紫苏质体型PfLPAT1B基因的克隆及其在油脂合成中的功能分析

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

摘要/Abstract

摘要：

目的溶血磷脂酸酰基转移酶（lysophosphatidic acid acyltransferase, LPAT）在植物生长发育和脂质代谢等过程中发挥重要作用，探究紫苏PfLPAT1B基因的生物学功能，为紫苏及其他油料作物的遗传改良和新品系培育提供科学依据。方法从紫苏基因组数据库鉴定PfLPAT1B基因序列，利用组学工具分析其序列特征及系统进化，通过RT-qPCR检测PfLPAT1B基因在紫苏不同组织及种子不同发育时期表达特性，利用LPAT缺陷型菌株SM2-1检测PfLPAT1B蛋白的活性，通过酿酒酵母与烟草遗传转化分析PfLPAT1B在油脂合成中的功能。结果 PfLPAT1B共编码369个氨基酸，为碱性不稳定亲水蛋白；PfLPAT1B具有典型的LPAT酰基转移酶活性保守结构域。PfLPAT1B基因在紫苏不同组织及种子不同发育时期均有表达，在花中的表达量最高，且随着种子发育PfLPAT1B的表达量逐渐升高。亚细胞定位结果表明紫苏PfLPAT1B定位于叶绿体。SM2-1菌株互补酶活检测证明紫苏PfLPAT1B具有LPAT酶活性。酵母与烟草转化结果表明，过表达PfLPAT1B基因导致转基因酵母及烟草总油脂含量显著上升，且C16：0和C16：1含量显著上升。此外，过表达PfLPAT1B促进转基因烟草淀粉含量显著上升，可溶性糖含量显著降低，表明PfLPAT1B过表达可能促进转基因烟草碳通量的分配，使其从糖代谢途径进入油脂代谢途径。结论紫苏PfLPAT1B基因编码蛋白具有LPAT酰基转移酶活性，异源过表达PfLPAT1B可显著提高宿主组织油脂产量，并改变主要脂肪酸组分的含量。

关键词: 紫苏, 溶血磷脂酸酰基转移酶, 油脂合成积累, 序列特征分析, 表达特性分析, 大肠杆菌互补功能测试, 酿酒酵母转化, 烟草异源表达

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

黄旭升, 周雅莉, 柴旭东, 闻婧, 王计平, 贾小云, 李润植. 紫苏质体型PfLPAT1B基因的克隆及其在油脂合成中的功能分析[J]. 生物技术通报, 2025, 41(7): 226-236.

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.

图/表 8

表1 本研究所用引物序列

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

图1 PfLPAT1B蛋白结构域（A）、保守氨基酸序列（B）及系统进化树（C）分析

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

图2 紫苏PfLPAT1B基因克隆（A）及其组织特异性表达分析（B）M：DNA marker DL 2 000；1：PfLPAT1B基因PCR检测。不同小写字母表示在P<0.05水平差异显著，下同

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

图3 PfLPAT1B蛋白的亚细胞定位

Fig. 3 Subcellular localization of PfLPAT1B protein

图4 紫苏PfLPAT1B蛋白酶活性检测

Fig. 4 Enzymatic activity assay of PfLPAT1B protein from perilla

图5 紫苏PfLPAT1B蛋白分子对接

Fig. 5 Molecular docking of PfLPAT1B protein in perilla

图6 PfLPAT1B转基因酵母油滴（A）、总油脂（B）及脂肪酸组分含量（C）INVSc1：酿酒酵母野生型菌株；EV：转化pYES2.0空载酵母；PfLPAT1B：酿酒酵母野生型菌株转化PfLPAT1B基因

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

图7 PfLPAT1B转基因烟草油脂及其他代谢产物含量WT：野生型烟草株系；OE-1/-2/-3：PfLPAT1B转基因烟草不同株系

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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