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

Abstract: 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 ventilation tissue 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-1IL 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 （ 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 1 049 bp promoter sequence of SmPAD4 was amplified, which included cis-acting elements such as light response element, hypoxia response element, methyl jasmonate response element, dry early, auxin response element and binding site of WRKY transcription factor.（4）Real-time quantitative fluorescence analysis showed that SmPAD4 gene was expressed in roots, stems and leaves, and the expression level was the highest in leaves. Under ultraviolet and hypoxia stress, the expression of SmPAD4 gene was up-regulated in leaves and stems, and down-regulated in roots. （ 5 ） Subcellular localization showed that SmPAD4 is 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 stress, 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 S. medusa.