垂序商陆PaAACT基因克隆和表达分析

Cloning and Expression Analysis of PaAACT Gene from Phytolacca americana

目的:从垂序商陆根中克隆了商陆皂苷甲生物合成过程中关键酶乙酰乙酰辅酶A转移酶(acetoacetyl-CoA transferase,AACT)基因,进行生物信息学分析和原核表达。方法:提取垂序商陆根的总RNA,然后逆转录合成c DNA,在分析垂序商陆转录组数据的基础上,设计Pa AACT基因的特异性引物,聚合酶链式反应(PCR)扩增Pa AACT基因的c DNA序列,通过构建原核表达载体p ET-32a-PaAACT,诱导表达并且纯化目的蛋白。结果:Pa AACT基因开放阅读框为1 254 bp,编码417个氨基酸。生物信息学分析显示Pa AACT蛋白的分子式为C1 914H3 120N538O576S17,推测其相对分子质量为43. 43 k Da,理论等电点8. 90,不稳定系数32. 27,属于稳定蛋白质。根据生物信息学分析结果,Pa AACT蛋白属于硫解酶家族成员,在C末端含有硫解酶家族的1个保守位点和1个活性位点。Pa AACT蛋白可能位于细胞质中、不含信号肽、没有跨膜区。系统进化分析显示Pa AACT蛋白与甜菜等廖科植物AACT蛋白亲缘关系较近。经IPTG诱导,在大肠埃希菌BL21 (DE3)菌株中表达了Pa AACT重组蛋白,利用Ni2+亲和色谱获得了纯化的目的蛋白。结论:该研究从垂序商陆中克隆Pa AACT基因,为下一步测定Pa AACT酶催化活性、制备抗体奠定基础,也为研究其在商陆皂苷甲生物合成途径中的作用提供理论依据。

Objective:To clone the key enzyme gene involved in the biosynthesis of esculentoside A(EsA),acetoacetyl-CoA transferase(AACT)gene was cloned from Phytolacca americana for bioinformatics analysis and prokaryotic expression.Method:Total RNA was extracted from the root of P.americana,and then cDNA was synthesized through the reverse transcription.Based on analysis of the transcriptome data of P.americana,the specific primers of PaAACT gene were designed,and the cDNA sequence of PaAACT gene was amplified by polymerase chain reaction(PCR)method.Prokaryotic induction,expression and purification of the target protein were induced through the construction of the prokaryotic expression vector pET-32 a-PaAACT.Result:The open reading frame(ORF)of PaAACT gene was 1 254 bp,and encoded 417 amino acid residues.Bioinformatic analysis showed that the molecular formula of Pa AACT protein was C1 914 H3 120 N538 O576 S17,inferring that its molecular weight was 43.43 kDa,the theoretical isoelectric point was 8.90,and the instability index of PaAACT protein was 32.27,which was a stable protein.According to bioinformatics analysis,PaAACT protein was a member of the thiolase family and contained one conserved site and one active site of the thiolase family at the C-terminal.PaAACT protein may be located in the cytoplasm,without a signal peptide or transmembrane domain.The phylogenetic analysis indicated that Pa AACT protein showed the highest homology with AACT protein from polygonaceae plants(such as Beta vulgaris).The recombinant PaAACT protein was successfully expressed in Escherichia coli BL21(DE3)strain through IPTG induction,and the purified target protein was obtained by Ni2+affinity chromatography.Conclusion:In this study,the PaAACT gene was cloned from P.americana,which lays a foundation for further determination of enzyme activity assay of PaAACT and preparation of antibody,and provides the theoretical basis for studying its role in the biosynthesis pathway of EsA.