Oil [2,8?11]. These enzymes have unique expression patterns in a variety of

Oil [2,8?11]. These enzymes have unique expression patterns in a variety of plant tissues [7] and may have other roles besides seed oilaccumulation, such as FA mobilization [12] and leaf senescence [13]. The process by which such enzymes are regulated 22948146 in developing seeds and other tissues remains poorly understood. Much research has focused on DGATs because of their important roles in TAG synthesis. Overexpression of such genes can greatly increase the oil content of transgenic organisms. For example, overexpression of the Arabidopsis DGAT1 gene in tobacco and yeast greatly enhanced the TAG content of the transformed lines [14?5]. Interestingly, Ricinus communis DGAT2 (RcDGAT2) has a strong preference for hydroxyl FAs containing diacylglycerol (DAG) substrates, the levels of which increased from 17 to nearly 30 when E7449 manufacturer RcDGAT2 was expressed in Arabidopsis [10]. In Ricinus seeds, RcDGAT2 expression was 18-fold higher than in leaves, whereas RcDGAT1 expression differed little between seeds and leaves. Hence, RcDGAT2 probably plays a more important role in castor bean seed TAG biosynthesis than RcDGAT1 [2]. In addition, OeDGAT1 from the olive tree Olea europaea is responsible for most TAG deposition in seeds, while OeDGAT2 may be a key mediator of higher oil yields in ripening mesocarps [16].Peanut Diacylglycerol Acyltransferase 2 ExpressionRecombinant proteins can be used as alternatives to endogenous ones to study their structures and functions or to make hightiter antibodies that recognize them. Because most DGATs are integral membrane proteins, they are difficult to express and purify in heterologous expression systems [17,18]; thus far, only limited success has been achieved in this area [18?0]. Weselake et al. expressed the N-terminal 116 amino acid residues of Brassica napus (oilseed rape) DGAT1 as a His-tagged protein in Escherichia coli [16]. The resulting recombinant BnDGAT1(1?16)His6 interacted with long chain acyl-CoA and displayed enhanced affinity for erucoyl (22:1cisD13)-CoA over oleoyl (18:1cisD9)-CoA [18]. Subsequently, the amino terminal 95 residues of mouse DGAT1 were expressed in E. coli with similar results [19]. Encouragingly, fulllength DGAT1 expression from the tung tree (Vernicia fordii) in E. coli has been achieved [20]. In this case, the recombinant protein was mostly targeted to the membranes, and there were insoluble fractions with extensive degradation from the carboxyl end as well as association with other proteins, lipids, and membranes. Arachis hypogaea (peanut, Fabaceae) is one of the most economically-important oil-producing crops, so the fact that peanut DGATs have not been extensively studied is surprising. Saha et al. identified a soluble DGAT3 from immature peanut eFT508 cotyledons and expressed its full length in E. coli, where the recombinant protein had high levels of DGAT activity but no wax ester synthase activity [5]; this is the only published report on peanut DGATs thus far. Here, we identified two isozymes of DGAT2 in peanut and expressed both of them as full-length recombinant proteins in E. coli. This is the first time that a full-length recombinant DGAT2 protein from peanut has been successfully expressed in E. coli, and the first evaluation of its effects on the growth and FA content of the transformed E. coli strains studied.39). PCRs were performed according to the manufacturer’s protocol. The fragments were sequenced and assembled into a full-length sequence. Based on the full-length sequence.Oil [2,8?11]. These enzymes have unique expression patterns in a variety of plant tissues [7] and may have other roles besides seed oilaccumulation, such as FA mobilization [12] and leaf senescence [13]. The process by which such enzymes are regulated 22948146 in developing seeds and other tissues remains poorly understood. Much research has focused on DGATs because of their important roles in TAG synthesis. Overexpression of such genes can greatly increase the oil content of transgenic organisms. For example, overexpression of the Arabidopsis DGAT1 gene in tobacco and yeast greatly enhanced the TAG content of the transformed lines [14?5]. Interestingly, Ricinus communis DGAT2 (RcDGAT2) has a strong preference for hydroxyl FAs containing diacylglycerol (DAG) substrates, the levels of which increased from 17 to nearly 30 when RcDGAT2 was expressed in Arabidopsis [10]. In Ricinus seeds, RcDGAT2 expression was 18-fold higher than in leaves, whereas RcDGAT1 expression differed little between seeds and leaves. Hence, RcDGAT2 probably plays a more important role in castor bean seed TAG biosynthesis than RcDGAT1 [2]. In addition, OeDGAT1 from the olive tree Olea europaea is responsible for most TAG deposition in seeds, while OeDGAT2 may be a key mediator of higher oil yields in ripening mesocarps [16].Peanut Diacylglycerol Acyltransferase 2 ExpressionRecombinant proteins can be used as alternatives to endogenous ones to study their structures and functions or to make hightiter antibodies that recognize them. Because most DGATs are integral membrane proteins, they are difficult to express and purify in heterologous expression systems [17,18]; thus far, only limited success has been achieved in this area [18?0]. Weselake et al. expressed the N-terminal 116 amino acid residues of Brassica napus (oilseed rape) DGAT1 as a His-tagged protein in Escherichia coli [16]. The resulting recombinant BnDGAT1(1?16)His6 interacted with long chain acyl-CoA and displayed enhanced affinity for erucoyl (22:1cisD13)-CoA over oleoyl (18:1cisD9)-CoA [18]. Subsequently, the amino terminal 95 residues of mouse DGAT1 were expressed in E. coli with similar results [19]. Encouragingly, fulllength DGAT1 expression from the tung tree (Vernicia fordii) in E. coli has been achieved [20]. In this case, the recombinant protein was mostly targeted to the membranes, and there were insoluble fractions with extensive degradation from the carboxyl end as well as association with other proteins, lipids, and membranes. Arachis hypogaea (peanut, Fabaceae) is one of the most economically-important oil-producing crops, so the fact that peanut DGATs have not been extensively studied is surprising. Saha et al. identified a soluble DGAT3 from immature peanut cotyledons and expressed its full length in E. coli, where the recombinant protein had high levels of DGAT activity but no wax ester synthase activity [5]; this is the only published report on peanut DGATs thus far. Here, we identified two isozymes of DGAT2 in peanut and expressed both of them as full-length recombinant proteins in E. coli. This is the first time that a full-length recombinant DGAT2 protein from peanut has been successfully expressed in E. coli, and the first evaluation of its effects on the growth and FA content of the transformed E. coli strains studied.39). PCRs were performed according to the manufacturer’s protocol. The fragments were sequenced and assembled into a full-length sequence. Based on the full-length sequence.