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 lysophosphatidylethanolamine acyltransferase(LPEAT) in the green microalga Myrmecia incisa Reisigl

缺刻缘绿藻三酰甘油(TAG)中花生四烯酸(Ar A)占其总脂肪酸含量的68.0%。为了弄清Ar A是如何被优先地用于合成TAG,鉴于Lands循环是通过改变膜脂的脂肪酸组成进而影响TAG的脂肪酸组成,本研究选择在Lands循环中起关键作用的溶血磷脂酰乙醇胺酰基转移酶(LPEAT)作为突破口。运用反转录PCR与3′-及5′-c DNA末端快速扩增技术,自缺刻缘绿藻(Myrmecia incisa Reisigl)中克隆到MiLPEAT;它的c DNA序列全长1303 bp,其中,5′-非翻译区(UTR)长129 bp,3′-UTR长193 bp,开放阅读框长981 bp,编码326个氨基酸残基;以缺刻缘绿藻基因组DNA为模板扩增得到该基因长为1871 bp的DNA序列;这两序列的比对结果显示,MiLPEAT含有6个内含子,将其开放阅读框(ORF)分割成7个外显子。通过多序列比对及生物信息学分析,发现MiLPEAT存在一个磷酸酰基转移酶结构域Pls C,并含有LPEAT家族所具有的NH(x)4D、FPEGT等4个特征性模体;Wolfpsort等在线预测结果以及MiLPEAT羧基端存在的双赖氨酸模体"KKxx",暗示它可能位于内质网并参与分泌途径。基于不同植物LPEAT的氨基酸序列所构建的邻接聚类图表明,MiLPEAT因与2型LPEAT有不同的序列特征而形成不同的分支,但却与1型LPEAT聚类在一起,推测它们的功能可能更近似。通过荧光定量PCR检测,发现MiLPEAT的基因转录量在氮饥饿8 h时显著(P<0.05)增加;与其变化相应的溶血磷脂酰乙醇胺(LPE)相对丰度却极显著地(P<0.01)减少49%,但磷脂酰乙醇胺(PE)相对丰度的增加却不显著;推测在氮饥饿过程中增加的磷脂酰乙醇胺(PE)可能被磷脂:二酰甘油酰基转移酶作用以合成为TAG,致使缺刻缘绿藻TAG含量的增加。

Myrmecia incisa Reisigl,a coccoid green microalga,is an oleaginous alga that can accumulate an unprecedentedly 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 phospholipids,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 carboxyl 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 phylogenetically with LPEAT1,suggesting their similar functions in the acylation of phospholipids.Quantitative realtime 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 lysophosphatidylethanolamine(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 acyltransferase(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.