Molecular Cloning and Expression Analysis of Cu / Zn SOD Gene from Gynura bicolor DC

Superoxide dismutase is an important antioxidant enzyme extensively existing in eukaryote, which scavenges reactive oxygen species (ROS) and plays an essential role in stress tolerance of higher plants. A full-length cDNA encoding Cu/Zn SOD was cloned from leaves of Gynura bicolor DC. by rapid amplification of cDNA ends (RACE). The full-length cDNA of Cu/Zn SOD is 924 bp and has a 681 bp open reading frame encoding 227 amino acids. Bioinformatics analysis revealed that belonged to the plant SOD super family. Cu/Zn SODs of theHelianthus annuus,Mikania micrantha, and Solidago canadensis var. scabra all have 86% similarity to the G. bicolor Cu/Zn SOD. Analysis of the expression of Cu/Zn SOD under different treatments revealed that Cu/Zn SOD was a stress-responsive gene, especially to 1-MCP. It indicates that the Cu/Zn SOD gene would be an important gene in the resistance to stresses and will be helpful in providing evidence for future research on underlying molecular mechanism and choosing proper postharvest treatments for G. bicolor.


Introduction
Reactive oxygen species (ROS) is a group of substances which have oxidation properties to obtain electron from adjacent materials.ROS is classified into two groups in botanical system: inorganic radical, including hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ), and organic radical, which mainly are the products of membrane lipid peroxidation.The generation and scavenging of ROS are in balance during the plant growth [1].Nevertheless, the ROS level will be enhanced when plant is exposed to biotic and abiotic stresses, which result in the inactivation of enzymes and breakage of DNA strands and membrane lipids [2,3].A whole set of mechanisms have been evolved in plant cells to cope with environment stress, scavenge overmuch ROS in time, and adjust internal environment.Scavenging mechanisms include enzymatic and nonenzymatic systems.The former mainly include superoxide dismutase (SOD), ascorbate peroxidase (APX), catalyse (CAT), and glutathione peroxidase (GPX).
Superoxide dismutase (SOD) is an antioxidant enzyme, which plays an important role in process of scavenging ROS through catalyzing the dismutation of superoxide anion to H 2 O 2 and molecular oxygen [4].It is also a sort of important active substances that enhance human health and a kind of important ingredients of functional foods.According to the metal cofactor of catalytic site, SODs are classified into at least three distinct groups: iron SOD (Fe-SOD) in chloroplast, manganese SOD (Mn-SOD) in mitochondrion and peroxisome, and copper/zinc SOD (Cu/ Zn SOD ) in chloroplast and cytosol or in the extra cell which represent 90% of total SOD.Plant SODs are known to make contribution to defence against toxic oxygen species and are important for stress tolerance consequently.Different types of SODs have their own expression and regulation.The plant SOD gene has been first cloned from corn [5]; then the SOD gene of cassava [6], rice [7], sugarcane [8], and cantaloupe [9] has been cloned and applied to improve the plant resistance in recent years.The cDNA library construction, 5  -and 3  -rapid amplification of cDNA ends (RACE), and homology cloning are main methods of gene cloning.
Gynura bicolor DC. (G.bicolor) is a perennial plant of the composite Gynura Cass and rich in microelement, anthocyanins [10,11], amino acid, essential oil [12], and flavonoid [13,14].Its leaves are distinctive reddish-purple in color on the abaxial side, contrasting with typical green color on the adaxial side (due to the presence of anthocyanins) [15].It has potent antioxidant capacity with high SOD activity; therefore it has high nutritional and medicinal value and is beneficial for human health.Since the living organism with high metabolic activity after harvest is prone to losing nutrients and appearance during senescence, proper postharvest treatments are in need for G. bicolor production [16].Most studies focused on the physiological and biochemical of postharvest treatments, while less gene expression was reported.Therefore we explore effects of 1methylcyclopropene (1-MCP) treatment, nitric oxide (NO) treatment, controlled atmosphere treatment, and hypobaric treatment to G. bicolor at the molecular level.
We herein described the molecular cloning and characterization of Cu/Zn SOD gene from G. bicolor.The bioinformatics analysis and expression of Cu/Zn SOD under different treatments were also studied, which will be helpful in providing evidence for future research on underlying molecular mechanism and improving yields of postharvest treatments of G. bicolor and other leafy vegetables.

Plant Materials and
Treatments.G. bicolor leaves were obtained from a commercial farmland in Shanghai, China.Harvested leaves were precooled to 2-5 ∘ C and delivered to the laboratory immediately.Then the leaves without visual defects (pest-damaged, bruised, and defective) were randomly divided into six groups and subjected to the following treatments: (1) Being stored in room temperature: (a) control group (CK): without any treatment; (b) 1-MCP treatment (MT): leaves were closed up in desiccators in the presence of 50 L/L 1-MCP (Agrofresh, USA) for 6 h.KOH (Sinopharm Chemical Reagent Beijing Co., Ltd., China) solution (1% (w/v)) was placed inside it to absorb CO 2 during treatment; (c) nitric oxide (NO) treatment (NT): 10 L/L nitric oxide (Source Gas Co., Ltd., Nanjing, China) was injected into desiccators; then leaves were closed up in it for 3 h.Then these groups were stored in a room held at 20-25 ∘ C for 7 days; leaves from each treatment group were frozen in liquid nitrogen and stored at −80 ∘ C on days 0, 1, 3, 5, and 7. (2) Being stored in low temperature: (d) control group (CKL): without any treatment; (e) controlled atmosphere treatment (CA): leaves were placed in normal polyethylene plastic bags with a controlled atmosphere of 3% O 2 and 97% N 2 (controlled CO 2 < 1%); the gas compositions were continuously controlled constant throughout the storage; (f) hypobaric treatment (HT): leaves were put in a plastic basket and placed into a hypobaric chamber, whose pressure was 61.33 kPa.These groups were treated at 4 ∘ C for 20 days; leaves from each treatment group were frozen in liquid nitrogen and stored at −80 ∘ C on days 0, 5, 10, 15, and 20.

RNA Extraction.
The G. bicolor leaves were pulverized with liquid nitrogen firstly; then total RNA was isolated from the leaves using a modified CTAB method.The total RNA concentration and purity were measured by microultraviolet spectrophotometer (Thermo Electron, America), and RNA integrity was checked by agarose gel electrophoresis.
PCR was conducted using the following procedure: the template was denatured at 95 ∘ C for 5 min followed by 30 cycles at 94 ∘ C for 1 min, 60 ∘ C for 40 s, and 72 ∘ C for 1 min and extended for 10 min at 72 ∘ C. Amplicons of expected sizes were recovered from the agarose gel and purified using AxyPrep6 DNA Gel Extraction Kit (Axygen, America), then subcloned into the pMD 19-T cloning vector (TaKaRa, Japan), and transformed into competent Escherichia coli DH5 cells (Trans, China).The bacterium solution as template performed PCR using the same procedure as that above, and the positive clones were sequenced in Nanjing GenScript Biological Technology Co., Ltd.(Nanjing, China).

Bioinformatics Analysis.
The nucleotide sequence, deduced amino acid sequence, and open reading frame (ORF) of Cu/Zn SOD cDNA were analyzed using the software Vector NTI Advance 11.5.1.Homology search was carried out and Cu/Zn SOD sequences from different organisms were obtained using the NCBI BLAST search program.MEGA6 software was used for phylogenetic analysis with neighbourjoining method.
Protein analyses of physicochemical properties were performed using ProtParam and ProtScal program of ExPASy tools.Signal peptide, transmembrane domain, subcellular localization, and conserved domain were predicted by the Signal P 4.1, TMHMM, ProtComp Version 9.0, and Batch CD-search program, respectively.The secondary structure was predicted by SOPMA program and the presumed tertiary structure was established using the SWISS-MODEL workspace.

Expression Analysis by
Quantitative RT-PCR.mRNA relative expression of Cu/Zn SOD in G. bicolor was analyzed by the method of real-time quantitative RT-PCR with a Bio-Rad CFX96 Real-Time System (C1000 Thermal Cycler) (Bio-Rad, USA).RNA treated with 50 L/L 1-MCP and 10 L/L nitric oxide, controlled atmosphere (3% O 2 and 97% N 2 ), and hypobaric (61.33 kPa) were extracted.Total RNA was reverse-transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan).The PCR reaction was performed in 20 L volume containing 10 L SYBR premix Ex Taq, 0.8 L each primer solution, and 1 L cDNA solution as a template, and then RNase-free water was added to a total 20 L volume.The specific qRT-PCR primers were Cu/ Zn SOD-real-F (5  -ACCGCAAAGTTCACCATTGTTGATAAG-3  ) and Cu/ Zn SOD-R (5  -TGCCCTGGATAGGACTGGAACG-3  ), and the primers of internal control were Actin-real-F (5  -AGGAAT-GGTGAAGGCAGGGTTTG-3  ) and Actin-real-R (5  -TTG-TCCCATACCAACCATAACACCAG-3  ).The amplification was performed by the following procedure: initial denaturation at 95 ∘ C for 30 s followed by 40 cycles of amplification (95 ∘ C for 5 s and 60 ∘ C for 30 s).Samples were normalized with Actin, and the relative expression levels were calculated using the 2 −ΔΔCt method.Each assay was repeated in triplicate.

Statistical Analysis.
All data of expression analysis were average values of three replicates with standard errors (SE).Standard errors (SE) were calculated by Microsoft Excel and the figures revealed differences between experimental treatments that were performed with the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA).The data were treated by analysis of variance using Duncan's test.A 95% confidence level ( < 0.05) was used for all analyses.of G. bicolor Cu/Zn SOD and other plant Cu/Zn SODs to investigate the evolutionary relationships.The result reveals that G. bicolor Cu/Zn SOD has the same evolutionary origin with other analyzed plants.G. bicolor and Compositae plant such as Helianthus annuus, Mikania micrantha, and Solidago canadensis are a cluster and share a close relationship with Salicaceae and Solanaceae plant.Thus it can be seen that Cu/Zn SOD has a certain genus characteristic in phylogenetic relationships (Figure 3).

Molecular Characteristics of Deduced G. bicolor Cu/Zn
SOD Amino Acid Sequence 3.3.1.The Primary Structure Analysis.The results of physicochemical properties with ProtParam program of ExPASy are as follows.The calculated molecular mass is 24.52 kDa, and the theoretical isoelectric point (pI) is 6.66.The polypeptide consists of 20 types of amino acid, the highest content of it is Gly (13.7%), acidic amino acid (D, E) is 10.1%, basic amino acid (K, R) is 9.2%, hydrophobic amino acid (A, I, L, F, W, and V) is 34.4%, polar amino acid (N, C, Q, S, T, and Y) is 20.6%, and charged residue (R, K, H, Y, C, D, and E) is 28.5%.The instability index is computed to be 28.40; this classifies the protein as stable (index < 40).Grand average of hydropathicity (GRAVY) is −0.132, classified as hydrophilia protein (hydrophobic protein: 0 < GRAVY < 2; hydrophilia protein: −2 < GRAVY < 0), the analysis of hydropathy profile using ProtScal program of ExPASy has the same result, and most of amino acid residues have stronger hydrophilicity, whereas N-end and C-end regions have hydrophobicity.
Predicted by the Signal P 4.1 program, result indicates that N-end of the deduced protein has no signal peptide, classified as nonsecretory protein.Analyzed by the TMHMM server, result shows that the protein is located in the outside of membrane and has no transmembrane domain; therefore the protein can be deduced in cytoplasmic matrix.Based on the predicted subcellular localization, it is most probably located in the cytoplasm with prediction value of 3.8.In summary, the protein belongs to nonsecretory protein located in cytoplasmic matrix.

Secondary Structure.
Protein secondary structure prediction analyzed by SOPMA program indicates that the protein contains -helix (h), extended strand (e), -turn (t), and random coil (c), which accounted for 12.78%, 29.96%, 11.45%, and 45.81% of total amino acids, respectively.On the whole structure, random coil and extended strand are the main structural element, and -helix and -turn are distributed in the protein.

Tertiary Structure.
According to the prediction method of homology modeling, the potential tertiary structure of the Cu/Zn SOD from G. bicolor was established using the SWISS-MODEL workspace, based on the A-chain model of Cu/Zn SOD from Potentilla atrosanguinea (PDB code: 2Q2L) with identity of 80.26% (Figure 4).The tertiary structure showed  that the Cu/Zn SOD contained a novel eight antiparallel strands forming a flattened cylinder, which was similar to the Cu/Zn SOD in Brassica campestris [17].Ramachandran plot generated by Procheck shows that amino acids of the predicted model in most favoured regions, additional allowed regions, and generously allowed regions are 89.3%, 9.9%, and 0.8%, respectively, which reveals that structure of the predicted model has good stability.Overall average of G-Factors is −0.22, which shows that the structure belongs to the ordinary range.

Expression Analysis of Cu/Zn SOD Gene under Different
Treatments.The expression of Cu/Zn SOD gene under different treatments including 1-MCP, NO, controlled atmosphere, and hypobaric pressure in G. bicolor was performed by realtime RT-PCR.The results showed that the four treatments caused the upregulation of Cu/Zn SOD gene with different degree (Figures 5 and 6).
After 1-MCP treatment the expression of Cu/Zn SOD gene was elevated to 18.58-fold at peak time point 1 d and then declined and decreased to 1.1 times at 7 d.The same variation trend was observed in the SOD activity of G. bicolor [18]: G. bicolor was treated in the presence of 0.25 ppm, 0.50 ppm, and 1.00 ppm 1-MCP for 6 h, 12 h, and 24 h respectively, and then stored in 0 ± 1 ∘ C. The results showed that the SOD activity level first increased and then decreased slowly and peaked at 5 d.The SOD activity of Brassica oleracea [19], Ficus carica [20], and kiwifruit [21] also had the same variation trend.So we can deduce that, in the earlier storage of some leafy vegetables and fruits, 1-MCP treatment can induce the expression of Cu/Zn SOD gene and then upregulated the activity level In NO treatment, the expression level increased continuously with a slight reduction at 5 d and peaked at 7 d (2.89fold).The variation trend was similar to the SOD activity of rape [22].While the variation trend of SOD activity and gene expression was different in banana [23], the activity of SOD in NO treatment increased gradually, reaching a maximum at day 3. Thereafter, a decline occurred with a slight increase at day 9.The expression levels are consistent with the changes of SOD activity.So we deduced that the induction mechanism of NO treatment in vegetables was perhaps different in fruits.
Under hypobaric treatment the expression pattern has similar variation trend to 1-MCP; the highest level was achieved at 10 d, which was 2.71 times, and then declined, which was consistent with the SOD activity of blueberry [24] and tomato [25].
The expression of Cu/Zn SOD gene in controlled atmosphere treatment has two peaks, 3.43 times at 5 d and 2.65  times at 15 d, respectively, while the SOD activity of G. bicolor first decreased and then increased, reaching a maximum at day 15 and thereafter a slight decline at day 20 [18].So we speculated that, in this treatment, other factors participated in the induction of expression of Cu/Zn SOD gene, and regulation of gene to activity was time-lapse.
In this study, we also found that a low temperature could affect the expression of Cu/Zn SOD gene, and the expression level of CKL group was elevated (3.82-fold) at 5 d, while the expression of CK group decreased (0.63-fold) at the same time.So we speculated that a low temperature could induce expression of Cu/Zn SOD gene.
The results suggested that the four treatments were effective for induction of expression of Cu/Zn SOD gene, and 1-MCP treatment was the most obvious.Different expression patterns of treatments result from different regulatory mechanism.The expression amount of 1-MCP, controlled atmosphere, and hypobaric treatments dropped to the level comparable to the original level at the last time point.It was indicated that the influence of the three treatments on the expression of Cu/Zn SOD gene was time-controlled.In the group stored at 4 ∘ C, the expression amount of controlled atmosphere and hypobaric treatments was less than control group in the same time point before 15 d and more than at 15 d, because low oxygen concentration and low pressure can inhibit accumulation of superoxide anion [26][27][28]; therefore the expression of Cu/Zn SOD gene reduced before 15 d, and inhibiting effect was not obvious at 15 d.

Conclusions
A full-length cDNA of Cu/Zn SOD from leaves of G. bicolor was isolated and characterized in this paper.Multiple alignments and bioinformatics analysis results indicated that the deduced Cu/Zn SOD had high similarity to other plant Cu/Zn SODs, which further proves the cloned gene is Cu/Zn SOD gene.The result supported the hypothesis that Cu/Zn SOD may play an important role in bioprocess and thus are conserved in evolution.Expression profiles of Cu/Zn SOD under different treatments suggest that Cu/Zn SOD was a stress-responsive gene, especially to 1-MCP.The molecular cloning, characterization, and expression analysis of Cu/Zn SOD will be helpful to choose proper postharvest treatments for G. bicolor and other leafy vegetables.

Figure 1 :
Figure 1: The full-length cDNA sequence and deduced amino acid sequence of G. bicolor Cu/Zn SOD.The start codon (ATG) is boxed and the stop codon (TAA) is underlined.

Figure 4 :
Figure 4: Prediction of protein tertiary structure of G. bicolor Cu/Zn SOD (the tertiary structure based on the A-chain model of Cu/Zn SOD from Potentilla atrosanguinea (PDB code: 2Q2L) showed that the Cu/Zn SOD contained eight antiparallel -strands forming a flattened cylinder).

Figure 5 :
Figure 5: Expression profile by qRT-PCR with 1-MCP and NO treatments (CK: control group; MT: 1-MCP treatment; NT: nitric oxide (NO) treatment).(a and b) Differences between experimental treatments that were performed with the SPSS 20.0 software.

Figure 6 :
Figure 6: Expression profile by qRT-PCR with controlled atmosphere and hypobaric treatments (CKL: control group; CA: controlled atmosphere treatment; HT: hypobaric treatment).(a,b, and c) Differences between experimental treatments that were performed with the SPSS 20.0 software.
-end, 5  -end, and conserved sequences.The full-length sequence of cDNA consists of 924 bp nucleotides, and it contains 103 bp in the 5  untranslated region, an open reading frame (ORF) of 681 bp at position 104-784 bp, encoding a deduced protein of 227 amino acid residues, and 140 bp in the 3  untranslated region (Figure1).
3.1.Sequence of G. bicolor Cu/Zn SOD cDNA.The full-length cDNA of G. bicolor Cu/Zn SOD was deduced by aligning and assembling the 3 sequence alignment of 12 amino acid sequences which have high identity to G. bicolor Cu/Zn SOD finds many high conserved domains; therefore the amplified sequence is a typical Cu/Zn SOD sequence (Figure2).With the MEGA6 program, a neighbour-joining (NJ) tree was constructed based on the deduced amino acid sequence