Objective To clone the cytochrome P450 gene (CaCYP2) involved in C-6β hydroxylation modification of triterpenoid saponins in Centella asiatica, systematically analyze its molecular characteristics, tissue expression pattern and subcellular localization, and achieve prokaryotic soluble expression, so as to provide experimental basis for clarifying its function in triterpenoid biosynthesis. Methods The full-length sequence of CaCYP2 was cloned from C. asiatica cDNA and subjected to bioinformatics analysis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the gene specific expression in different tissues. Seamless cloning technology was employed to construct yeast two-hybrid vector, prokaryotic expression vector and subcellular localization vector for self-activation detection, recombinant protein expression and tobacco transient localization analysis, respectively. Results The CaCYP2 gene was successfully cloned. Its open reading frame (ORF) was 1 443 bp, encoding 480 amino acids, and it belonged to the CYP716 family, hence named CYP716E116. The predicted protein had a relative molecular mass of 54 430 and a theoretical isoelectric point of 8.98, showing hydrophilicity. It contained a typical CYP90-like conserved domain and one transmembrane domain. Subcellular localization results indicated that this protein was localized in the endoplasmic reticulum. qRT-PCR analysis showed that the expression level of CaCYP2 was highest in the leaves, which was 6.2 times higher than that in the roots (P < 0.000 1), showing significant differences compared to other tissues. Prokaryotic expression results indicated that a soluble recombinant protein with a relative molecular mass of approximately 91 300 (including a 40 300 tag protein) was obtained after isopropyl β-D-thiogalactoside (IPTG) induction. The yeast two-hybrid experiment confirmed that the yeast two-hybrid vector had no self-activating activity and could be used for subsequent screening of interacting proteins. Conclusion This study successfully cloned the CaCYP2 gene and characterized its molecular structure, tissue-specific expression pattern, and endoplasmic reticulum localization. It also confirmed that the protein can be expressed solubly in a prokaryotic system. These findings lay an important foundation for further investigation into its catalytic function and regulatory role in the biosynthetic pathway of triterpenoid saponins in C. asiatica.

Centella asiatica (L.) Urban.  /  CaCYP2  /  gene cloning  /  prokaryotic expression  /  subcellular localization