Genome-Wide Identification and Transcriptional Expression Analysis of Cucumber Superoxide Dismutase (SOD) Family in Response to Various Abiotic Stresses

Abstract

Superoxide dismutase (SOD) proteins are widely present in the plant kingdom and play important roles in different biological processes. However, little is known about the SOD genes in cucumber. In this study, night SOD genes were identified from cucumber (Cucumis sativus) using bioinformatics-based methods, including 5 Cu/ZnSODs, 3 FeSODs, and 1 MnSOD. Gene structure and motif analysis indicated that most of the SOD genes have relatively conserved exon/intron arrangement and motif composition. Phylogenetic analyses with SODs from cucumber and several other species revealed that these SOD proteins can be traced back to two ancestral SODs before the divergence of monocot and dicot plants. Many cis-elements related to stress responses and plant hormones were found in the promoter sequence of each CsSOD gene. Gene expression analysis revealed that most of the CsSOD genes are expressed in almost all the tested tissues. qRT-PCR analysis of 8 selected CsSOD genes showed that these genes could respond to heat, cold, osmotic, and salt stresses. Our results provide a basis for further functional research on SOD gene family in cucumber and facilitate their potential applications in the genetic improvement of cucumber.

Figure 1

Unrooted neighbor-joining phylogenetic tree and conserved motif analysis of CsSOD proteins. (a) Phylogenetic tree was generated based on the protein sequences of CsSOD proteins. (b) Conserved motif analysis of CsSOD proteins. Different color boxes represent different types of motifs.

Figure 2

Multiple sequence alignment of deduced amino acid sequences of CsSOD proteins. Cu/ZnSOD signatures (GFH[VLI]H[EA][FL]GDTT and GNAG[GAE]R[VLI]ACG) are boxed in blue. The metal-binding sites for Cu²⁺ and Zn²⁺ are highlighted with yellow and red, respectively. The conserved metal-binding domain (DVWEHAYY) for Fe-MnSOD is in black box. The signature ([AE][QL][VI]WNH[TD]F[YFL]W[EH][CS]) responsible for the recognition of iron ion by FeSODs is boxed in pink. The conserved metal-binding motifs (MxCxxC and CxC) of CsCSD3 are highlighted in green. Six metal-binding sites in MnSOD are marked with cyan, and three residues (His, Gln, and Asp) that are present only in MnSOD and absent in FeSODs are marked with cyan in red box.

Figure 3

Chromosomal locations of 9 CsSOD genes on chromosomes of cucumber. The chromosome numbers are indicated at the top of chromosomes, and the size of chromosome is represented with a vertical scale.

Figure 4

Exon-intron structures of CsSOD genes based on the phylogenetic relationship. Exons and introns are shown as green boxes and thin lines, respectively. Intron phases 0, 1, and 2 are indicated by numbers 0, 1, and 2.

Figure 5

Phylogenetic tree of SOD proteins from cucumber and other plants. Deduced protein sequences of SOD proteins from different plant species were analyzed by ClustalW and then redrawn with the Geneious Pro software. Five colors (yellow, orange, cyan, blue, and green) represent five subgroups (a, b, c, d, and e), respectively. CsSOD proteins were highlighted in red. The amino acid sequences used to build the phylogenic tree were listed in Table S4.

Figure 6

RT-PCR analysis of cucumber SOD genes in different tissues. RNA was isolated from different tissues, including root (R), stem (S), leaf (L), flower (F), and fruit (Fr).

Figure 7

qRT-PCR analysis of cucumber SOD genes in response to various abiotic stresses including heat (a), cold (b), PEG (c), and NaCl (d). Error bars indicate SD based on three biological replicates.