Cloning and Functional Characterization of the Lysophosphatidic Acid Acyltransferase Gene AsLPAAT5 in Avena sativa

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

Abstract

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

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, 2026, 42(10): 88-97.

Figures/Tables 7

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

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

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

Fig. 3 Subcellular localization of AsLPAAT5 protein

Fig. 4 Oil-bodies staining of transgenic yeast

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

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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