绿色巴夫藻脂肪酸去饱和酶的克隆和初步研究(英文)
THE CLONING AND PREL IM INARY CHARACTERIZATION OF A C18 ∶0Δ9 DESATURASE GENE FROM MARINE M ICROALGAE PAVLOVA VI RID IS
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摘要: 在高等植物中，Δ9脂肪酸去饱和酶引入第一个双键到饱和的脂肪酸链中，导致单不饱和脂肪酸的形成。我们通过RT-PCR、RNA ligase mediated RACE(RLM-RACE)and Overlap-PCR方法从海洋微藻绿色巴夫藻中克隆到一个命名为PvfadA的脂肪酸去饱和酶候选基因。通过将PvfadA基因在大肠杆菌表达系统中成功表达，PvFadA可以特异性地将C18∶0脂肪酸转变成C18∶1脂肪酸。PvFadA的氨基酸序列中存在一个存在于acyl-ACP去饱和酶的特异性金属离子结合区段(D／E)X2HX～100(D／E)X2H。通过同源模建PvFadA的3D结构显示，其包含了11个α螺旋，其中α3、α4、α6和α7组成了一个4个螺旋桶的核心结构，预测其可能是酶的活性中心。PvFadA的3D结构类似于蓖麻和结核分枝杆菌H37Rv的acyl-ACP去饱和酶。
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关键词:
- 硬脂酰-ACP /
- Δ9去饱和酶 /
- RLM-RACEPCR /
- 绿色巴夫藻 /
- GC-MS
Abstract: In higher plant, Δ9 desaturase introduces the first double bond into saturated fatty acids, resulting inthe corresponding monounsaturated fatty acids.We have cloned gene, designated as PvfadA, for C18∶0Δ9 desaturase catalytic activity from Pavlova viridis by RT-PCR, RNA ligase mediated RACE (RLM-RACE) and Overlap-PCR strategy. This desaturase, when expressed in Escherichia coli, desaturated stearic acid to yield oleic acid, but did not desaturate other fatty acids in E. coli. These results indicate that PvFadA is specific to stearic acid. The deduced amino acid sequences of PvFadA show that a putative (D /E) X2HX～100(D /E) X2 H metal binding motif, which specifically existed in acyl-ACP desaturase. Moreover, Amino acids of PvFadA are similar in part to those of acyl-ACP desaturases from higher plant, such as A rabidopsis thaliana, Oryza sativa and Glycine max, but not to those of acyl-lipid desaturases of Cyanobacteria, and acyl-CoA desaturases of higher plant. In addition, the 3D2model structure of PvFadA is composed of 11 helixes, moreover, α3, α4, α6 andα7 form a core 42helix bundle to regards as an active center in this enzyme, similar as acyl-ACP desaturase of Ricinus communis and M ycobacterium tuberculosis H37Rv.-
Keywords:
- Stearoyl-ACP /
- Δ9 desaturase /
- RLM-RACE PCR /
- Pavlova viridis /
- GC-MS
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