Five-year-old plants of ginseng (Panax ginseng C.A. Meyer) were harvested from Fusong County (Jinlin, China). The freshly collected material was prepared for gene cloning.

Phytopathogenic fungal species used in this study were Rhizoctonia solani and Cylindrocarpon destructans (Hyphomycetes); Phytophthora cactorum (Oomycetes); Fusarium solani, Alternaria panax and Botrytis cinerea (Fungi imperfecti); and Sclerotinia sp. (Discomycetes). All the fungal species were obtained from Jilin Agricultural University (Changchun, China).

Total RNA was seperated from ginseng leaves. The pgCyP gene was obtained by carrying out polymerase chain reaction (PCR) amplification from leaf cDNA with synthetic nucleotide primers. The PCR product was inserted into pMD18-T (Takara, Dalian, China). After digestion with BamHI and NotI, the generated DNA fragment was cloned in a pGEX-6p1 vector.

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to express pgCyP His₆. Transformed cells were cultured in Luria-Bertani medium supplemented with 50 µg/ml ampicillin at 37°C on a rotary shaker at 200 rpm. When the OD₆₀₀ value of the cell culture reached 0.6–0.8, protein overexpression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 0.5 mM, and the culture was grown for a further 5 h. Cells were harvested by centrifugation at 10,000 × g for 2 min. Protein expression levels were analyzed by SDS-PAGE and visualized with Coomassie Blue staining (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The transformed cell pellets were resuspended in wash buffer [20 mM Tris (pH 8.0), 100 mM NaCl and 3 M urea]. After centrifugation, the cells were lysed in lysis buffer [50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl (pH 8.0), 0.13 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml lysozyme and 1.33 mg/ml sodium deoxycholate] and sonicated at 4°C for 30 min. DNase I was then added to a concentration of 2,000 U/ml and the solution was incubated at 37°C with shaking at 200 rpm for 1 h. The lysate was then centrifuged at 10,000 × g for 20 min at 4°C. The resulting inclusion bodies were washed twice with a solution consisting of 50 mM Tris, 100 mM NaCl, 2 M urea, 0.5% Triton-X and 10 mM EDTA at pH 8.0. The washed inclusion bodies were dissolved by stirring for 1 h in extraction buffer [1.5% sarkosyl, 25 mM triethanolamine amine and 1 mM EDTA (pH 8.0)] at 4°C. The solubilized inclusion bodies were centrifuged at 10,000 × g for 10 min at 4°C and then refolded by dialysis in binding buffer [10 mM NaH₂PO₄·2H₂O, 10 mM Na₂HPO₄·12H₂O (pH 7.8), 150 mM NaCl and 10 mM imidazole]. The refolded protein was purified by Ni-chelating Sepharose Fast Flow chromatography (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The adsorbed proteins were eluted with elution buffer containing 100 mM imidazole. Eluates were pooled together and the samples were renatured on a Sephadex G-25 column. Protein concentrations were determined by the Bradford Protein Assay kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China).

A mixture of the following components was incubated on ice for 10 min: 930 µl Assay Buffer [50 mM HEPES and 100 mM NaCl (pH 7.8)], 30 µl of 200 µM α-chymotrypsin (Sigma-Aldrich) and either 10 µl of GST-pgCyP-His₆ (1 µM) or the GST (1 µM) negative control. Each sample was placed in a spectrophotometer (Thermo Fisher Scientific, Waltham, ΜΑ, USA), which was pre-cooled to 8°C. After the addition of 30 µl of 7.8 mM Suc-Ala-Ala-Pro-Phe-NA (Sigma-Aldrich), absorbance at 390 nm was immediately recorded every second for 5 min at 8°C.

GST-pgCyP-His₆ and GST protein were tested for possession of antifungal activity. Fungi were grown in potato dextrose agar (PDA) for 48 h at 28°C. The fungi were then spread onto PDA plates. Sterilized blank paper disks were placed on the plates and dotted with an aliquot of protein at different concentrations. The plates were incubated and monitored for up to 3 days. To determine the IC₅₀ of proteins against various fungal pathogens, the fungal spores were collected and placed in 96-well microtiter plates. Recombinant pgCyP (20 µl) or the negative control was then added to each well. After 12–36 h of incubation at 28°C, fungal growth was evaluated microscopically. The turbidity of each well was also measured by recording absorbance at 595 nm using a microtiter reader (Emax, Molecular Devices, Sunnyvale, CA, USA).

The nucleotide sequence of pgCyP in the present study has an accession no. KX034081 in GenBank.

As shown in Fig. 1A (lane 3), pgCyP cDNA was successfully generated by reverse transcription-polymerase chain reaction (RT-PCR) amplification from cDNA of ginseng leaves. The corresponding nucleotide sequence, consisting of 525 bp, was predicted to be 174 a.a. in length, as well as a theoretical isoelectric point of 7.67 and a molecular weight of 18.7 kDa. As it lacked a transit peptide, this predicted protein was probably localized in the cytosol (24). The pgCyP protein contained a PPIase domain and a divergent loop (48-VSGKPLH-54). Two CyPs (positioned at 40 and 168) as well as a Glu (positioned at 83) were conservatively kept similar to the other members in the CyPs family. The predicted secondary structure of pgCyP comprised 6.32% α helices, 21.26% extended strands and 72.42% random coils. On the basis of protein sequence alignment, pgCyP was closely associated with Ziziphus jujuba CyP, Citrus sinensis CyP19-3 and Ricinus communis CyP proteins. pgCyP shares 90% identity with its homolog in Ziziphus jujuba (Fig. 1D).

In the phylogenetic tree of CyP-like proteins shown in Fig. 2, pgCyP may be classified, as expected, into the clade of CyP proteins from dicot species, where it is most closely related to Ziziphus jujuba belonging to the same family. A 3D pgCyP model was created through primary protein sequence (25) using the SWISS-MODEL server (Fig. 1C).

cDNA of the pgCyP isolated from this study, which differs from the pgCyP previously reported (23), and possibly belongs to a different ginseng CyP subfamily, was cloned into vector pMD18-T. The resulting plasmid was then digested with BamHI and NotI. After target fragments were cloned into pGEX-6p1, the protein were carrying a tandem His tag and GST at both ends (Fig. 1B).

Escherichia coli BL21 (DE3) competent cells were transformed with the pGEX-6p1/pgCyP-His₆ plasmid. Overexpression of recombinant protein was induced at different IPTG concentrations for different lengths of time. The optimal IPTG concentration and induction time was 0.5 mM and 5 h, respectively. The induced protein was resolved in SDS-PAGE. As expected, a protein bank of 46 kDa was evident (Fig. 3). Moreover, the analysis indicated GST-pgCyP-His₆ present as inclusion bodies (Fig. 3).

After solubilizing the inclusion bodies in extraction buffer, pgCyP was purified using Ni-NTA affinity chromatography as described in Materials and methods. The target protein with His tag was fractionated with 100 mM imidazole. GST-pgCyP-His₆ was confirmed in SDS-Page (lane 9, Fig. 3).

After purification and renaturation, the recombinant protein was tested for its ability to inhibit fungal growth in vitro. Pathogenic fungus included Sclerotinia sp., R. solani, P. cactorum, F. solani, C. destructans, A. panax and B. cinerea. The fusion protein at concentrations of 1.28 and 2.14 µM had pronounced effects on the growth of P. cactorum (Fig. 4A), but had no effect on the growth of the other fungi. The IC₅₀ of pgCyP against P. cactorum, was 2.55 µΜ (Fig. 4B). GST, as a negative control, was inactive. Hereby we demonstrate in vitro resistance activity against fungi of pgCyP.

In the present study, we showed pgCyP has PPIase activity. PgCyP is capable of accelerating isomerization of imide between the Ala and Pro peptide bonds in contrast to spontaneous inter-conversion in the negative control. GST protein, the control, had no isomerize activity.