Cloning, Expression, and Preliminary Functional Analysis of Oleosin Gene in Paeonia delavayi
QIU Wei-Wei1, LI Hong-Yan1, CHEN Jing-Lei1, LI Xin-Ru1, WANG Juan2,*, ZHANG Peng-Yuan1,*
1 College of Biology and Food Engineering, Southwest Forestry University, Kunming 650224, China; 2 College of Forestry, Southwest Forestry University, Kunming 650224, China
Abstract：Oleosin (OLE) plays a crucial role in the synthesis and storage of plant oils. Using Paeonia delavayi as the experimental material, this study conducted gene cloning, bioinformatics analysis, expression pattern analysis, and subcellular localization analysis to initially clarify the possible role of PdOLE gene in the oil synthesis of P. delavayi. The results showed that the PdOLE gene (GenBank No. PX564745) sequence was 411 bp in length and could encode 136 amino acids. Bioinformatics analysis indicated that the protein contained an OLE superfamily domain and 13 phosphorylation sites, lacked a signal peptide, and exhibited the highest proportion of α-helix in its secondary and tertiary structures. Sequence alignment results showed that the PdOLE protein had relatively low identity with OLE homologs from the closely related species P. ostii and P. lactiflora. Notably, it showed significantly higher homology with OLE proteins derived from 4 representative oil crops: Theobroma cacao, Olea europaea, Sesamum indicum, and Helianthus annuus. Consistent with the results, phylogenetic tree construction further demonstrated that the PdOLE protein formed distinct clade separated from the OLE proteins of the closely related species mentioned above, and clustered more closely with those from the selected oil crops, indicating a closer evolutionary affinity. The qRT-PCR results indicated that the expression level of the PdOLE gene in seeds was significantly higher than that in other organs (P<0.05), reaching its peak at 90 DPF (day post flowers). Nile red staining of paraffin sections demonstrated that the number of seed lipid droplet increased significantly at mid-development (after 60 DPF), accounting well for the PdOLE expression trend. Subcellular localization results indicated that the PdOLE protein was localized to the cell membrane. This study would provide a scientific basis for future investigations aimed at elucidating OLE gene function and the oil biosynthesis pathway of P. delavayi.
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