French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boker Campus, Israel.
FEBS J. 2011 Oct;278(19):3651-66. doi: 10.1111/j.1742-4658.2011.08284.x. Epub 2011 Sep 6.
We have identified and isolated a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)1-like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full-length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63-bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid-deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C(16) and C(18) fatty acids into TAG.
我们从硅藻微藻三角褐指藻（Phaeodactylum tricornutum）中鉴定并分离出一种 cDNA，它编码一种新型酰基辅酶 A：二酰基甘油酰基转移酶（DGAT）1 样蛋白（PtDGAT1）。PtDGAT1 全长 cDNA 序列表明，该基因转录两种类型的 mRNA，即 PtDGAT1short 和 PtDGAT1long。PtDGAT1short 编码一个 565 个氨基酸的序列，与几种功能明确的高等植物 DGAT1 蛋白同源，与假定的硅藻塔玛亚历山大藻的 DGAT1 有 55%的相同性，但与其他可用的假定和克隆藻类 DGAT 序列的同源性很小。PtDGAT1long 由于在包含终止密码子的 mRNA 中缺失 63bp 核苷酸，因此缺少几个催化结构域。内含子保留的选择性剪接似乎调节了活性 DGAT1 的产生量，为三角褐指藻在氮饥饿条件下增加三酰基甘油（TAG）生物合成提供了一种可能的分子机制。DGAT 介导 TAG 生物合成的最后一个关键步骤，因此我们研究了在氮饥饿诱导 TAG 积累时两种类型 mRNA 表达水平的变化。在氮饥饿下，两种转录物的丰度都明显增加，但 PtDGAT1short 的增加幅度要小得多。PtDGAT1short 的 PtDGAT1 活性在一个异源酵母转化系统中得到了证实，该系统通过恢复 Saccharomyces cerevisiae 中性脂缺陷的四重突变体菌株（H1246）的 DGAT 活性，导致脂滴形成。只有在表达 PtDGAT1short 而不是 PtDGAT1long 时，才会恢复脂滴的形成。重组酵母似乎更倾向于将饱和的 C(16)和 C(18)脂肪酸掺入 TAG 中。
