燕麦溶血磷脂酸酰基转移酶AsLPAAT5基因克隆与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0470

摘要/Abstract

摘要：

目的溶血磷脂酸酰基转移酶（lysophosphatidic acid acyltransferase, LPAAT）是三酰甘油（triacylglycerol, TAG）组装途径中的关键限速酶。研究燕麦AsLPAAT5基因在油脂合成积累中的功能，为燕麦及其他作物油脂产量与品质改良提供新思路。方法从燕麦中克隆AsLPAAT5基因（Gene ID：AVESA.00001b.r1.1Ag0003121.1），生物信息学方法分析其结构特征，通过LPAAT缺陷型大肠杆菌突变株SM2-1、酿酒酵母INVSc1和拟南芥转化体系研究AsLPAAT5酶的活性及生物学功能。结果燕麦AsLPAAT5基因编码蛋白具有LPAAT的典型保守结构域。AsLPAAT5基因在燕麦根、茎、叶、穗以及开花后9、17、25 d的种子中均有表达，在开花后25 d的种子中表达量最高。大肠杆菌功能互补试验证实AsLPAAT5能够恢复突变株SM2-1的细胞膜脂生物合成功能，具备LPAAT酶活性。在酿酒酵母中过表达AsLPAAT5导致转基因酵母总脂含量相较于野生型酵母显著升高2.11%，C16∶1含量增加14.25%。在拟南芥中过表达AsLPAAT5使转基因拟南芥种子千粒重增加2.2%-2.8%，总油脂含量提高1.9%-2.0%，C18∶2含量显著升高25.49%-26.0%。结论 AsLPAAT5是定位于内质网且具备典型LPAAT催化活性的酶蛋白，其异源表达能够显著促进宿主总油脂积累，并对不饱和脂肪酸（尤其是C18∶2）的富集具有特异性调控作用。

关键词: 燕麦, 溶血磷脂酸酰基转移酶, 油脂合成, 亚油酸, 功能分析

Abstract:

Objective Lysophosphatidic acid acyltransferase (LPAAT) is a key rate-limiting enzyme in triacylglycerol (TAG) assembly pathway. This study aimed to investigate the function of the oat (Avena sativa L.) AsLPAAT5 gene in lipid biosynthesis and accumulation, providing new insights for improving oil yield and quality in oat and other crops. Method The AsLPAAT5 gene (Gene ID: AVESA.00001b.r1.1Ag0003121.1) was cloned from oat, and its structural characteristics were analyzed using bioinformatics tools. The enzymatic activity and biological function of AsLPAAT5 were evaluated using the Escherichia coli LPAAT-deficient mutant strain SM2-1, Saccharomyces cerevisiae wild-type strain INVSc1, and Arabidopsis thaliana transformation systems. Result The protein encoded by AsLPAAT5 possessed the typical conserved domains of LPAAT. AsLPAAT5 was expressed in roots, stems, leaves, panicles, and seeds at 9, 17, and 25 days after flowering, with the highest expression level detected in seeds at 25 days after flowering. Functional complementation assays in E. coli confirmed that AsLPAAT5 can restore membrane lipid biosynthesis in the mutant strain SM2-1, demonstrating LPAAT enzymatic activity. Overexpression of AsLPAAT5 in S. cerevisiae resulted in a significant 2.11% increase in total fatty acid content compared with wild-type yeast, along with a 14.25% increase in C16:1 content. Overexpression of AsLPAAT5 in A. thaliana increased thousand-seed weight by 2.2%–2.8%, enhanced total oil content by 1.9%–2.0%, and significantly elevated C18:2 content by 25.49%–26.0%. Conclusion AsLPAAT5 is an endoplasmic reticulum-localized enzyme with typical LPAAT catalytic activity. Heterologous expression of AsLPAAT5 significantly promotes total lipid accumulation in hosts and exerts a specific regulatory effect on the enrichment of unsaturated fatty acids, particularly C18:2.

Key words: Avena sativa L., lysophosphatidic acid acyltransferase, oil biosynthesis, linoleic acid, functional analysis

胡荣芳, 丁家豪, 马菊花, 谭辉进, 宋亚楠, 薛金爱. 燕麦溶血磷脂酸酰基转移酶AsLPAAT5基因克隆与功能分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0470.

HU Rong-fang, DING Jia-hao, MA Ju-hua, TAN Hui-jin, SONG Ya-nan, XUE Jin-ai. Cloning and Functional Characterization of the Lysophosphatidic Acid Acyltransferase Gene AsLPAAT5 in Avena sativa[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0470.

图/表 7

表 1 本研究所用的引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Sequence（5′-3′）	用途 Purpose
1302-AsLPAAT5-F	acgggggactcttgaccatggATGTGGTTAGCCATGTTTCCATT	载体构建
1302-AsLPAAT5-R	aagttcttctcctttactagtTCAGAGTTTCTTGGCATGTGCG	载体构建
pYES2.0-AsLPAAT5-F	gggaatattaagcttggtaccATGTGGTTAGCCATGTTTCCATT	载体构建
pYES2.0-AsLPAAT5-R	tacatgatgcggccctctagaTCAGAGTTTCTTGGCATGTGCG	载体构建
1300-AsLPAAT5-F	gcccttgctcaccatggtaccATGTGGTTAGCCATGTTTCCATT	载体构建
1300-AsLPAAT5-R	atacaccaaatcgactctagaGAGTTTCTTGGCATGTGCGC	载体构建
pET28A-AsLPAAT5-F	gcaagcttgtcgacggagctcATGTGGTTAGCCATGTTTCCATT	载体构建
pET28A-AsLPAAT5-R	ggataacaattcccctctagaTCAGAGTTTCTTGGCATGTGCG	载体构建
AsLPAAT5-qRT-F	ATACAAGCATCGGCTGCCAACT	qPCR
AsLPAAT5-qRT-R	CCTGAACCACACGGACGAGAAC	qPCR
AsEP-F	GCACAAGTGATGCCAGAATAGC	内参基因
AsEP-R	CGAGATGCATTAGATTCGTTGG	内参基因

图1 AsLPAAT5基因克隆及其序列特征分析A：燕麦AsLPAAT5基因的克隆，M：DNA marker DL 2000；B：不同物种LPAATs蛋白系统进化树；C：AsLPAAT5蛋白结构域；D：LPAAT5蛋白保守氨基酸序列

Fig. 1 Cloning and sequence characterization of AsLPAAT5 geneA: Cloning of AsLPAAT5, M: DNA marker DL 2000. B: Phylogenetic tree of LPAAT proteins from different plant species. C: Protein domain of AsLPAAT5. D: Conserved amino acid sequences of LPAAT5 proteins

图2 AsLPAAT5蛋白酶活验证A：AsLPAAT5基因表达分析；B：AsLPAAT5在SM2-1菌株中的功能互补测试。不同小写字母表示差异显著（P<0.05），下同

Fig. 2 Enzyme activity validation of AsLPAAT5 proteinA: Expression analysis of AsLPAAT5. B: Functional complementation test of AsLPAAT5 in the SM2-1 strain. Different lowercase letters indicate significant differences (P<0.05). The same below

图3 AsLPAAT5蛋白的亚细胞定位

Fig. 3 Subcellular localization of AsLPAAT5 protein

图4 转基因酵母油滴BODIPY染色

Fig. 4 Oil-bodies staining of transgenic yeast

图5 AsLPAAT5转基因酵母油脂含量分析A：AsLPAAT5转基因酵母总脂含量；B：AsLPAAT5转基因酵母脂肪酸含量

Fig. 5 Analysis of lipid content in AsLPAAT5 transgenic yeastA: Total fatty acid content of AsLPAAT5 transgenic yeast. B: Fatty acid content of AsLPAAT5 transgenic yeast

图6 转基因拟南芥表型及油脂分析A：拟南芥种子千粒重；B：总脂含量；C：脂肪酸组分

Fig. 6 Phenotypic and lipid analysis of transgenic ArabidopsisA: Thousand-seed weight of A. thaliana. B: Total fatty acid content. C: Fatty acid composition

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