Zhang Donghui, Jasieniecka-Gazarkiewicz Katarzyna, Wan Xia, Luo Ling, Zhang Yinbo, Banas Antoni, Jiang Mulan, Gong Yangmin
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan, 430062, China.
Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822, Gdansk, Poland.
PLoS One. 2015 Dec 18;10(12):e0144653. doi: 10.1371/journal.pone.0144653. eCollection 2015.
In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid acyltransferases from N. benthamiana.
在磷脂酰胆碱（PC）合成的重塑途径中，酰基辅酶A依赖性溶血磷脂酰胆碱（lysoPC）酰基转移酶（LPCAT）催化lysoPC的再酰化反应。近年来，已从多种植物中克隆并鉴定了多个编码LPCAT的基因。利用拟南芥和其他植物的LPCAT序列筛选本氏烟草的基因组数据库，我们鉴定出两个编码假定烟草LPCAT（NbLPCAT1和NbLPCAT2）的cDNA。预测它们均编码一个463个氨基酸的蛋白质，与其他植物的LPCAT具有高度相似性。蛋白质序列特征，如至少存在八个假定的跨膜区域、四个高度保守的特征基序和几个不变残基，表明NbLPCAT属于膜结合O-酰基转移酶家族。通过在缺乏内源性LPCAT酶活性的酵母突变体Y02431（lca1△）中异源表达每个全长cDNA，测试溶血血小板活化因子（lysoPAF）敏感性，证实了NbLPCAT的溶血磷脂酰基转移酶活性。对补充了多不饱和脂肪酸的表达NbLPCAT的酵母突变体Y02431培养物中磷脂的脂肪酸谱分析表明，与携带空载体的酵母突变体相比，更多的亚油酸（18:2n6，LA）和α-亚麻酸（18:3n3，ALA）掺入到PC中。体外酶活性测定表明，NbLPCAT1具有高lysoPC酰基转移酶活性，明显偏好α-亚麻酰辅酶A（18:3），而NbLPCAT2对α-亚麻酰辅酶A表现出高溶血磷脂酸（lysoPA）酰基转移酶活性，对lysoPC酰基转移酶活性较弱。组织特异性表达分析表明，NbLPCAT1和NbLPCAT2在根、茎、叶、花和种子中普遍表达，在发育中的花中强烈表达。这是关于本氏烟草溶血磷脂酰基转移酶克隆和鉴定的首次报道。
