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
发布时间：2018-12-18 01:19
【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Chlorella variabilis 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 k Da,p I 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Ss PDAT和At PDAT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]:In order to elucidate the role of diacylglyceryl transferase (DGAT) in the synthesis of Chlorella oil, the prokaryotic expression and function of diacylglyceryl transferase (Chlorella variabilis NC64A) of Chlorella sp. Chlorella variabilis NC64A were studied. The results showed that the encoding sequence was 894 BP, encoding 297 amino acids, and the apparent molecular weight of the expressed protein was 33k Da,p I 9.48. The conserved domain analysis showed that the protein belongs to the Lysophospholipid acyltransferases (LPLATs) superfamily and has the activity of diacylglyceryl transferase. Can bind acyl groups? Acyl carrier protein (ACP) or acyl coenzyme A catalyzes the last step in the synthesis of triacylglycerol. The sequence similarity between the expressed protein and Ss PDAT and At PDAT were 32% and 24% respectively, which indicated that the protein might have the activity of phosphatidyl glyceryl transferase (PDAT) and could synthesize triacylglycerol from phospholipid acyl group. Therefore, using L- 伪 -phosphatidylcholine and 1- 2-dioleyl-sn- glycerol as substrates, it was found that L- 伪 -phosphatidylcholine and 1 ~ (2 +) -dioleyl-sn- had PDAT activity by thin-layer chromatography. The results showed that the PDAT activity of DGAT in NC64A could promote the synthesis of lipide-coupled triglycerides under nitrogen limit.
【作者单位】： 农业生物技术国家重点实验室中国农业大学生物学院;
【基金】：863计划(2013AA065802) 农业生物技术国家重点实验室2014年开放课题(2014SKLAB06)资助
【分类号】：Q943.2
本文编号：2385073
[Abstract]:In order to elucidate the role of diacylglyceryl transferase (DGAT) in the synthesis of Chlorella oil, the prokaryotic expression and function of diacylglyceryl transferase (Chlorella variabilis NC64A) of Chlorella sp. Chlorella variabilis NC64A were studied. The results showed that the encoding sequence was 894 BP, encoding 297 amino acids, and the apparent molecular weight of the expressed protein was 33k Da,p I 9.48. The conserved domain analysis showed that the protein belongs to the Lysophospholipid acyltransferases (LPLATs) superfamily and has the activity of diacylglyceryl transferase. Can bind acyl groups? Acyl carrier protein (ACP) or acyl coenzyme A catalyzes the last step in the synthesis of triacylglycerol. The sequence similarity between the expressed protein and Ss PDAT and At PDAT were 32% and 24% respectively, which indicated that the protein might have the activity of phosphatidyl glyceryl transferase (PDAT) and could synthesize triacylglycerol from phospholipid acyl group. Therefore, using L- 伪 -phosphatidylcholine and 1- 2-dioleyl-sn- glycerol as substrates, it was found that L- 伪 -phosphatidylcholine and 1 ~ (2 +) -dioleyl-sn- had PDAT activity by thin-layer chromatography. The results showed that the PDAT activity of DGAT in NC64A could promote the synthesis of lipide-coupled triglycerides under nitrogen limit.
【作者单位】： 农业生物技术国家重点实验室中国农业大学生物学院;
【基金】：863计划(2013AA065802) 农业生物技术国家重点实验室2014年开放课题(2014SKLAB06)资助
【分类号】：Q943.2
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