br Materials and methods br Results

Materials and methods
Results
Discussion The first DGAT1 gene was described in the mouse in 1998 [28], and a few years later DGAT2 was identified from the oleaginous fungus Mortierella ramanniana[29]. Although both of them are known as the primary enzymes for de novo TAG biosynthesis, they share no similarities in amino Apamin receptor sequence and differ in their biochemical, cellular and physiological functions [30]. In contrast to DGAT1 that is structurally related to sterol: acyl-CoA acyltransferase, DGAT2 has more homology with monoacylglycerolacyl transferases (MGATs) and acyl-CoA wax-alcohol acyltransferases. According to the phylogenetic tree shown in Fig. 3, MiDGAT1 and 2 are clearly separated into two distinct families, indicating the convergent evolution between them. So far, both DGAT1 and 2 have been found in various living organisms [6], [24], but there are very few studies on DGATs in microalgae in spite of the availability of increasing full genomic sequences of microalgae. For example, PtDGAT1 from the diatom Phaeodactylum tricornutum is the only algal DGAT1 that has been biochemically characterized [27]. In Chlamydomonas reinhardtii, a gene for a DGAT1-type enzyme was identified after transcript-based correction of gene models [31]. In this study, three putative DGAT genes, including one MiDGAT1 and two MiDGAT2s, from the green alga M. incisa were reported for the first time. In general, algae have multiple DGAT2 homologous genes, which is contrary to most living organisms that contain only a single DGAT2 gene. For example, C. reinhardtii has 5 type-2 DGATs, namely DGAT2A-E (or DGTT1-5) [32]. It could be resulted from the lineage-specific duplication events [33]. Also, those algae-derived DGAT2s may be very divergent from each other, even among isoforms within the same species [7]. Prediction results showed that MiDGAT1 had 9 transmembrane domains, whereas MiDGAT2A and 2B had two. This is in line with previous observation that DGAT1s are bigger proteins with more transmembrane domains than DGAT2s [24]. Besides, an ER retrieval motif was also found in both MiDGAT1 and MiDGAT2s. These structural features supported the role of MiDGATs as integral membrane proteins localized to the ER [7], [16]. It is worth noting that MiDGAT1 had a special region, namely PH domain located in the region of amino acids 47–153. PH domain is a small protein module consisting of 100–200 amino acids, which is commonly found in a variety of proteins involved in cell signaling and cytoskeletal rearrangement [34], however they have been rarely reported in algae. Results (Fig. 4) showed that the mount of TAGs produced by the yeast H1246 carrying pY-MiDGAT1-ΔPH was significantly reduced, suggesting the important roles of PH domain for MiDGAT1. An important feature of PH domain is that it binds with high affinity to proteins or phosphoinositides on the membrane to respond to lipid messengers [35], and according to this characteristic, we speculate that PH domain may promote the binding of MiDGAT1 to membranes. As a result, DGAT1 could bind to membranes via PH domain which functions as an anchor to fully exert the enzymatic activity. For MiDGAT2s, multiple sequence alignment of amino acids indicated that both MiDGAT2A and 2B contained a conserved HPHG motif, which is a characteristic sequence found in DGAT2 members as well as other closely related families [7]. Stone et al. [16] created a triple mutation of ‘HPH’ to ‘AGA’ in murine DGAT2 and observed an 80% reduction in catalytic activity, indicating the crucial roles of HPHG for the full enzymatic function. Previous studies also suggested this conserved motif may have variations among algal DGAT2s or even between the same species. For example, among five putative DGAT2s in C. reinhardtii, only one had the conserved ‘HPHG’ sequence (DGAT2A had CPHSA; DGAT2B had HPHG; DGATC, D, E had XPHG) [7]. Based on the results of using the heterologous host system, we further analyzed the expression patterns of MiDGATs. Nitrogen starvation stress was used as an inducer, because TAG synthesis in microalgae is highly dependent on the varying environmental conditions. Of the three MiDGAT genes, MiDGAT2A was the only one responsive to nitrogen deficiency, whilst MiDGAT1 and MiDGAT2B were not significantly affected. Furthermore, the up- and down-regulation of MiDGAT2A was consistent with the TAG accumulation within algal cells. These results suggested that among three MiDGAT genes, the regulation of MiDGAT2A alone might be sufficient to induce TAG accumulation under the nitrogen starvation stress. Similar studies were carried out using other algae: in C. reinhardtii, both CrDGAT1 and CrDGAT2B (DGTT1) were found to contribute to the TAG synthesis in response to nitrogen deficiency, whilst other members of CrDGAT2, i.e. DGTT 2-5 were not involved or detected [26], [30], [31]. In P. tricornutum, PtDGAT1 was highly responsive to nitrogen deprivation, whereas all PtDGAT2s did not get regulated [27]. These results indicated that the expression patterns of DGATs in microalgae were species-dependent.