Identification and Characterization of Papaya (Carica papaya, L.) Promoters by Heterologous Expression as eGFP Fusions in Arabidopsis thaliana

Abstract

Promoters are essential upstream genetic switches that activate and repress gene expression. Promoter characterization is a key prerequisite before using in downstream biotechnology applications. The model system, Arabidopsis thaliana, was utilized here to efficiently hasten the time to verify and characterize the functionality and strength of promoters from the more complex tropical tree, Carica papaya. Four putative promoter regions and their 5’UTRs were isolated from the genes encoding peroxidase (Cp9), β-1,3-glucanase (Cp29), ferulate-5-hydroxylase (Cp35) and hypersensitive-induced response protein (Cp45) and fused to eGFP. In silico analysis predicted the presence of several cis-elements associated with regulatory functions in stress and defense responses. The Arabidopsis transcriptional machinery readily recognized the promoters, as determined by qualitative and quantitative measurements of eGFP expression (fluorescence, mRNA and protein levels). The eGFP was expressed in a variety of tissues of the transgenic plants (vasculature, shoot apex, cotyledon, cotyledon petioles, hypocotyls, and root). The Cp29 and Cp45 promoters showed the highest and most promising overall expression. Comparison of eGFP mRNA and protein levels indicated post-transcriptional regulation. Identifying the precise transcription start sites (TSSs) demonstrated transcription fidelity and mapped the length of the 5′-UTR. Predicted mRNA 5′-UTR secondary structures potentially affected the translational efficiency of the mRNAs during development, most notably for Cp9 and Cp35. This work demonstrates the utility of using Arabidopsis to quickly evaluate and identify useful promoters (Cp29, Cp45) from complex tropical plants for future biotechnology goals, and to analyze 5′UTR regulation, cis-elements, and trans-acting factors.