Molecular cloning and analysis of pyrophospomevalonate decarboxylase gene from Paris polyphylla var. yunnanensis / 中草药

ABSTRACT

Objective: Saponin is the primary effective component in Paris polyphylla var. yunnanensis. The aims of this study are to obtain the full-length cDNA sequence of a key gene (i.e. MVD) in the saponin synthesis pathway and to analyze its bioinformatics and expression in different tissues. Methods Conserved fragments of MVD cDNA in P. polyphylla var. yunnanensis were obtained by homologous cloning, and its full-length cDNA sequence was acquired by RACE, which was temporarily named PpMVD. Bioinformatics analysis was then conducted and the expression of PpMVD in different tissues of P. polyphylla var. yunnanensis was detected by real-time fluorescence quantitative PCR. Results The total length of PpMVD cDNA was 1 583 bp, and the length of ORF was 1 269 bp, which encoding 422 amino acids. The molecular weight of this protein was 46 820, isoelectric point was 5.69, and the instability index was 45.40. It contained 159 alpha-helix (accounted for 37.68%), 19 β-pleated sheet (accounted for 4.50%), 69 extended chains (accounted for 16.35%), 175 irregular curly (accounted for 41.47%) in the secondary structure of PpMVD protein which had no transmembrane helical region and signal peptide. This gene contained a conserved structural domain of MVD1-Superfamily which belonged to GHMP kinase protein family. The qRT-PCR results showed that the expression of PpMVD gene was significantly different in seed, leaves, stems, and tubers, i.e. tubers > leaf > seed > stem. The highest expression wasobserved in tuber, which was about 3.3 times of that in stem. Conclusion In this study, we first cloned the full-length PpMVD cDNA sequence, and found that it was highly expressed in tuber, which was the major medicinal organ of P. polyphylla var. yunnanensis. This study laid a foundation for the further study on the effect of PpMVD gene on the saponin biosynthesis, and facilitated the efficient and sustainable utilization of P. polyphylla var. yunnanensis.