Characterization of a Mn-SOD from the desert beetle Microdera punctipennis and its increased resistance to cold stress in E. coli cells

Insect treatments, total RNA extraction and cDNA synthesis

The beetles were collected from the wild field in Wujiaqv (N44°29′, E87°31′, 410 m), which is about 100 km northeast of the geological center of Asia. The samples were returned to the laboratory and kept in large plastic beakers containing dry sands at 30 ± 0.5 °C, 16:8 h (light: dark) photoperiod and relative humidity (RH) of 30 ± 6%. Adults were fed with wheat bran and fresh cabbage leaves.

Beetles were dissected in cold 1 ×PBS (phosphate belanced solution) to isolate different tissues, such as head, midgut, hindgut (containing Malpighian tubule), fat body and carcass (whole body after head, gut and fat body were removed). The dissected tissues were immediately frozen in liquid nitrogen for RNA extraction.

As 4 °C is the low temperature at which the insect begins to respond to cold stress (Hou et al., 2010), the beetle individuals were exposed at 4 °C for different time periods (0.5 h, 1 h, 1.5 h, 2 h, 3 h, 5 h, 7 h, 9 h and 11 h, respectively, three replicates per treatment group). The individuals at room temperature (about 25 °C) without any cold treatment were used as control (0 h). After the cold treatment, beetles were immediately frozen in liquid nitrogen for RNA extraction. Total RNA extraction was performed by using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. RNA concentration was quantified by using a Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). The cDNA was synthesized from 1.0 µg total RNA based on Reverse Transcriptase M-MLV (Takara, China) according to the manufacturer’s instructions.

Cloning of the full-length MpmMn-SOD cDNA

MpmMn-SOD fragment (transcriptomic ID c41919) which had the up-regulated expression at 4 °C was selected from the transcriptomic data of M. punctipennis (Tusong et al., 2017). The lacked 3′-sequence were obtained by SMARTer™ RACE cDNA Amplification Kit (Clontech, Beijing, China). Primers used in this experiment were detailed in Table 1. The PCR program was 95 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 63 °C for 30 s, 72 °C for 1 min and a final extension at 72 °C for 10 min. For verification, PCR products were purified, and cloned into pMD18-T vector (Trans GenBiotech, Beijing, China), and then were transformed into competent E. coli cells (DH5a) for Sanger sequencing by Quintarabio, Urumqi, China.

The deduced amino acid domains in MpmMn-SOD were analyzed using the BLAST search program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The physicochemical properties were predicted by using AntPASy’s ProtParam Online Tool. Multiple sequence alignments among insect species in different orders were created with DNAMAN 6.0 software (http://www.lynnon.com). The signal peptide cleavage site was examined with SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) program. TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) was used to predict presence of a putative mitochondrial targeting sequence (MTS). Phylogenetic analysis was performed by IQTREE 1.6.2. The phylogenetic tree was constructed based on predicted amino acid sequences using the Maximum Likelihood (ML) method with 5,000 replicates bootstrap. Mn-SOD sequences in different insect species were downloaded from the database in NCBI website.

Detection of the mRNA level of MpmMn-SOD by Fluorescent real-time quantitative PCR (RT-qPCR)

The expression of MpmMn-SOD transcript was assayed on a 7500 Real Time PCR System (Applied Biosystems, USA) using SYBR Green Mix to determine the expression profiles of MpmMn-SOD gene in different tissues and in the beetle at 4 °C for 0∼11 h as described above. Translation elongation factor (EF-α) was used as a reference gene to normalize the target gene expression levels among samples (Xikeranmu et al., 2019). Primers for RT-qPCR are detailed in Table 1. The qPCR amplification conditions were 94 °C for 2 min, followed by 40 cycles at 94 °C for 30 s and 62 °C for 30 s. The relative expression of the target gene was calculated using the comparative 2^−△△CT method. The change of the gene expression levels at 4 °C was normalized to the gene in the control (0 h). The value at each time point was given as mean ± S.E. (n = 3).

The expression of MpmMn-SOD mRNA in head, midgut, hindgut, fat body and carcass were separately detected by RT-qPCR. The mRNA levels of MpmMn-SOD in different tissues were normalized to that of the head which had the lowest expression level. The value was given as mean ± S.E. (n = 3).

Prokaryotic expression and Western blot analysis of the fusion protein Trx-His-MpmMn-SOD

To obtain the recombinant MpmMn-SOD protein and examine whether it possesses antioxidant activity, DNAMAN was used to design primers containing Bam HI and Xho I restriction sites (Table 1) to amplify the coding sequence (CDS) of this gene. The amplified fragments were digested with the endonucleases, and subcloned into a pET-32a (+) expression vector that was digested with the same enzymes. The constructed plasmid denoted as pET-32a (MpmMn-SOD) was transformed into competent cells of E. coli BL21 (DE3). The parent vector pET-32a without inserts gene was transformed into BL21 (DE3), and used as a control. The two transformed bacteria, BL21(pET-32a-MpmMn-SOD) and BL21(pET-32a), were induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 25 °C for 10 h to overexpress fusion proteins Trx-His-MpmMn-SOD (41 kDa) and Trx-His (the tag protein on the vector, 18.5 kDa) respectively in E. coli. LB (Luria-Bertani) broth was used for bacterial culture medium. Expression efficiency of different transformants was assessed by analysis of the target protein band in sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). The correct expressions of these proteins were further confirmed by Western blotting with anti-His antibody (Zsbiotech, Beijing, China).

Antioxidant activity assay by the Oxford cup method

To test whether MpmMn-SOD has antioxidant activity, the tolerance of the Trx-His-MpmMn-SOD-overexpressed E. coli cells to hydroperoxide was determined by the Oxford cup method (Liu et al., 2018). The bacteria BL21(pET-32a-MpmMn-SOD) and BL21(pET-32a) were grown overnight at 37 °C in LB broth containing ampicillin (Amp⁺) (50 mg/L), and then diluted 1: 100 in LB medium. The diluted cells were further incubated at 37 °C until a final optical density of 0.4∼0.6 at 595 nm. These cells were induced with 0.3 mM IPTG at 25 °C for 10 h. Then, 100 µL BL21(pET-32a-MpmMn-SOD) and BL21(pET-32a) were, respectively, added to fresh LB (Amp⁺) solid medium in plates. After the medium is solidified, five Oxford cups were placed on the plate, then 100 µL of different concentrations (100, 75, 50, 25 and 0 mmol/L) of H₂O₂ were respectively added to the top of the cups. The plates were incubated overnight at 37 °C with three replicates per treatment group. BL21(pET-32a) cells were used as the control. The agent diffused into the surrounding area through the cup to form a decreasing concentration gradient. Observe the zone of inhibition formed around the cup and record the diameter of the zone. The inhibition zones were measured as described by Burmeister et al. (2008).

Measurement of SOD activity and O₂^•− content in the MpmMn-SOD overexpressed bacteria at −4 °C

As 4 °C is not enough to influence bacteria survival within short time, we treated the MpmMn-SOD-overexpressed bacteria under −4 °C. BL21(pET-32a) were set as the control. Cultures of the two bacteria were induced to produce proteins Trx-His-MpmMn-SOD and Trx-His separately by addition of IPTG described above, and five mL cultures of the bacteria were exposed to −4 °C for 0 h, 2 h, 4 h and 6 h, respectively. At the end of the cold treatments, the bacteria in each group were recovered at 37 °C for 1 h, and OD595 was determined for making survival curve. The control was −4 °C for 0 h no cold treatment. Each treatment had three replicates.

Then, the cells were harvested by centrifugation (12,000 rmp,10 min, at 4 °C). The collected cells were sonicated in an ice bath after suspension in PBS. The supernatants were collected as crude enzyme liquids and were quantified using the BCA Protein Assay Kit (Thermo Scientific Pierce, IL, USA). O₂^•− content was measured according to the hydroxylamine oxidation method described by Wang & Luo (1990). The SOD activity was determined using the CuZn/Mn-SOD Assay Kit (Jiancheng, Nanjing, China) following the manufacturer’s protocol with minor modification (Meng et al., 2013).

The relative increase of SOD activity was calculated by subtracting the SOD activity of BL21(pET-32a-MpmMn-SOD) from that of the control BL21(pET-32a), then dividing the difference by the SOD activity of BL21(pET-32a-MpmMn-SOD), so did for calculating the relative decrease of O₂^•− content.

Measurement of relative electrical conductivity (REC) and malondialdehyde (MDA) content of bacteria BL21 (pET-32a-MpmMn-SOD) under −4 °C

The influence of low temperature on cell membrane permeability was determined by measuring the relative electrical conductivity (REC) in bacteria BL21(pET-32a-MpmMn-SOD) and BL21(pET-32a) respectively. BL21(pET-32a) was used as the control. After the recombinant protein was over-expressed by IPTG induction, five mL of the bacteria were centrifuged at 6,000 rpm for 10 min, then the cells were collected and washed with 5% dextrose solution until the bacterial solution’s REC (denoted as L1) was comparable to that of 5% glucose solution. Then, five mL of the isotonic bacterial solutions was stored at −4 °C for 0 h, 2 h, 4 h and 6 h, respectively, following which REC (denoted as L2) was measured for each time point. After boiling for 5 min, the REC (denoted as L0) of the treated bacteria solution was measured again. The final relative conductivity was calculated as: REC (%) = 100 × (L2 -L1)/L0.

For MDA determination, five mL of the cultures were exposed at −4 °C for 0 h, 2 h, 4 h and 6 h respectively after IPTG induction. Then, 400 µL MDA extract solution (MDA Assay Kit, Solarbio, Beijing, China) were added to the bacteria cells (about two million) to lyse the cells. The mixture was centrifuged at 8,000 rpm for 10 min at 4 °C. The supernatant was collected and set on ice bath. The MDA content was determined by using MDA Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s protocol.

Statistical analysis

One-way analysis of variance and Tukey’s multiple comparison test were conducted for data analysis for gene expression, SOD activity measurement and O₂•⁻ content determination. Paired t-test was employed for analyzing data from experiments measuring diameter of death zone, relative conductivity and MDA content. Spearman’s correlation analysis was used for correlation analysis of SOD activity and O₂•⁻ content. Data were shown as mean ± S.E.

Commonly used acronyms

ecCuZn-SOD: extracellular copper/zinc superoxide dismutase; GO: gene ontology; icCu-Zn/SOD: intracellular copper/zinc superoxide dismutase; IPTG: Isopropyl β-D-thiogalactoside; Mn-SOD: manganese SOD; mMn-SOD: mitochondrial Mn-SOD; MTS: mitochondrial targeting sequence; MDA: malondialdehyde; PBS: phosphate balanced solution; REC: relative electrical conductivity; SDS-PAGE: sodium dodecyl sulfate- polyacrylamide gel electrophoresis; Trx-His: thioredoxin–histone.

Identification and characterization of the MpmMn-SOD sequence

We obtained the MpmMn-SOD sequence from the transcriptomic data of M. punctipennis, and then confirmed this sequence by cDNA cloning. The full-length cDNA was 1,359 bp including an open reading frame (ORF) of 609 bp, a 3′-UTR of 750 bp with a poly (A) tail and a single polyadenylation signal (AATAAA). The ORF encoded a protein of 202 amino acids (GenBank accession no. MK676072.1), the calculated molecular mass was 22 kDa, and the estimated pI was 6.54, no signal peptide was predicted. The protein was predicted as a Mn-SOD containing one N-glycosylation site (NGTL) and a putative N-terminal mitochondrial targeting sequence (MTS) which was consisted of 12 amino acids (Fig. 1), suggesting this protein may exist in mitochondria. We designated this sequence MpmMn-SOD.

Comparison of the predicted amino acids of MpmMn-SOD with Mn-SODs from different insect species revealed the high conservation of four manganese binding sites (His26, His76, Asp162 and His166). One signature of Mn-SOD from 162 to 169 (DV/IWEHAYY) was also conserved across these insect species (Fig. 2). MpmMn-SOD was most like the yellow meal worm Mn-SOD, the two sequences both had a shortened N-terminal sequence compared to Mn-SODs from insects in other taxonomic order. However, the identity of these two sequences was only 35.27%, suggesting that MpmMn-SOD was a novel insect Mn-SOD.

To further analyze MpmMn-SOD sequence with SOD sequences in other insects at evolutionary perspective, phylogenetic analysis was conducted. The results revealed two separate clusters, Mn-SOD and Cu/Zn-SOD, in the phylogenetic tree with strong bootstrap (100%) support, in accordance with their distinct metal cofactor requirements (Fig. 3). MpmMn-SOD was clustered with Mn-SODs. Within Cu/Zn-SOD clade, ecCu/Zn-SOD and icCu/Zn-SOD were classified as two subgroups with strong bootstrap support (98%), and ecCu/Zn-SOD subgroup was the basic form. MpmMnSOD was closed to the Mn-SOD from the yellow meal worm Tenebrio molitor.

Expression of the MpmMn-SOD gene in different tissues

To examine the tissue distribution profile of MpmMn-SOD expression, the mRNA levels from head, midgut, hindgut, fat body and carcass were measured by using RT-qPCR. The results showed that MpmMn-SOD expressed in all these tissues, but the expression levels varied greatly among these tissues. The highest was in hindgut followed by fat body, midgut and carcass; the lowest was in head (Fig. 4A). The expression levels in hindgut, fat body, midgut and carcass were 57-fold, 17-fold, 5.3-fold and 3.5-fold of the head, respectively (F_(4,10) = 111.645, P < 0.01), suggesting a tissue specific expression pattern.

Temporal expression of MpmMn-SOD in M. punctpennis at 4 °C

To test whether MpmMn-SOD expression is cold inducible and how does cold affect MpmMn-SOD expression in the insect, the overall mRNA expression profile at 4 °C for different time periods was determined by RT-qPCR. As 4 °C is the low temperature at which the insect begins to respond to cold stress, we exposed the beetles to 4 °C for 0 h ∼11 h. The results showed that the mRNA level of MpmMn-SOD was significantly increased after the cold exposure compared with the control (0 h, no cold treatment), and this simulative effect was very significant (F_(9,18) = 80.07, P < 0.001) (Fig. 4B). It was approximately 9.9-fold, 22.5-fold and 125-fold of the control after cold exposure for 0.5 h,1 h and 1.5 h (P < 0.01) respectively. The second large expression peak appeared at 11 h, which was about 67.3-fold of the control (0 h). From 2 h to 9 h the levels slightly fluctuated with a small peak of 16-fold of the control at 3 h. This cold expression profile presented a stress-responsive pattern. The large fluctuation of MpmMn-SOD expression during the cold treatment indicated that cells could adjust the level of the enzyme timely and precisely.

Prokaryotic expression and Western blot analysis of the fusion protein Trx-His-MpmMn-SOD

To study the enzyme activity of MpmMn-SOD, we inserted MpmMn-SOD into pET32a expression vector and transformed E. coli BL21 with the recombinant plasmid. pET32a alone was also transformed into E. coli BL21 as the control. The fusion protein Trx-His-MpmMn-SOD and the tag protein Trx-His were separately over-expressed in the two transformants through IPTG induction. The expression of these two proteins were analyzed on SDS-PAGE (Fig. 5A). A clear thick band of about 41 kDa appeared in lane 4 after IPTG induction, it matched the calculated size of the molecular mass of Trx-His-MpmMn-SOD; and a clear thick band of about18.5 kDa appeared in lane 2 after IPTG induction, it matched the calculated size of the molecular mass of Trx-His. The two proteins were absent in the un-induced samples. We further confirmed the two fusion proteins by Western blotting using anti-His antibody (Fig. 5B). The results indicated that Trx-His-MpmMn-SOD and Trx-His both were correctly expressed in their host E. coli cells.

Oxidative tolerance of the MpmMn-SOD overexpressed BL21 (pET32a-mMn-SOD)

Antioxidant activity assay was performed to evaluate whether the overexpress mMpMn-SOD could enhance the tolerance of BL21 (pET32a-mMn-SOD) to oxidative stress. The results showed that the death zones of each concentration on the BL21 (pET32a-mMn-SOD) plate were smaller than those on the control BL21 (pET32a) plate (Fig. 6A). The quantified diameters of the death zones on the BL21 (pET32a-mMn-SOD) plates were significantly smaller than those of the control bacteria (Fig. 6B), demonstrating that the mMn-SOD-overexpressing E. coli cells were more tolerant to H₂O₂-mediated oxidative damage than the control BL21 (pET32a).

Survival curve of the MpmMn-SOD overexpressed BL21 (pET32a-mMn-SOD) under cold stress at −4 °C

As E. coli is not significantly harmed by exposure to 4 °C for a limited time, we exposed the MpmMn-SOD overexpressed bacteria BL21(pET32a-mMn-SOD) to −4 °C to test the protective function of MpmMn-SOD for the bacteria under cold stress. After the bacteria were exposed to −4 °C for 0 h, 2 h, 4 h, 6 h and 8 h, respectively, they were recovered at 37 °C for 1 h, and OD595 was determined for making survival curve. The survival curve of BL21(pET32a-mMn-SOD) was a convex type, while it was a rough negative line for the control bacteria (Fig. 7), indicating the cold resistance of BL21(pET32a-mMn-SOD) was significantly increased compared to the control BL21(pET32a). At 4 h of the cold treatment the OD595 of BL21(pET32a-mMn-SOD) was 0.63, significantly higher than the control of 0.52.

SOD activity and O₂^•− content of the MpmMn-SOD overexpressed BL21 (pET32a-mMn-SOD) under −4 °C

We detected the changes of the SOD activity and O₂•⁻ content in the MpmMn-SOD overexpressed bacteria after −4 °C treatment. The results showed that the cold stress significantly stimulated SOD activity of BL21(pET32a-Mn-SOD) compared to BL21(pET32a) (Fig. 8A), suggesting the over-expressed MpmMn-SOD in bacteria not only increased SOD activity overall, but also enhanced the response of SOD activity of the cells to cold stress. Accordingly, the O₂•⁻ content in BL21(pET32a-Mn-SOD) was significantly lower than the control bacteria, suggesting that the over-expressed MpmMn-SOD effectively scavenged O₂•⁻ in cells (Fig. 8B). Pearson’s correlation analysis showed that the SOD activity and O₂•⁻ content of BL21(pET32a-Mn-SOD) under −4 °C treatment were strong negatively correlated, and the correlation coefficient was −0.995 (P < 0.05). The relative increase of SOD activity at 2 h and 6 h of the cold treatment was 2.3-fold and 2-fold of those of the control respectively (Fig. 8C). Correspondingly, at 2 h and 6 h the relative decrease of O₂•⁻ content (Fig. 8D) was high. The two indexes had similar changing trends, suggesting that the more increase in SOD activity, the more decrease in O₂•⁻ content under −4 °C temperature.

Relative electrical conductivity (REC) and Malondialdehyde (MDA) content of the MpmMn-SOD overexpressed BL21 (pET32a-mMn-SOD) at −4 °C

The excessive accumulation of ROS under cold stress may cause lipid peroxidation which leads to damage of cell membranes. Therefore, electrolyte leakage and MDA level in the BL21 (pET32a-mMn-SOD) and the control BL21 (pET32a) under −4 °C were determined. After the cold stress, the two groups of bacteria showed increased REC and MDA content (Fig. 9), indicating that −4 °C caused cells damage to both groups. However, the increasing trends of the two indexes in BL21(pET32a-mMn-SOD) were slower than those of the control BL21(pET32a) during the cold treatment. At 4 h and 6 h of the cold treatment, both REC and MDA content in the control group were significantly higher than the experimental group (Figs. 9A, 9B), indicating that the cell membrane injury in BL21(pET32a-mMn-SOD) was less serious than in the control bacteria. These results suggested that the overexpressed MpmMn-SOD conferred cold tolerance to cells via increasing their ability for ROS-scavenging thus reducing membrane damage.