优异靶基因。基于栅藻在CO₂和光传质效率高、用水量少的生物膜培养条件下的转录组测序(RNA sequencing,RNA-seq)数据，克隆1条高表达且功能注释为ME的基因。应用生信工具系统解析SqME蛋白序列和理化特性。运用本氏烟草(Nicotiana benthamiana)叶片瞬时转染方法分析SqME蛋白亚细胞定位。通过遗传转化普通烟草(Nicotiana tabacum)鉴定SqME的生物学功能，并评估其在高等植物遗传改良中的应用价值。栅藻SqME基因的开放阅读框(open reading frame,ORF)为1 770 bp，编码1个590个氨基酸的蛋白质，该蛋白定位于叶绿体。蛋白结构和进化树分析表明，SqME属于NADP-ME型，具有典型的ME酶蛋白结构特征。转SqME烟草株系的苹果酸酶活性是对照株系的1.8倍。异源表达SqME使转基因烟草株系叶绿素a、叶绿素b和总叶绿素含量分别提升了20.9%、26.9%和25.2%。与对照植株相比，转基因烟草叶片荧光参数非光化学猝灭系数(non-photochemical quenching,NPQ)提高54.0%，初始荧光(fluorescence origin,Fo)降低30.1%。转基因烟草叶片生物量、总脂和可溶性糖含量分别提高20.5%、25.7%和9.5%。相反，转基因烟叶的淀粉和蛋白质含量分别降低22.4%和12.2%。研究结果表明，栅藻SqME基因编码的酶蛋白具有较强的酶活性。异源过表达SqME能显著增强宿主光合保护机制、光合作用和生物量。SqME还可驱动宿主碳代谢重构，促使更多碳代谢通量导向油脂合成。因此，SqME可应用于高等植物遗传改良，以增进植物的光合固碳能力并促进油脂富集。本研究为栅藻功能基因挖掘及其在高等植物遗传改良的应用提供了参考。

Abstract:

Microalgae possess high photosynthetic efficiency, robust adaptability, and substantial biomass, serving as excellent biological resources for large-scale cultivation. Malic enzyme (ME), a ubiquitous metabolic enzyme in living organisms, catalyzes the decarboxylation of malate to produce pyruvate, CO₂, and NAD(P) H, playing a role in multiple metabolic pathways including energy metabolism, photosynthesis, respiration, and biosynthesis. In this study, we identified the Scenedesmus quadricauda malic enzyme gene (SqME) and its biological functions, aiming to provide excellent target genes for the genetic improvement of higher plants. Based on the RNA-seq data from S. quadricauda under the biofilm cultivation mode with high CO₂ and light energy transfer efficiency and small water use, a highly expressed gene (SqME) functionally annotated as ME was cloned. The physicochemical properties of the SqME-encoded protein were systematically analyzed by bioinformatics tools. The subcellular localization of SqME was determined via transient transformation in Nicotiana benthamiana leaves. The biological functions of SqME were identified via genetic transformation in Nicotiana tabacum, and the potential of SqME in the genetic improvement of higher plants was evaluated. The ORF of SqME was 1 770 bp, encoding 590 amino acid residues, and the encoded protein was located in chloroplasts. SqME was a NADP-ME, with the typical structural characteristics of ME. The ME activity in the transgenic N. tabacum plant was 1.8 folds of that in the wild-type control. Heterologous expression of SqME increased the content of chlorophyll a, chlorophyll b, and total chlorophyll by 20.9%, 26.9%, and 25.2%, respectively, compared with the control. The transgenic tobacco leaves showed an increase of 54.0% in the fluorescence parameter NPQ and a decrease of 30.1% in Fo compared with the control. Moreover, the biomass, total lipids, and soluble sugars in the transgenic tobacco leaves enhanced by 20.5%, 25.7%, and 9.5%, respectively. On the contrary, the starch and protein content in the transgenic tobacco leaves decreased by 22.4% and 12.2%, respectively. Collectively, the SqME-encoded protein exhibited a strong enzymatic activity. Heterologous expressing of SqME could significantly enhance photosynthetic protection, photosynthesis, and biomass accumulation in the host. Additionally, SqME can facilitate carbon metabolism remodeling in the host, driving more carbon flux towards lipid synthesis. Therefore, SqME can be applied in the genetic improvement of higher plants for enhancing photosynthetic carbon fixation and lipid accumulation. These findings provide scientific references for mining of functional genes from S. quadricauda and application of these genes in the genetic engineering of higher plants.