发菜β-酮脂酰ACP合成酶Ⅱ(FabF)的基因克隆表达与生物信息学分析

Gene Cloning,Expression and bioinformatics analysis ofβ-ketoacyl-(acyl carrier protein)SynthaseⅡ(FabF)from Nostoc flagelliforme

FabF基因编码的β-酮脂酰ACP合成酶Ⅱ是脂肪酸合成循环反应中的关键酶之一。为了解发菜Fab F分子信息,本研究通过设计特异性引物克隆发菜Fab F基因,对Fab F的核苷酸序列及推译的氨基酸序列进行生物信息学分析并进行原核表达。结果表明:①发菜Fab F全长为1251 bp(Gen Bank的登录号为KX421859),氨基酸序列中包含Thr磷酸化位点11个、Tyr磷酸化位点7个、Ser磷酸化位点15个;第72位的赖氨酸(Lys)亲水性最强,第358位的缬氨酸(Val)疏水性最强。②二级结构和三级结构预测结果表明Fab F蛋白主要是由随机卷曲、α螺旋、β折叠和β转角构成。③将Fab F基因在大肠杆菌中表达,得到一个43.9 k D的外源蛋白,对此蛋白进行MALDI-TOF/TOF-MS鉴定,证明该蛋白为Fab F蛋白。研究结果为进一步探索发菜Fab F基因的功能及其在逆境条件下的表达调控和脂肪酸合成途径奠定了基础。

Theβ-ketoacyl acyl ACP synthaseⅡwhich is one of the key enzymes in the synthesis of fatty acids is encoded by FabF gene.In this study,FabF gene was cloned by specific primers in order to study its molecular information.Bioinformatics methods were conducted on the sequences of DNA and ammonia acid of FabF gene which was expressed in prokaryotic system.The results showed that①the length of FabF gene was 1251 bp with accession number being KX421859 in Gen Bank.The amino acid sequence included 11 Thr phosphorylation sites,7 Tyr phosphorylation sites and 15 Ser phosphorylation sites.The Lys in site 72 displayed the strongest hydrophilicity,while Val in site 358 was found to have the strongest hydrophobicity.②Prediction by secondary and tertiary structures indicated that FabF protein was mainly composed of random coil,α-helix,β-sheet andβ-turn.③A 43.9 k D exogenous protein was obtained by the expression of FabF gene in Escherichia coli.Based on MALDI-TOF/TOF-MS,the mass spectrum analysis proved that this obtained exogenous protein was FabF protein.The results lay a foundation for further study on expression and regulation of FabF gene and fatty acid synthesis pathway of Nostoc flagelliforme.