Cloning and expression analysis of CpPMM gene in Codonopsis pilosula

Abstract

GDP-mannose is an important precursor for the synthesis of Codonopsis pilosula polysaccharides and involved in the synthesis of sugar chains. Phosphomannomutase(PMM)catalyzes the conversion of mannose-6-phosphate(Man-6-P)to mannose-1-phosphate(Man-1-P)to synthesize GDP-mannose. In this study, specific primers were designed based on the PMM gene sequence information in transcriptome data, and the full length of the C. pilosula PMM gene was cloned and named CpPMM. The correlation between the CpPMM gene expression and C. pilosula polysaccharide synthesis was analyzed by a series of bioinformatics analysis, prokaryotic expression and qRT-PCR. The results show that the CpPMM gene contains a 741 bp open reading frame(ORF), encoding 246 amino acids, which is highly similar to the PMM of other species and highly homologous to the Helianthus annuus from the Asteraceae family. It was predicted to be a hydrophilic non-transmembrane protein without signal peptide, which was predicted to be located in the cytoplasm with multiple phosphorylation sites. Combined with predictive analysis of conserved domains, this protein belongs to the HAD(haloacid dehalogenase)superfamily; prokaryotic expression studies show that the size of the CpPMM fusion protein is about 29 kDa, which is consistent with the relative molecular mass predicted. The target protein is an inclusion body and is partially soluble. The qRT-PCR results showed that the CpPMM gene exerted spatiotemporal expression patterns, and the expression level in fruiting period was significantly higher than that in the other three periods such as the flowering period. Along with the growth period of C. pilosula, the polysaccharide content of C. pilosula showed a gradual increase trend, reaching the highest during the harvest time. And there are significant differences in the polysaccharide content of C. pilosula in each period. In this study, the CpPMM gene was cloned from the root of C. pilosula, at the same time, the prokaryotic expression system was constructed. In addition, its gene expression level is highly correlated with the polysaccharide content of C. pilosula. It lays the foundation for further studying the function of CpPMM gene and the analysis of biosynthetic pathways of polysaccharides in medicinal plants.