穿心莲糖苷水解酶基因ApGH的克隆、生物信息学分析及表达研究

Cloning,Bioinformatics Analysis,and Expression of A Glucoside Hydrolase Gene ApGH from Andrographis paniculata

目的:对穿心莲糖苷水解酶(ApGH)基因进行克隆、生物信息学分析、原核表达和相对表达量分析。方法:取穿心莲新鲜叶片,提取RNA并反转录为互补脱氧核糖核酸(cDNA)作为模板,以穿心莲转录组中筛选到的糖苷水解酶ApGH基因开放阅读框(ORF)设计特异性引物,进行聚合酶链式反应(PCR)扩增,经测序后获得的基因序列利用生物信息学软件预测基因编码蛋白特征,构建原核表达载体在大肠埃希菌中诱导表达目的蛋白质,并利用实时荧光定量PCR分析ApGH基因的表达模式。结果:克隆所得ApGH基因的ORF全长1734 bp,编码577个氨基酸(GenBank登录号:OR887606)。ApGH为稳定亲水蛋白质,无信号肽和跨膜结构域,属于糖苷水解酶家族;系统进化分析表明,ApGH归属于GH1家族。将ApGH基因构建到原核表达载体HIS-MBP-pET28a上,在大肠埃希菌Transetta(DE3)中成功表达出重组蛋白质。实时荧光定量PCR检测结果表明,ApGH基因在叶中表达量最高,茎和根中表达量较低。结论:克隆得到穿心莲ApGH基因并对其蛋白质序列特征进行了系统分析,证明其在大肠埃希菌中成功表达重组蛋白质,且主要在穿心莲叶中表达。研究结果为进一步探索穿心莲糖苷水解酶的催化功能提供了依据。

Objective:The glycoside hydrolase gene(ApGH)from Andrographis paniculata was cloned,and its bioinformatics,prokaryotic expression,and relative expression were analyzed.Methods:RNA was extracted from the fresh leaves of A.paniculata and reversely transcribed into complementary deoxyribonucleic acid(cDNA)as templates.Specific primers were designed according to the open reading frame(ORF)of the ApGH gene screened from the transcriptome of A.paniculata,after which polymerase chain reaction(PCR)amplification was performed.The gene sequence obtained after sequencing was used to predict the protein features encoded using bioinformatics software.Prokaryotic expression vectors were constructed to induce the expression of interest proteins in Escherichia coli.The expression of the ApGH gene was examined by real-time fluorescence quantitative PCR(real-time PCR).Results:The ORF of the ApGH gene was 1734 bp in length and encoded 577 amino acid residues(GenBank accession number:OR887606).It was a stable hydrophilic protein without signal peptides or transmembrane domains and belonged to the glycoside hydrolase family.Phylogenetic analysis revealed that the ApGH gene was grouped into the GH1 family.The ApGH gene was ligated to the prokaryotic expression vector HIS-MBP-pET28a and subsequently transformed into Escherichia coli Transetta(DE3)for the expression of recombinant protein.The analysis of real-time PCR revealed that the expression level of the ApGH gene was the highest in leaves but lower in stems and roots.Conclusion:In this study,the glycoside hydrolase gene,ApGH,from A.paniculata is cloned,and its protein sequence characteristics are systematically analyzed.The recombinant protein is successfully expressed in Escherichia coli,and the ApGH gene is predominantly expressed in the leaves of A.paniculata.This study provides a basis for further study of the catalytic function of glycoside hydrolases from A.paniculata.