小球藻Chlorella variabilis NC64A二酰甘油酰基转移酶基因的克隆表达与功能研究
Clone, Expression and Function Analysis of Diacylglycerol Acyltransferase Gene from Chlorella variabilis NC64A

摘要 为了揭示二酰甘油酰基转移酶(DGAT)在小球藻油脂合成过程中的作用, 对单细胞小球藻Chlorell avariabilis NC64A 的二酰甘油酰基转移酶进行原核克隆表达及功能初步研究。结果表明, 其编码序列为894bp, 编码 297 个氨基酸, 表达蛋白的表观分子量为 33 kDa, pI 9.48。保守结构域分析表明, 该蛋白属于Lysophospholipid acyltransferases (LPLATs)超家族, 具有二酰甘油酰基转移酶活性, 序列位点H68, L71, F76, R94, I97 和GAA (144？146)组成特定的酰基受体结合口袋, 能够结合酰基？酰基载体蛋白(ACP)或者酰基辅酶A上的酰基, 催化三酰甘油合成的最后一步。表达蛋白与SsPDAT 和AtPDAT 的序列相似性分别为32%和24%, 表明该蛋白可能具有磷脂酰甘油酰基转移酶(PDAT)活性, 能利用磷脂上的酰基合成三酰甘油。因此, 采用薄层层析方法以L-α-磷脂酰胆碱和 1,2-二油酰-sn-甘油为底物, 检测到其确实具有PDAT 活性, 表明限氮条件下NC64A 中DGAT 的PDAT 活性可能促进了膜脂降解耦合三酰甘油的合成。
Abstract To reveal the function of diacylglycerol acyltransferase (DGAT) on algae lipid production, DGAT from Chlorella variabilis NC64A was cloned and expressed by E.coli BL21 (DE3). The results showed that the DGAT gene was 894 bp and was coded 297aa. The apparent molecular weight of DGAT was 33 kDa and the pI was 9.48. Conserved domain analysis showed that it belonged to the Lysophospholipid acyltransferases (LPLATs) super family and the amino acids of H68, L71, F76, R94, I97 and GAA (144–146) could form special acyl acceptor binding pocket to bind acyl of ACP or CoA and served as the catalyst in the synthesis of TAG. Sequence analysis showed that DGAT shared a 32% and 24% homology with SsPDAT and AtPDAT respectively. Thin layer chromatography showed that DGAT had PDAT enzyme activity, which may promote the membrane lipid degradation and couple TAG synthesis under nitrogen starvation.