Differential expression of copper-zinc superoxide dismutase gene of Polygonum sibiricum leaves, stems and underground stems, subjected to high-salt stress

Abstract

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5' untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3' untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1-16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO(3). The different mRNA levels' expression of PS-CuZnSOD show the gene's different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.

Keywords: P. sibiricum Laxm.; PS-CuZnSOD; RACE; gene expression; real-time PCR.

Figure 1.

Nucleotide and deduce amino acid sequences of PS-CuZnSOD cDNA from P. sibiricum Laxm. The PCR products of PS-CuZnSOD cDNA were sequenced. The full length was 669 bp, with a 5′ untranslated region of 87 bp, a 3′ untranslated region of 123 bp and an open reading frame (ORF) encoding 152 amino acid (459 bp). The cDNA sequence of PS-CuZnSOD has been submitted to GenBank (accession No. GQ472846). The rectangle indicates the active site of the PS-CuZnSOD; ellipse indicates the Cu²⁺ binding site; * indicates the Zn²⁺ binding site; # indicates the stop codon. Two conserved CuZnSOD signatures are shown in broken line. The signal peptide is shown in solid line. Two cysteines (Cys56 and Cys145) form a disulfide bond for this gene.

Figure 1.

Nucleotide and deduce amino acid sequences of PS-CuZnSOD cDNA from P. sibiricum Laxm. The PCR products of PS-CuZnSOD cDNA were sequenced. The full length was 669 bp, with a 5′ untranslated region of 87 bp, a 3′ untranslated region of 123 bp and an open reading frame (ORF) encoding 152 amino acid (459 bp). The cDNA sequence of PS-CuZnSOD has been submitted to GenBank (accession No. GQ472846). The rectangle indicates the active site of the PS-CuZnSOD; ellipse indicates the Cu²⁺ binding site; * indicates the Zn²⁺ binding site; # indicates the stop codon. Two conserved CuZnSOD signatures are shown in broken line. The signal peptide is shown in solid line. Two cysteines (Cys56 and Cys145) form a disulfide bond for this gene.

Figure 2.

Alignment of the deduced amino acid sequences of PS-CuZnSOD and the known copper-zinc superoxide dismutase from GenBank. Identities compared with those from Melastoma malabathricum, Mesembryanthemum crystallinum, Fagus sylvatica, Populus suaveolens, Gossypium hirsutum, Codonopsis lanceolata, Ricinus communis, Litchi chinensis, Citrus limon were 92, 89, 83, 90, 91, 90, 89, 87 and 87 respectively.

Figure 3.

The phylogenetic tree of PS-CuZnSOD from plants and animals. Clustal W was used to establish the phylogenetic tree, and the result was displayed using Treeview software.

Figure 4.

The change of PS-CuZnSOD after 3% NaHCO₃ exposure in leaves, stems, underground stems’ organs. Total RNA was prepared using SDS reagent for all P. sibiricum Laxm. samples taken at 0, 4, 8, 24, 48, 72, 144 h, independent of 3% NaHCO₃ stress condition. After digested with DNase I to eliminate the genome contamination, the cDNA was synthesized using the oligo d (T) primer and random 6 primer. Real-time PCR was performed with the DNA Engine Opticon™-II sequence detection system. SYBR green Real-time PCR mix (TaKaRa) was used for PCR. (A) The expression of PS-CuZnSOD gene in leaves, stems, underground stems’ organ without stress comparison; (B) The levels of PS-CuZnSOD mRNA in leaves tissues; (C) The level of PS-CuZnSOD mRNA in stems tissues; (D) The levels of PS-CuZnSOD mRNA in underground stems tissues. A multiple comparisons test was conducted to compare significant differences in PS-CuZnSOD expression between leaves, stems and underground stems using the SPSS software. A significant level of p = 0.05 was chosen.