Abstract：
The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands\' cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.
摘要：
磷脂酶A2（PLA2）通过土地循环在磷脂的酰基重塑中起关键作用，并因此改变三酰甘油(TAG)中的脂肪酸组成。在这项研究中,使用快速扩增cDNA末端的技术克隆了编码Myrmeciaincisa磷脂酶A2（MiPLA2）的全长cDNA序列。将1082bpcDNA与其相应的克隆DNA序列进行比较，发现MiPLA2包含3个内含子。成熟的MiPLA2（mMiPLA2）具有保守的Ca2结合环和催化位点基序，已在植物分泌PLA2（sPLA2）蛋白中得到识别。相应地,系统发育分析表明，MiPLA2聚集在植物sPLA2蛋白的GroupXIA中。为了确定MiPLA2的功能，将编码mMiPLA2的cDNA亚克隆到载体pET-32a中，以促进在大肠杆菌中产生重组mMiPLA2。纯化重组mMiPLA2并用于体外酶反应。重组mMiPLA2产生的催化产物的薄层色谱谱表明，从磷脂中裂解sn-2酰基链具有特异性，从而在功能上表征MiPLA2。尽管重组mMiPLA2表现出对磷脂酰乙醇胺的强烈偏好，它优先水解磷脂酰胆碱sn-2位的花生四烯酸（ArA）。p1300-sp-EGFP-mMiPLA2融合表达的结果表明，MiPLA2位于洋葱表皮的细胞间隙中。此外，MiPLA2转录与游离ArA水平呈正相关。因此,阐明了mMiPLA2在富含ArA的TAG的生物合成中的作用。这项研究有助于了解M.incisa如何优先使用ArA合成TAG。
