Cloning, characterisation and salt tolerance analysis of EuHPPa1 gene, encoding inorganic pyrophosphatase in Eucommia ulmoides Oliv

Eucommia ulmoides Oliver is dioecious plant that is prized for its Eucommia gum-rich pericarp, leaves, and barks, which makes it a valuable source of collagen with adaptability across various regions. It not only has significant medicinal value but also has advantageous traits such as drought and salt tolerance. Membrane-bound pyrophosphatase functions as an H+ proton pump, and its related genes have been successfully cloned in various plant species. The study focused on investigating the functional role of EuHPPa1 in E. ulmoides using gene cloning, bioinformatics analysis, RT-PCR, subcellular localization, transformation methods, and other approaches. The EuHPPa1 gene with 2298 bp was successfully cloned, showcasing significant advancements in understanding plant stress resistance, growth, and development. The protein encoded by the gene is present in both the cell membrane and the nuclear membrane. Analysis of the spatio-temporal expression pattern of the EuHPPa1 gene indicated a degree of tissue-specificity, with predominant expression in the roots. The promoter of the EuHPPa1 gene was cloned to predict its response elements including GA3, ABA, MeJA, and light. Through tobacco transformation, the highest expression was observed in the root, indicating regulation by the promoter. Wild-type tobacco and transgenic tobacco were both exposed to salt stress treatment, with subsequent analysis of protective enzyme activities and stress-induced gene expression levels. Consequently, this study aimed to explore the response of EuHPPa1 to salt stress in E. ulmoides by evaluating alterations in growth and physiological indicators. Furthermore, we confirmed the gene's function and showed how the EuHPPa1 gene affects the plant's response to salt stress, which may improve the plant's ability to withstand salt stress.