Zheng Ling, Shockey Jay, Bian Fei, Chen Gao, Shan Lei, Li Xinguo, Wan Shubo, Peng Zhenying
College of Life Science, Shandong University, Jinan, China.
Shandong Provincial Key Laboratory of Genetic Improvement, Ecology and Physiology of Crops, Department of Bio-Tech Research Center, Shandong Academy of Agricultural Sciences, Jinan, China.
Front Plant Sci. 2017 Oct 16;8:1751. doi: 10.3389/fpls.2017.01751. eCollection 2017.
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triacylglycerol (TAG) biosynthesis via the acyl-CoA-dependent acylation of diacylglycerol. This reaction is a major control point in the Kennedy pathway for biosynthesis of TAG, which is the most important form of stored metabolic energy in most oil-producing plants. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14.' Sequence analysis of 11 different peanut cultivars revealed a gene family of 8 peanut DGAT2 genes (designated AhDGAT2a-h). Sequence alignments revealed 21 nucleotide differences between the eight ORFs, but only six differences result in changes to the predicted amino acid (AA) sequences. A representative full-length cDNA clone (AhDGAT2a) was characterized in detail. The biochemical effects of altering the AhDGAT2a sequence to include single variable AA residues were tested by mutagenesis and functional complementation assays in transgenic yeast systems. All six mutant variants retained enzyme activity and produced lipid droplets in vivo. The N6D and A26P mutants also displayed increased enzyme activity and/or total cellular fatty acid (FA) content. N6D mutant mainly increased the content of palmitoleic acid, and A26P mutant mainly increased the content of palmitic acid. The A26P mutant grew well both in the presence of oleic and C18:2, but the other mutants grew better in the presence of C18:2. AhDGAT2 is expressed in all peanut organs analyzed, with high transcript levels in leaves and flowers. These levels are comparable to that found in immature seeds, where DGAT2 expression is most abundant in other plants. Over-expression of AhDGAT2a in tobacco substantially increased the FA content of transformed tobacco seeds. Expression of AhDGAT2a also altered transcription levels of endogenous tobacco lipid metabolic genes in transgenic tobacco, apparently creating a larger carbon 'sink' that supports increased FA levels.
二酰甘油酰基转移酶（DGAT）通过二酰甘油的酰基辅酶A依赖性酰化作用催化三酰甘油（TAG）生物合成的最后一步。该反应是TAG生物合成的肯尼迪途径中的一个主要控制点，而TAG是大多数产油植物中储存代谢能量的最重要形式。在本研究中，从花生品种‘鲁花14’中克隆了花生2型DGAT（AhDGAT2）基因。对11个不同花生品种的序列分析揭示了一个由8个花生DGAT2基因组成（命名为AhDGAT2a - h）的基因家族。序列比对显示8个开放阅读框之间有21个核苷酸差异，但只有6个差异导致预测的氨基酸（AA）序列发生变化。对一个代表性的全长cDNA克隆（AhDGAT2a）进行了详细表征。通过转基因酵母系统中的诱变和功能互补试验，测试了改变AhDGAT2a序列以包含单个可变AA残基的生化效应。所有6个突变变体均保留酶活性并在体内产生脂滴。N6D和A26P突变体还表现出酶活性和/或总细胞脂肪酸（FA）含量增加。N6D突变体主要增加了棕榈油酸的含量，而A26P突变体主要增加了棕榈酸的含量。A26P突变体在油酸和C18:2存在的情况下生长良好，但其他突变体在C18:2存在的情况下生长更好。AhDGAT2在所有分析的花生器官中均有表达，在叶和花中的转录水平较高。这些水平与未成熟种子中的水平相当，而在其他植物中DGAT2在未成熟种子中的表达最为丰富。在烟草中过表达AhDGAT2a显著增加了转基因烟草种子的FA含量。AhDGAT2a的表达还改变了转基因烟草中内源烟草脂质代谢基因的转录水平，显然形成了一个更大的碳“汇”，从而支持了FA水平的增加。
