中国水产科学2018，Vol.25Issue(2)：251-262,12.DOI:10.3724/SP.J.1118.2018.17357
缺刻缘绿藻溶血磷脂酰乙醇胺酰基转移酶(LPEAT)的基因克隆与特征分析
Cloning and characterization of a gene encoding lysophosphatidyleth-anolamine acyltransferase(LPEAT)in the green microalga Myrmecia incisa Reisigl
摘要
Abstract
Myrmecia incisa Reisigl, a coccoid green microalga, is an oleaginous alga that can accumulate an un-precedentedly high level of arachidonic acid (ArA, 20:4ω6)-rich triacylglycerols (TAG) under the growth stress of nitrogen starvation. ArA accounts for 68.0% of total fatty acids in TAG. How is ArA preferentially utilized for the biosynthesis of TAG? The Lands' cycle plays an important role in the composition change of fatty acids of phos-pholipids, thus altering the composition of fatty acids in TAG. Lysophosphatidylethanolamine acyltransferase (LPEAT),a key enzyme in the Lands'cycle,was the focus of the present study.Myrmecia incisa(Mi)LPEAT was cloned using reverse transcription-PCR and 3'- and 5'-cDNA rapid amplification of cDNA ends technique. The full-length cDNA was 1303 bp long, and contained a 129-bp 5'-untranslated region (UTR) and 193-bp 3'-UTR. The length of the open read frame was 981 bp that encoded a 326-amino acid protein. The DNA sequence of MiLPEAT was also cloned from M.incisa with the isolated algal genomic DNA as a template,and it was 1871 bp long.Comparison of the cDNA and DNA sequences showed that MiLPEAT possessed 6 introns that separated the coding sequence into 7 exons. Multiple sequence alignment and bioinformatics analysis of LPEATs from different species demonstrated that MiLPEAT possessed a phosphate acyltransferase domain, PlsC, thus suggesting that it was one member of the lysophospholipid acyltransferase (LPLAT) superfamily. MiLPEAT also had the 4 typical motifs, NH(x)4D, GCxYVxR, FPEGT, and PVxPVx, which are characterized in the LPLAT superfamily. Both the prediction, as analyzed online by Wolfsport and Protein Prowler, and the presence of a dilysine motif at the car-boxyl terminus of MiLPEAT, implied that MiLPEAT might reside at the algal endoplasmic reticulum and possibly participate in the secretion pathway. A neighbor-joining phylogeny was constructed on the basis of deduced amino acids of LPEATs from different species of plants. It illustrated that MiLPEAT was so different from LPEAT2 that they located at different clades due to their various characteristics of sequences. MiLPEAT was clustered phy-logenetically with LPEAT1, suggesting their similar functions in the acylation of phospholipids. Quantitative real-time PCR detection pointed out that MiLPEAT transcripts increased at a statistically significant level(P<0.05)at 8 h after treatment with nitrogen starvation in M.incisa. By coincidence, the relative abundance of lysophosphati-dylethanolamine (LPE) in the microalgal cells reduced by approximately 49% at an extreme significance level (P<0.01) under the nitrogen starvation stress. The corresponding phosphatidylethanolamine (PE) generated from LPE as catalyzed by MiLPEAT, however, did not increase significantly. It is assumed that the net increase of PE under nitrogen starvation in M.incisa was possibly utilized for TAG biosynthesis by the phospholipid DAG acyl-transferase (PDAT), so that the content of TAG has been reported to increase. This research lays a foundation for us to understand the TAG and phospholipid biosynthetic pathway and how to regulate TAG synthesis in M.incisa.关键词
缺刻缘绿藻/溶血磷脂酰乙醇胺酰基转移酶(LPEAT)/三酰甘油(TAG)/磷脂/氮饥饿Key words
Myrmecia incisa/lysophosphatidylethanolamine acyltransferase (LPEAT)/triacylglycerol (TAG)/phospholipid/nitrogen starvation分类
农业科技引用本文复制引用
周志刚,包虹,欧阳珑玲..缺刻缘绿藻溶血磷脂酰乙醇胺酰基转移酶(LPEAT)的基因克隆与特征分析[J].中国水产科学,2018,25(2):251-262,12.基金项目
国家自然科学基金项目(31772821,31402274) （31772821,31402274）
国家海洋局可再生能源专项基金项目(SHME2011SW02) （SHME2011SW02）
上海高校水产科学高峰学科建设项目. （）
