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OACSTPCD

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.