Microalgae represent a rich and naturally occurring source of triacylglycerol (TAG), and the microalgae-derived TAG has enormous potential in bioenergy, bio-based materials and other agricultural innovations. Glycerol-3-phosphate acyltransferase (GPAT) is a key enzyme that catalyzes the biosynthesis of TAG. Previously our lab has reported the first GPAT in the oleaginous alga Myrmecia incisa, and in the present study, a new GPAT was further identified from the same alga, which was designated as MiGPAT2. A full-length cDNA of MiGPAT2 consisting of a 1374-bp ORF, a 153-bp 5′-UTR and a 271-bp 3′-UTR was cloned. The putative protein composed of 457 amino acids, possessing the HX4D motif that acts as the activity center of GPAT catalysis. Emphasis was put on the comparison between two MiGPATs regarding subcellular localization and TAG synthesis function. A polyclonal and monoclonal antibody were separately prepared for MiGPAT1 and MiGPAT2, and the following immunogold labeling showed they were localized on chloroplast and endoplasmic reticulum (ER), respectively. This was further confirmed by the GFP fusion studies. Both MiGPATs were expressed heterologously in a GPAT-deficient yeast mutant, and it was found that MiGPAT2 played key roles in the de novo TAG biosynthesis, whereas MiGPAT1 was more involved in the phospholipid formation, contributing to TAG production via an indirect way. Findings of the present study expanded our knowledge on functional and evolutionary relationships of GPAT members in M. incisa, suggesting the possibility of genetic manipulation of GPAT in this alga for enhanced TAG production.

微藻代表了三酰基甘油（TAG）的丰富和天然来源，而微藻衍生的TAG在生物能源，生物基材料和其他农业创新方面具有巨大潜力。3-磷酸​​甘油酰基转移酶（GPAT）是催化TAG生物合成的关键酶。以前，我们的实验室已经报道了含油藻Myrmecia incisa中的第一个GPAT ，在本研究中，从同一藻中进一步鉴定出了新的GPAT，称为MiGPAT2。MiGPAT2的全长cDNA克隆了由1374-bp ORF，153-bp 5'-UTR和271-bp 3'-UTR组成的序列。推测的蛋白质由457个氨基酸组成，具有作为GPAT催化活性中心的HX4D基序。重点放在两个MiGPAT之间关于亚细胞定位和TAG合成功能的比较上。分别为MiGPAT1和MiGPAT2制备了多克隆抗体和单克隆抗体，随后的免疫金标记表明它们分别位于叶绿体和内质网（ER）上。GFP融合研究进一步证实了这一点。两种MiGPATs均在GPAT缺陷型酵母突变体中异源表达，并且发现MiGPAT2在从头TAG生物合成中起关键作用，而MiGPAT1更参与磷脂的形成，通过间接方式为TAG的生产做出贡献。本研究的发现扩展了我们对GPAT成员在功能和进化关系方面的认识。M. incisa，暗示了在该藻类中进行GPAT基因操作以增强TAG产生的可能性。