High-Tech Research Center, Shandong Academy of Agricultural Science, Jinan, China.
PLoS One. 2013 Apr 11;8(4):e61363. doi: 10.1371/journal.pone.0061363. Print 2013.
Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.
二酰甘油酰基转移酶（DGAT）是真核生物三酰基甘油生物合成的限速酶。三酰基甘油是花生、大豆和油菜等植物中重要的储能油。本研究采用同源基因序列法和快速扩增 cDNA 末端法，从花生品种‘鲁花 14’中克隆出花生型 2 型 DGAT（AhDGAT2）基因。为了了解 AhDGAT2 在三酰基甘油生物合成中的作用，我们将两个氨基酸序列差异 3 个氨基酸的 AhDGAT2 核苷酸序列表达为大肠杆菌 Rosetta（DE3）中的谷胱甘肽 S-转移酶（GST）融合蛋白。经 IPTG 诱导后，同工酶（AhDGAT2a 和 AhDGAT2b）以 64.5 kDa GST 融合蛋白的形式表达。AhDGAT2a 和 AhDGAT2b 均存在于宿主细胞质和包涵体中，包涵体中含量较多。与非转化细胞相比，AhDGATs 的过表达会降低宿主细胞的生长速率，但携带空载体、AhDGAT2a-GST 或 AhDGAT2b-GST 的细胞生长速率没有明显差异。有趣的是，诱导 AhDGAT2a-GST 和 AhDGAT2b-GST 蛋白的表达使宿主细胞的大小增加了 2.4-2.5 倍（诱导后）。AhDGAT2a-GST 和 AhDGAT2a-GST 转化体的总脂肪酸（FA）水平以及 C12:0、C14:0、C16:0、C16:1、C18:1n9c 和 C18:3n3 FA 水平显著升高，而 C15:0 和 C21:0 水平低于非转化细胞或携带空载体的细胞。此外，两个转化株系之间的一些 FA 水平存在差异，表明这两种同工酶在花生中的功能可能不同。这是首次在细菌表达系统中产生全长重组花生 DGAT2，并首次分析其对大肠杆菌中脂肪酸含量和组成的影响。我们的结果表明，AhDGAT2 是大肠杆菌中高效 FA 生产的候选基因。
