E. coli DH5α was used as the host for subcloning and plasmid amplification. E. coli Rosetta (DE3) was used for the expression of the recombinant protein. Two prokaryotic expression vectors with different tags, pET-28a (+) and pGEX-4T-1, were used as the expression plasmid. The pMD-19T vector, BamHI, EcoRI, XhoI and T4 DNA ligase were purchased from Takara (Dalian, China). All other chemical reagents were of analytical grade and manufactured in China.

We cloned the protein coding region of NbNAC1 using two pair primers NbNAC1-F (5'-CGGGATCCATGATCAAAGGCGTACAC-3') and NbNAC1-R (5'- CGCTCGAGGTAAGGTTTCTGCATGTATA-3'), where the underlined bases represent BamHI and XhoI endonuclease sites, respectively, plus NbNAC1-F’ (5'-CCGGAATTCATGATCAAAGGCGTA-3') and NbNAC1-R’ (5'-CCGCTCGAGGTAAGGTTTCTGCATGTATA-3'), where underlined bases represent EcoRI and XhoI endonuclease sites, respectively. The RT-PCR products were purified and recovered using a TIANgel Midi Purification Kit (Tiangen, Beijing, China).

The resulting PCR products were digested with BamHI and XhoI, and ligated into the pET-28a plasmid at the corresponding restriction sites. The resulting PCR products were digested with EcoRI and XhoI, and ligated into the pGEX-4T-1 plasmid at the corresponding restriction sites. The ligation mixture was transferred into E. coli DH5a competent cells for propagation of the recombinant plasmids. The recombinant plasmids pET-28a-NbNAC1 and pGEX-4T-1-NbNAC1 were confirmed by plasmid PCR, restriction endonucleases digestion, and sequencing.

Cultivation of the recombinant strain was carried out in 50 mL Luria-Bertani (LB) media and cultured at 37°C. We used various concentrations of IPTG (0.5 mM, 1 mM, 1.5 mM, and 2 mM) for inducing the recombinant protein. There were no significant differences with varying IPTG concentration. Cell growth at 1 mM IPTG was not affected; so, this concentration was used extensively in the prokaryotic expression experiments. When the optical density of the culture solution at 600 nm reached approximately 0.6, a final concentration of 1.0 mM isopropyl-β-d-thiogalactoside (IPTG) was added. The culture solution was then separated to culture at 18°C, 28°C, or 37°C. After 4 h, the induction was stopped, the cultures were centrifuged at 7992 g for 1 min, and the proteins were extracted from the collected bacteria cells. Finally, these protein samples were assayed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).

The recombinant strain was cultured in 1 L of LB medium at 37°C until the optical density of the culture solution at 600 nm reached 0.6. IPTG was added to prepare a 1 mM final concentration, which was cultured for 4 h. The induced culture was next centrifuged at 5550 g for 15 min at 4°C. A 50 mL of protein suspension buffer (200 mM Tris-HCl, 500 mM NaCl, 10% glycerol) was added to resuspend the 35 grams of precipitate. The resuspended culture was sonicated for 10 min (the sonication time was 2 s and the intermittent time was 3 s) with an ultrasonic cell breaker. The sonicated culture was centrifuged at 22733 g for 1 h. The supernatant and precipitate were collected for future use. Finally, the supernatant and precipitate samples were assayed with SDS-PAGE.

The His-tagged NbNAC1 proteins from inclusion bodies were purified using 5 mL of Ni-NTA resin affinity chromatography (GenScript, Nanjing, China). After disruption and centrifugation, the precipitate was resuspended with a dialysate that was a solution prepared with 8 M urea and protein suspension buffer (200 mM Tris-HCl, 500 mM NaCl, 10% glycerol). The insoluble protein pellet was dialysed. Before purification, insoluble protein pellet needed to be completely dissolved and renatured by dialysis. Further processing was performed according to the manufacturer’s instructions. Each wash fraction was analyzed by SDS–PAGE using 12.5% polyacrylamide separation gel. The collected fractions were then analyzed by SDS–PAGE.

Pre-treatment of the dialysis bag followed the manufacturer’s instructions (Solarbio, Beijing, China) with the prepared 1L for each concentration of dialysis solutions containing 6 M, 4 M, 2 M, 1 M, 0.5 M, 0.2 M, 0.1 M and 0 M urea, respectively. The 10 ml of eluate containing the target protein was placed into the dialysis bag, which was placed into the dialysis solutions from high to low concentrations at 4°C with the dialysis time for each dialysis solutions being over 2 h. Finally, the dialyzed protein was quick-frozen in liquid nitrogen and stored at –80°C.

The GST-tagged soluble NbNAC1 proteins were purified using Glutathione Resin Affinity chromatography (GenScript, Nanjing, China) and processed according to the manufacturer’s instructions. First of all, add 5 mL of GST purification medium to the chromatography column to allow the preservation solution in the purification medium to flow out, and then 5 column volumes of protein suspension buffer were added to process the chromatography column. After that, PMSF was added with a final concentration of 1 mM to the supernatant sample obtained from a large amount of protein induction. Then pass the sample through the column and collect the effluent, moreover repeat the passage through the column 2–3 times. Besides, add 5 column volumes of protein suspension buffer (200 mM Tris-HCl, 500 mM NaCl, 10% glycerol) to the column in order to wash the column to remove the contaminants bound in the GST purification medium. Then use 10 mL of the configured glutathione eluent (10 mM reduced glutathione, 5 mM dithiothreitol) to elute the target protein. Each wash fraction was analyzed by SDS–PAGE using 12.5% polyacrylamide separation gel. The collected fractions were then analyzed by SDS–PAGE.

The purified proteins were concentrated using Amicon® Ultra-15 Centrifugal Filter Devices (Millipore) following the manufacturer’s protocol. The molecular weight cut off level of Amicon® Ultra-15 Centrifugal Filter is 3 kDa. The particle diameter of this ultrafiltration centrifuge tube is between 1.5 nm and 3 nm. The concentration of purified NbNAC1 protein was determined according to the BCA method.

New Zealand big-ear rabbits were first immunized subcutaneously with 1 mg/mL purified His-tagged NbNAC1 proteins using the same volume as Freund’s complete adjuvant. The remaining procedures followed Zhu et al. (2012).

Purified NbNAC1 proteins were analyzed in 12.5 % (v/v) SDS-PAGE. A western blot analysis was performed following Zhu et al. (2012).

The protein coding region of NbNAC1 gene (GenBank accession No. MF033266) had been cloned by RT-PCR from N. benthamiana in our previous study (Zhu et al., 2019). To study the effects of two prokaryotic expression vectors on the expression of the NbNAC1 transcription factor, two pair primers (NbNAC1-F, NbNAC1-R, and NbNAC1-F’, NbNAC1-R’) were respectively used to amplified the NbNAC1 gene with different endonuclease sites. As shown in Fig. 1A, the NbNAC1 gene with BamHI and XhoI endonuclease sites was successfully amplified by the plasmid PCR. In addition, we successfully obtained the NbNAC1 gene with EcoRI and XhoI endonuclease sites (Fig. 1B).

The PCR product NbNAC1 with BamHI and XhoI endonuclease sites was digested with BamHI and XhoI, and ligated into the pET-28a plasmid containing the His-tags at the corresponding restriction sites (Fig. 2A). Another PCR product with EcoRI and XhoI endonuclease sites was digested with EcoRI and XhoI, and ligated into the pGEX-4T-1 plasmid with GST-tags at the corresponding restriction sites (Fig. 2B). The ligation mixture was transformed into E. coli DH5a competent cells for propagation of the recombinant plasmid. The recombinant plasmids pET28a-NbNAC1 were confirmed by plasmid PCR (Fig. 3A). Furthermore, the recombinant plasmids pET28a-NbNAC1 were confirmed by restriction endonucleases digestion (Fig. 3B). As shown in Fig. 3B, the restriction endonucleases digestion assay had two bands appearing in the agarose gel. Similar, the recombinant plasmids pGEX4T-1–NbNAC1 were confirmed by plasmid PCR (Fig. 3C) and restriction endonucleases digestion (Fig. 3D). Both pET28a-NbNAC1 and pGEX4T-1–NbNAC1 were transformed into E. coli Rosetta (DE3). The recombinant plasmids pET28a-NbNAC1 and pGEX4T-1-NbNAC1 were further confirmed by sequencing in E. coli Rosetta.

The expression of the His-tagged NbNAC1 protein at different temperatures was initially investigated using a small-scale expression system. The molecular weight of the His-tagged NbNAC1 protein is approximately 41 kDa band according to the ORF of the NbNAC1 protein sequence. As shown in Fig. 4A, the SDS-PAGE analysis revealed the target proteins expressed an approximately 41 kDa band for NbNAC1 (IPTG lanes 2, 4, and 6 were induced). However, without the IPTG induction the target proteins were not observed without IPTG induction (lanes 1, 3, and 5). Furthermore, the expression of the recombinant NbNAC1 protein was maximized at 37°C (lane 2), which was much higher than the induced expressions at 28°C (lane 4) and 18°C (lane 6) (Fig. 4A).

We next investigated the expression of the GST-tagged NbNAC1 protein at different temperatures using a small-scale expression system. The molecular weight of the GST-tagged NbNAC1 protein is approximately 62 kDa according to the ORF of NbNAC1 protein sequence. As shown in Fig. 4B, SDS-PAGE analysis revealed that the GST-tagged NbNAC1 proteins expressed an approximately 62 kDa band for NbNAC1 (IPTG lanes 2, 4, and 6 were induced). However, without IPTG induction the GST-tagged NbNAC1 proteins were not observed (lanes 1, 3, and 5). Furthermore, the amount of recombinant NbNAC1 protein at 37°C (lane 6) was higher than at 28°C (lane 4) or 18°C (lane 2) (Fig. 4B).

To determine whether the recombinant NbNAC1 protein is in inclusion bodies, the solubility of the His-tagged NbNAC1 proteins and GST-tagged NbNAC1 proteins was investigated. As shown in Fig. 5A, the His-tagged NbNAC1 proteins were almost all in the pellet (lanes 2, 4, and 6), but not in the supernatant (lanes 1, 3, and 5). Thus, the His-tagged NbNAC1 proteins are insoluble and belong to inclusion bodies proteins. Likewise, as shown in Fig. 5C, most of the GST-tagged NbNAC1 proteins were still in the pellet (lane 1), but the supernatant also had some of the target proteins (lane 2). Therefore, the GST-tagged NbNAC1 proteins were partially soluble.

To obtain a large amount of target proteins, the His- and GST-tagged NbNAC1 proteins were induced to express in E. coli using a large-scale expression scheme. The His- and GST-tagged NbNAC1 proteins were purified by Ni-NTA Resin affinity chromatography and glutathione resin affinity chromatography, respectively. As shown in Fig. 5B, high levels of the His-tagged NbNAC1 proteins were observed in the eluent (lanes 3 and 4). Lower level of the GST-tagged NbNAC1 proteins were also found in the eluent (lane 4) (Fig. 5C).

The purified NbNAC1 protein was next enriched using Amicon® Ultra-15 Centrifugal Filter Devices (Millipore) and following the manufacturer’s protocol. As shown in Fig. 6A, the SDS-PAGE analysis revealed that an approximately 41 kDa single band (lanes 2 and 3) for the His-tagged NbNAC1 proteins. We successfully obtained high concentrations of the His-tagged NbNAC1 proteins (5 mg, 0.5 mg/mL). Similarly, the SDS-PAGE analysis revealed that an approximately 62 kDa band (lanes 1, 2, 3, and 4) for the GST-tagged NbNAC1 proteins (2.5 mg, 1.25 mg/mL) (Fig. 6B). However, there was an approximately 46 kDa contaminant of unknown nature under the target protein (Fig. 6B).

Since the GST-tagged NbNAC1 proteins were purified with contaminant of unknown nature, we selected the His-tagged NbNAC1 proteins to immunize rabbits and obtained serum antibodies. The antibodies in different dilutions were tested with the different concentration of the purified His-tagged NbNAC1 proteins. As expected, an approximately 41 kDa band was detected with different dilutions of anti-NbNAC1 antibodies and different concentration of the purified His-tagged NbNAC1 proteins (Fig. 7A). The strongest approximately 41 kDa bands were shown in the Lanes 1, 4, and 7 (loaded the highest concentration of His-tagged NbNAC1 protein) (Fig. 7A). Furthermore, NbNAC1 antibody was used to detect the E. coli crude extract protein after induction of NbNAC1 expression and N. benthamiana crude extract protein. The results suggest that NbNAC1 antibody can also detect the NbNAC1 protein from the E. coli crude extract protein after induction of NbNAC1 expression and N. benthamiana crude extract protein (Fig. 7B). Therefore, our results suggested the anti-NbNAC1 antibodies had a high degree of detectable sensitivity.