Sequence characteristics, expression and subcellular localization of PtCYP721A57 gene from cytochrome P450 family in Polygala tenuifolia willd

Experimental materials

P. tenuifolia seeds and plants were collected from the Pharmaceutical Botanical Garden of Shaanxi University of Traditional Chinese Medicine (Xianyang, Shaanxi Province) (108°74′E, 34°31′N). Three strains of 3-year-old P. tenuifolia with consistent growth were selected, and the roots, stems, and leaves were pooled for full-length transcriptome sequencing analysis. Additionally, different parts (roots, xylem, cortex) were collected under uniform conditions for further study, in conjunction with previous experimental results from our research group and related studies. Thirty-day-old seedlings of P. tenuifolia, showing robust growth and uniform developmental status, were selected for the following treatments:

Seedlings were exposed to 200 μmol·L⁻¹ chitosan (CHT), 200 μmol·L⁻¹ abscisic acid (ABA), 100 mmol·L⁻¹ NaCl, and 10% PEG6000 for durations of 0, 6, 12, 24 and 48 h. Untreated seedlings served as the blank control.

Amplification of the PtCYP721A57

The total RNA from P. tenuifolia seedlings was extracted using the Trizol extraction kit from Bioengineering Co., Ltd. (Shanghai, China), following the manufacturer’s protocol. The extracted RNA was then reverse transcribed into cDNA using the PrimeScript^™ II 1st strand cDNA Synthesis Kit with Reverse Transcriptase from Takara Co., Ltd. (Japan). Specific primers designed for the PtCYP721A57 gene sequence (CYP721A57-1-F/R, as listed in Table 1) were used for PCR amplification with the cDNA as a template. The resulting PCR products were analyzed by 1.0% agarose gel electrophoresis. The target DNA fragment was purified and recovered using a DNA gel recovery kit, ligated with pMD^™19-T vector and transformed into DH5α competent cells. Positive clones were selected and sent to Aoke Biotechnology Co., Ltd. (Yangling, China) for sequencing.

Bioinformatics analysis of the PtCYP721A57 protein

Bioinformatics analysis and prediction of PtCYP721A57 were conducted using online bioinformatics software tools (Table 2).

Prokaryotic expression analysis of PtCYP721A57 protein

For prokaryotic expression of PtCYP721A57, the primer pair CYP721A57-2-F/R (Table 2) was used for polymerase chain reaction (PCR) amplification, introducing BamHI and HindIII restriction sites upstream and downstream of the gene. After purification and recovery of the target fragment using a DNA gel recovery kit, the fragment was ligated into the pMD19-T vector and transformed into E. coli DH5α competent cells. Positive clones were selected and sequenced by Aoke Biotechnology Co., Ltd., Beijing, China. The PtCYP721A57 gene fragment and the pET28 vector were digested with BamHI and HindIII, respectively. The resulting gene and vector fragments were recovered and ligated overnight at 16 °C. The recombinant plasmid was then transformed into E. coli BL21 (DE3) cells. After confirmation by sequencing, monoclonal cells were selected and cultured at 37 °C with shaking at 200 rpm until reaching an OD600 of 0.6–0.8 (approximately 12 h). Subsequently, the recombinant protein and the corresponding empty vector bacterial solutions were divided into two groups and cultured at different temperatures (16 and 25 °C). Each temperature group included an empty vector control, a non-induced recombinant protein group, and groups induced with varying concentrations of Isopropyl β-D-1-thiogalactopyranoside (IPTG: 0.1, 0.2, 0.5, 1, 2 mM). Following induction, 1–2 mL samples were collected for subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The remaining bacterial solution was centrifuged at room temperature to collect cells (5,000 rpm, 6 min). The cells were then resuspended in 10 mL phosphate-buffered saline (PBS) and subjected to two additional rounds of centrifugation and resuspension in PBS. Cell disruption was achieved on ice for 10 min using a high-pressure disruptor. The lysate was then centrifuged at 4 °C (12,000 rpm, 20 min), and 1 mL of the supernatant was collected for SDS-PAGE analysis.

Quantitative real-time polymerase chain reaction (qRT‑PCR) analysis

The expression levels of the PtCYP721A57 gene in different parts of P. tenuifolia and its response to NaCl, PEG6000 stress, and exogenous CHT and ABA treatments were analyzed using qRT-PCR. The primer pair q-PtCYP721A57-F/R (Table 1) was used for PCR amplification. Relative expression of PtCYP721A57 in each plant part and under various treatment time points was determined based on the Ct value at the plateau stage of quantitative amplification curves. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was a normalization reference gene (Gao et al., 2012). The StepOnePlus^™ Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA) was employed for these analyses. The relative expression level of the PtCYP721A57 gene was calculated using the 2^−ΔΔCt method (Li et al., 2019).

Statistical analysis

Image processing and t-test analysis were conducted using Prism 9.0 (GraphPad Software, La Jolla, CA, USA) and SPSS 27.0 (IBM Corp., Armonk, NY, USA) software packages. A significance level of P < 0.05 was used to determine statistical significance.

Cloning of the PtCYP721A57 gene

Following the sequencing and annotation of the full-length transcriptome of P. tenuifolia, a PtCYP450 gene was identified. Subsequent PCR amplification, sequencing analysis, and NCBI alignment confirmed the presence of the P. tenuifolia gene CYP721A57. This gene possessed an open reading frame (ORF) of 1,521 base pairs, encoding a 506-amino acid protein and contained a conserved CYP450 domain, thereby confirming its classification within the CYP450 family (Figs. 1–3).

Structural analysis of PtCYP721A57 protein

The protein encoded by PtCYP721A57 underwent a comprehensive analysis, which included determination of its relative molecular mass, isoelectric point (pI), hydrophilicity, prediction of transmembrane domains, and identification of phosphorylation sites.

The protein encoded by PtCYP721A57 had a molecular formula of C₂₆₆₅H₄₁₄₉N₇₁₃O₇₂₈S₂₂, totaling 8,277 atoms, and a relative molecular mass of 58.54 kDa. It consisted of a polypeptide chain comprising 506 amino acids. The theoretical isoelectric point (pI) of the protein was calculated to be 9.30, indicating it was alkaline. Additionally, the protein exhibited a lipid index of 64.97 and an instability index (II) of 53.06. Analysis of amino acid hydrophobicity (Fig. S1) revealed an average hydrophilicity coefficient of −0.312 and an instability coefficient of 32.40, suggesting stability as a hydrophobic protein. Understanding the phosphorylation sites and levels of the target protein can provide an important reference for characterizing its properties (Cong, 2020). Prediction of phosphorylation sites in the PtCYP721A57 protein indicated multiple sites, with 23 serine, 31 threonine, and 15 tyrosine (Fig. S2).

The secondary structure of proteins, characterized by the arrangement of the polypeptide backbone atoms, encompassed α-helices, β-sheets, β-turns, and random coils. Predicting this structure provides insights into the relationship between protein structure and its function (Li et al., 2022). For the PtCYP721A57 protein, the predicted secondary structure includes 49.01% α-helix, 12.25% β-sheet, 5.34% extended chain, and 33.40% irregular coil structures, with α-helices being the predominant element (Fig. 4A). Additionally, the three-dimensional structure of PtCYP721A57 was analyzed using SWISS-MODEL online software, employing cytochrome P4504B1 (SMTL ID: 6c94.1) as a template. The analysis indicated 100% coverage of the protein structure (Figs. 4B and 4C).

Prokaryotic expression analysis

The PtCYP721A57 gene and the pET28a vector underwent double digestion, followed by ligation using T4 ligase. After PCR verification, the target gene was sequenced, and analysis confirmed that the sequence matched the original, indicating the successful construction of the prokaryotic expression vector pET28a-PtCYP721A57.

The pET28a-PtCYP721A57 recombinant vector was introduced into BL21(DE3) cells, with strains transformed using the empty pET28a vector serving as controls. Different concentrations of IPTG were used to induce protein expression. SDS-PAGE analysis following cell disruption (Fig. 5) revealed that compared to both the control groups (blank and non-induced), IPTG significantly stimulated the expression of the recombinant protein. Differences in IPTG concentrations did not significantly affect protein expression, confirming the successful expression of the recombinant plasmid. However, no protein expression was observed in the supernatant, suggesting the absence of soluble protein. The addition of 8M urea facilitated overnight dissolution of the precipitate at 4 °C, leading to detectable expression, and confirming the formation of inclusion bodies. Additionally, it was found that expression in inclusion bodies was higher at 25 °C compared to 16 °C.

Subcellular localization analysis

To explore the cellular role of the PtCYP721A57 protein, the recombinant plasmid pBWA(V)HS-PtCYP721A57-GLosgfp was constructed. The localization of the protein within cells was assessed using tobacco transient transformation. Sequencing confirmed the successful ligation of the target gene with the vector, validating the construction of the recombinant subcellular localization vector. Observation of tobacco leaf epidermal cells under a laser confocal microscope revealed green fluorescence signals distributed in the endoplasmic reticulum from the pBWA(V)HS-PtCYP721A57-GLosgfp recombinant vector (Fig. 6).

Phylogenetic tree analysis of protein sequences

In this study, the amino acid sequences of CYP450 family members from various plants, including Arabidopsis thaliana (a model plant), and those sharing homology with PtCYP721A57 from Teleostei were retrieved from the NCBI database. Using MEGA 11.0 software, a phylogenetic tree was constructed to analyze the relationship among PtCYP721A57 and other CYP450 family members (Fig. 7). The phylogenetic analysis revealed that proteins encoded by CYP72 subfamily genes formed a distinct branch. This branch showed high homology with sequences from P. tenuifolia, Juglans regia, and Quercus. This clustering suggests potential similar biological functions among the proteins within this branch.

Gene expression analysis

Total RNA was extracted from the roots, xylem, and cortex of P. tenuifolia, and qRT-PCR was used to evaluate the expression of the PtCYP721A57 gene in each tissue. The GADPH gene (housekeeping gene) served as a reference for normalizing PtCYP721A57 expression levels (Fig. 8). Using the equation y = 341,572x − 156,068, the tenuifolin content was calculated for each sample. The cortex exhibited the highest tenuifolin content, followed by the root, while the xylem showed the lowest content (9.594 ± 0.122 mg/g). When using the xylem sample as a reference, the relative expression of the PtCYP721A57 gene correlated with the change in tenuifolin content. Correlation analysis revealed a significant positive correlation between the relative expression of PtCYP721A57 and tenuifolin content, with a correlation coefficient of 0.900 (P < 0.01).

Figure 9A illustrates the expression dynamics of the PtCYP721A57 gene at different time points in P. tenuifolia seedlings under various stress and induction conditions, with 0 h (CK) representing the control. Under Mock treatment, PtCYP721A57 expression initially decreased within 6 h, followed by an increase that peaked at 48 h (1.18-fold of CK). In the case of CHT induction, PtCYP721A57 expression showed an initial decline, followed by an increase starting from 12 h, reaching its peak at 48 h (2.84-fold of CK), which was 2.50 and 1.63 times higher than that observed under Mock and CTS treatments at the same time point, respectively. Similarly, under ABA treatment, PtCYP721A57 expression initially decreased and then increased, peaking at 48 h (1.75-fold of CK), which was 1.54 and 0.61 times higher than that observed under Mock and CHT treatments at the same time point, respectively.

Furthermore, Fig. 9B depicts the expression profile of PtCYP721A57 under different stress treatments at various time points. Under PEG6000 treatment, the relative expression of PtCYP721A57 gradually increased over the first 24 h, reaching its peak at 48 h (1.29-fold of CK), which was 1.05 and 1.89 times higher than that observed under Mock and NaCl treatments at the same time point, respectively. In contrast, NaCl treatment for 6 h significantly induced PtCYP721A57 expression (3.16-fold of CK), which was 11.42 and 3.17 times higher than that observed under Mock and PEG treatments simultaneously.