Cloning and expression of aerenchyma formation-related gene SmPAD4 in Saussurea medusa

The aerenchyma is an adaptive structure of Saussurea medusa in response to extreme environments, and its formation is usually accompanied by programmed cell death(PCD). The death of cells and the formation of aerenchyma are typically regulated by the PAD4 gene(Phytoalexin Deficient 4). However, the mechanism by which SmPAD4 regulates the formation of aerenchyma in S. medusa remains unclear. In this study, S. medusa was used as the experimental material, and the gene SmPAD4 related to aerenchyma formation was cloned by homologous cloning and RACE technology, and its sequence, phylogenetic evolution, expression and subcellular localization were analyzed, and its promoter was amplified by hi-TAIL PCR technology to explore its function in environmental adaptation. The results were as follows:(1)The cDNA of SmPAD4 gene was successfully cloned with a total length of 2 047 bp(GenBank accession number OR766038), including an open reading frame of 1 866 bp, encoding 621 amino acids, a molecular formula of C3163H4906N848O910S26. The protein was an alkaline and hydrophilic unstable protein.(2)Phylogenetic tree analysis showed that SmPAD4 had high similarity with CcPAD4 of Cynara cardunculus.(3)A length of 1 049 bp promoter sequence of SmPAD4 was amplified, which included cis-acting elements such as light response element, hypoxia response element, dry and auxin response elements.(4)Real-time quantitative fluorescence(qRT-PCR)analysis showed that SmPAD4 gene was expressed in root, stem and leaf, and the expression level was the highest in leaf. Under ultraviolet and hypoxia stresses, the expression of SmPAD4 gene was up-regulated in leaf and stem, and down-regulated in root.(5)Subcellular localization showed that SmPAD4 was distributed in the nucleus, cell membrane, and chloroplast. The results show that SmPAD4 gene has a unique protein domain and it responds to hypoxia and ultraviolet environmental stresses, so it plays an important role in the formation of aerenchyma and the response to adversity stress. This study provides theoretical basis for further exploring the role of SmPAD4 gene in the environmental adaptation process of Saussurea medusa.