Cloning of Plastidial PfLPAT1B Gene from Perilla frutescens and Its Functional Analysis in Oil Biosynthesis

doi:10.13560/j.cnki.biotech.bull.1985.2024-1056

Abstract

Abstract:

Objective Lysophosphatidic acid acyltransferase (LPAT) plays a crucial role in plant growth, development, and lipid metabolism. This study aims to investigate the biological function of the PfLPAT1B gene of Perilla frutescens, providing a scientific foundation for the genetic improvement and breeding of new cultivars of Perilla and other oilseed crops. Method The PfLPAT1B gene sequence was identified from the Perilla genome database, and its sequence characteristics and phylogenetic relationships were analyzed using omics tools. The expression patterns of PfLPAT1B in various tissues and different developmental seeds were evaluated by RT-qPCR. The enzyme activity of PfLPAT1B protein was assessed using the Escherichia coli LPAT-deficient strain SM2-1. The function of PfLPAT1B in oil biosynthesis was performed via genetic transformation of Saccharomyces cerevisiae and Nicotiana tabacum. Result PfLPAT1B gene encodes a total of 369 amino acid residues and it is a basic unstable hydrophilic protein, containing a typical conserved domain of lysophosphatidic acid acyltransferase. PfLPAT1B gene was expressed in different tissues and seeds at different developmental stages of Perilla, with the highest expression in flowers and an increasing trend during seed development. Subcellular localization showed that PfLPAT1B is localized in chloroplast. Complementation assays in the SM2-1 strain demonstrated that PfLPAT1B possesses LPAT enzymatic activity. Overexpression of PfLPAT1B gene in S. cerevisiae and N. tabacum significantly enhanced the total oil content, accompanied by increased levels of C16:0 and C16:1. Moreover, transgenic tobacco showed a notable increase in starch content and a decrease in soluble sugar content. Conclusion The PfLPAT1B gene from Perilla encodes a functional LPAT enzyme. Heterologous overexpression of PfLPAT1B can significantly enhance oil biosynthesis and accumulation and alter the content of major fatty acids in host tissues.

Key words: Perilla frutescens, lysophosphatidic acid acyltransferase, oil biosynthesis and accumulation, sequence characteristic analysis, expression characteristic analysis, Escherichia coli complementation function test, yeast transformation, tobacco heterologous expression

HUANG Xu-sheng, ZHOU Ya-li, CHAI Xu-dong, WEN Jing, WANG Ji-ping, JIA Xiao-yun, LI Run-zhi. Cloning of Plastidial PfLPAT1B Gene from Perilla frutescens and Its Functional Analysis in Oil Biosynthesis[J]. Biotechnology Bulletin, 2025, 41(7): 226-236.

Figures/Tables 8

Table 1 Primer sequence used in this study

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）	酶切位点 Restriction site	用途 Purpose
PfActin-q-F	AGACCTTCAATGTGCCAGCCA		内参基因
PfActin-q-R	CACGACCAGCAAGATCCAACC		内参基因
PfLPAT1B-q-F	TTAGACAGCAGAAGCCAAT		实时荧光定量PCR
PfLPAT1B-q-R	CCAGTTCCAACCAGAGTAA		实时荧光定量PCR
PfLPAT1B-1300-F	ATACACCAAATCGACTCTAGAATGGAATTATCTCTTCATTTTAGCCA	Xba Ⅰ、 Spe Ⅰ	亚细胞定位表达载体构建
PfLPAT1B-1300-R	CATGGTACCGGATCCACTAGTGCCTTGACGAGTAAGTTCCTGAG	Xba Ⅰ、 Spe Ⅰ	亚细胞定位表达载体构建
PfLPAT1B-pET28a-F	GGATAACAATTCCCCTCTAGAATGGAATTATCTCTTCATTTTAGCCA	Xba Ⅰ、 BamH Ⅰ	大肠杆菌表达载体构建
PfLPAT1B-pET28a-R	ACGGAGCTCGAATTCGGATCCCTAGCCTTGACGAGTAAGTTCCTGA	Xba Ⅰ、 BamH Ⅰ	大肠杆菌表达载体构建
PfLPAT1B-pYES2.0-F	ATTAAGCTTGGTACCGAGCTCATGGAATTATCTCTTCATTTTAGCCA	Sac Ⅰ、 Xba Ⅰ	酵母表达载体构建
PfLPAT1B-pYES2.0-R	TACATGATGCGGCCCTCTAGACTAGCCTTGACGAGTAAGTTCCTGA	Sac Ⅰ、 Xba Ⅰ	酵母表达载体构建
PfLPAT1B-1303-F	AACCTGCAGGTCGACTCTAGAATGGAATTATCTCTTCATTTTAGCCA	Xba Ⅰ、 Sma Ⅰ	植物过表达载体构建
PfLPAT1B-1303-R	TAAACGAGCTCGGTACCCGGGCTAGCCTTGACGAGTAAGTTCCTGA	Xba Ⅰ、 Sma Ⅰ	植物过表达载体构建

Fig. 1 Analysis of PfLPAT1B protein domain (A), conserved amino acid sequence (B), and phylogenetic tree (C)

Fig. 2 Cloning of PfLPAT1B gene (A) and its expression patterns in different tissues of perilla (B)M: DNA marker DL 2 000; 1: PCR detection of PfLPAT1B gene. Different lower letters indicate significant differences at P<0.05 level. The same below

Fig. 3 Subcellular localization of PfLPAT1B protein

Fig. 4 Enzymatic activity assay of PfLPAT1B protein from perilla

Fig. 5 Molecular docking of PfLPAT1B protein in perilla

Fig. 6 Lipid droplets (A), total oil (B), and FA profiles (C) of the PfLPAT1B-transgenic yeastINVSc1: The wild-type yeast strain of S. cerevisiae. EV: Yeast strains carrying pYES2.0 empty vector. PfLPAT1B: Yeast strains expressing PfLPAT1B gene

Fig. 7 Contents of oil and other metabolites in PfLPAT1B-transgenic tobaccoWT: Wild type tobacco plant lines. OE-1/-2/-3: Transgenic tobacco plant lines overexpressing PfLPAT1B gene

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