摘要
溶血磷脂酸酰基转移酶基因(lysophosphatideacid acyltransferases gene,LPAAT)是油脂合成的关键基因之一,其催化溶血磷脂酸合成磷脂酸,是高等植物Kennedy途径中的关键酶。本研究从油葵(Helianthus annuus)中克隆得到3个HaLPAAT2基因,分别命名为HaLPAAT2.1,HaLPAAT2.2和HaLPAAT2.3。对这3个HaLPAAT2进行了生物信息分析并将其转入SM2-1大肠杆菌突变体中验证LPAAT活性。结果表明:HaLPAAT2.1、HaLPAAT2.2和HaLPAAT2.3基因全长分别为1158、1137、1149 bp,分别编码385、378、382个氨基酸。预测3个基因的编码蛋白都含有LPLAT superfamily酰基转移酶结构域。跨膜结构分析显示这3个HaLPAAT2蛋白都含有3个跨膜结构域。亚细胞定位预测这3个蛋白都位于内质网上。对其理化性质分析表明这3个HaLPAAT2均为不稳定性蛋白。构建了HaLPAAT2.1、HaLPAAT2.2和HaLPAAT2.3基因的原核表达载体,并转化大肠杆菌(Escherichia coli)功能缺失突变体SM2-1进行功能验证。互补试验表明,3个HaLPAAT2基因都具有大肠杆菌LPAAT活性,可以互补大肠杆菌LPAAT突变体SM2-1菌株的活性。
Lysophosphatideacid acyltransferases gene(LPAATs)is one of the key genes for lipid synthesis.It catalyzes the synthesis of phosphatidic acid(PA)by lysophosphatidic acid(LPA)and plays a critical role in the Kennedy pathway in higher plants.In this study,three HaLPAAT2 genes(HaLPAAT2.1,HaLPAAT2.2 and HaLPAAT2.3)were cloned from the Helianthus annuus L..Bioinformatics analysis was performed on the three HaLPAAT2 s and then transferred them into SM2-1 Escherichia coli mutants to verify the LPAAT activity.The results showed that the full lengths of HaLPAAT2.1,HaLPAAT2.2 and HaLPAAT2.3 genes were 1158 bp,1137 bp and1149 bp respectively,of which encoded 385,378 and 382 amino acids accordingly,and it was predicted that all three proteins contained LPLAT superfamily acyltransferase domain.Transmembrane structure analysis showed that all these HaLPAAT2 proteins contained three transmembrane domains.Subcellular localization predicted that the three proteins were located on the endoplasmic reticulum.Analysis of its physical and chemical properties showed that the three HaLPAAT2 belonged to unstable proteins.Prokaryotic expression vectors of HaLPAAT2.1,HaLPAAT2.2 and HaLPAAT2.3 genes were constructed and transformed into E.coli functional deletion mutant SM2-1 for functional verification.Complementary experiments showed that the three HaLPAAT2 genes had E.coli LPAAT activity,which could complement the activity of E.coli LPAAT mutant SM2-1 strain.
作者
王岚
达红玉
周至铭
张程
杨佳宝
孙黎
Wang Lan;Da Hongyu;Zhou Zhiming;Zhang Cheng;Yang Jiabao;Sun Li(College of Life Sciences,Shihezi University,Shihezi,832003)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第5期2208-2215,共8页
Genomics and Applied Biology
基金
国家自然科学基金项目(31760064,31360052)资助
