Cloning and expression analysis of CmFe-SOD gene related to autotoxicity stress in Cucumis melo L.

Abstract: 【Objective】The objective of this study was to clone Fe-SOD gene, an autotoxic response gene in melon. The characteristics of the gene sequence, prediction of protein structure and subcellular localization were analyzed. The role Fe-SOD gene was explored in melon autotoxicity mediated by root exudates.【Methods】The homologous sequence of SOD gene was screened out from the he seedlings of melon, using transcriptome sequencing data related to melon autotoxicity stressed. The Fe-SOD gene of melon was cloned by RT-PCR and named as CmFe-SOD. The full length sequence of CmFe-SOD was analyzed at the NCBI website. Multiple sequence alignments of the protein sequences encoded by the genes were performed using DNAMAN (Version 6) software. The NCBI online tool was used for predictive analysis of characteristic of protein encoded by CmFe-SOD. HNN and SWISS-MODEL software were used to predict the secondary and tertiary structure of CmFe-SOD protein. Phyre2 software was used to edit the tertiary structure. Phylogenetic trees were constructed using MEGA 5.0 software.Healthy melon seedlings with three true leaves were taken and treated with melon root exudatesm Hoagland's nutrient solution was used as a control. The leaves and roots of the plants were taken as analysis samples at 0 h, 6 h, 12 h, and 24 h after treatment to analyze the expression pattern of CmFe-SOD in the leaves and the roots of melon seedlings after autotoxicity by qRT-PCR. Fusion expression vector pfGFPCmFe-SOD was constructed and be used in the subcellular localization of CmFe-SOD. The onion inner epidermis was transformed with pfGFP-CmFe-SOD and observed by laser confocal microscopy (Olympus FV1000) .【Results】The total length of genomic DNA for CmFe-SOD was 1 255 bp with an open reading frame of 918 bp, encoding 305 amino acids. Bioinformatics analysis showed that CmFe-SOD was an acidic hydrophilic protein without signal peptides and transmembrane regions. The alpha helix structures had the highest proportion in the secondary structure of CmFe-SOD, which was consistent with predicted tertiary structure. The gene sequence contained a typical Fe-SOD protein structure domains, 85 to 140 were the N-terminus of Fe-SOD, and 147 to 260 were the C-terminus of Fe-SOD, belonging to the typical Fe-SOD family. The secondary structure prediction showed that among 305 amino acids composing CmFe-SOD, 119 amino acids might form α-helices, accounting for 39.02%. 38 amino acids may form β-sheets, accounting for 12.46%, and 148 amino acids might form irregular curls, accounting for 48.52%. Homology analysis showed that Fe-SOD had a high homology in amino acid sequence among different species. CmFe-SOD had the highest similarity with Cucumis sativus, which was 97.05%, and the similarity with Momordica charantia, Luffa aegyptiaca, Cucurbita moschata, and Cucurbita maxima was 87.93%, 86.23%, 74.10% and 74.10%, respectively. The GFP fluorescence localization of onion inner epidermis showed that CmFe-SOD was located in the cytoplasm. The results of qRT-PCR showed that the expression level of CmFe-SOD gene in the leaves of melon seedlings increased persistently after root exudates treatment, and the expression level was highest at 24 h. In the roots, the expression level of CmFe-SOD increased firstly followed by a downward trend and had a peak at 6 h after treating with root exudates.【Conclusion】The results showed that CmFe-SOD gene, cloned from melon in this study, belonged to the SOD gene family, which had the inherent characteristics of SOD family in plant. The CmFe-SOD could express both in the roots and the leaves of melon and could play an important role in the clearance of adverse effects when they were subjected to autotoxicity stress. The expression level of CmFe-SOD in the leaves increased continuously during the autotoxicity stress, but increased firstly and then decreased in the roots. This might be due to the affection of the roots by the root exudates. Root was the direct damage organ under autotoxicity stress. As the increase of the autotoxicity, the root tissue of melon migh tbe deeply damaged. The roots were difficult to cope with the autotoxicity stress through CmFe-SOD gene, and finally lead to plant damage. These results would provide important reference for utilization of melon Fe-SOD gene, and help to clarify the mechanism of the gene related to reactive oxygen species (ROS) metabolism in the root exudates mediated melon autotoxicity response.