Cloning and functional verification of Geraniol-10-Hydroxylase gene in Lonicera japonica

Materials

Cloning of the LjG10H gene

Based on the genomic sequencing results of Lonicera japonica, specific primers were designed (Table 1) for PCR amplification utilizing Lonicera japonica complementary DNA (cDNA) as a template. The amplification protocol was as follows: an initial denaturation at 95 °C for 3 min, followed by 34 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30, and extension at 72 °C for 1.5 min, concluding with a final extension at 72 °C for 5 min. The cloning products were analyzed using 1% agarose gel electrophoresis and subsequently purified with a gel recovery kit (TIANGEN, Beijing, China). The target fragments were ligated into a TA vector and transformed into E. coli (DH-5α; TaKaRa, Beijing, China). Positive colonies were selected, verified through colony PCR, and sequenced following overnight incubation at 37 °C on LB agar plates supplemented with kanamycin (Beijing Solarbio Science & Technology, Beijing, China).

Tissue-specific expression analysis of LjG10H

Total RNA was extracted from the roots, stems, leaves, flowers, and fruits of 3-year-old Lonicera japonica utilizing an RNA extraction kit (Vazyme, Nanjing, China). The integrity of the extracted RNA was assessed through electrophoresis on a 1% agarose gel, and the concentration was quantified using a Qubit fluorescence photometer and cDNA was synthesized employing a TaKaRa reverse transcription kit (TaKaRa, Beijing, China), and the cDNA was stored at −20 °C. Specific primers for qRT-PCR based on the cDNA of the LjG10H gene were designed (Table 1). The CFX96 Touch Real-Time PCR Detection System was utilized for the qRT-PCR analysis. The qRT-PCR reactions were conducted using a qRT-PCR kit (TaKaRa, Beijing, China) following the amplification protocol: 95 °C for 3 min; 95 °C for 10s, 54 °C for 30 s, and 72 °C for 1 min for a total of 40 cycles. Each sample was subjected to three technical replicates, and the relative expression of the LjG10H gene was analyzed using the 2^−ΔΔCT method.

Prokaryotic expression of LjG10H

The prokaryotic expression vector pET32a was linearized through double digestion with Bam H I and Xho I (TaKaRa, Beijing, China) and subsequently ligated with the amplified LjG10H gene fragment, resulting in the construction of the plasmid pET32a-LjG10H. The recombinant plasmid was then transformed into competent E. coli BL21 (TaKaRa, Beijing, China), and positive colonies were verified via PCR and subsequently sent for sequencing. The plasmid confirmed by sequencing was extracted using a plasmid extraction kit (TIANGEN, Beijing, China) and transformed into E. coli BL21. Positive strains were cultured overnight and then inoculated into LB liquid medium containing ampicillin at a dilution ratio of 1:100. The culture was incubated at 37 °C with shaking at 200 rpm until the A600 reached between 0.6 and 0.8. Following this, 0.1 mmol/L isopropyl β-D-thiogalactoside (IPTG; TaKaRa, Beijing, China) was added, and the culture was induced at 16 °C for 16 h. Following induction, the bacteria were harvested via centrifugation at 5,000 rpm for a duration of 5 min. The bacterial pellet was then resuspended in 40 mL of cold equilibrium buffer, which was prepared by dissolving 1.64 g of sodium acetate and 29.25 g of sodium chloride in deionized water. To adjust the pH of the solution to 7.4, 1.17 mL of concentrated hydrochloric acid was added, and the final volume was adjusted to 1 L. The resuspended bacteria were subjected to sonication under the following parameters: power set at 200 W, sonication for 2.5 s, followed by a 5 s pause, repeated for a total of 80 cycles. Subsequently, the supernatant and precipitate were separated by centrifugation at 4,000 rpm and 4 °C for 5 min, after which both fractions were analyzed using SDS-PAGE. The supernatant was further purified utilizing a nickel agarose column chromatography technique.

Enzyme activity assay of LjG10H

To further assess the enzymatic activity of the purified protein, an in vitro reaction method was utilized to assess the enzyme activity of the recombinant protein. Geraniol served as the substrate, while the purified recombinant protein LjG10H acted as the catalyst in the enzymatic reaction. The reaction system comprised 1 IU of glucose-6-phosphate dehydrogenase, 4.5 mmol/L of glucose-6-phosphate (Shanghai Aladdin Biochemical Technology, Shanghai, China), 1 mmol/L of NADPH, and 20 μg of LjG10H protein. The incubation buffer consisted of a 50 mmol/L potassium phosphate buffer (pH 7.6) supplemented with 1 mmol/L EDTA, 1 mmol/L DTT, 10 mmol/L FAD, and 10 mmol/L FMN (Shanghai Aladdin Biochemical Technology, Shanghai, China). In this reaction, DTT serves to protect the reducing groups on the enzyme molecule and stabilize the activity of the enzyme. FMN functions as a crucial hydrogen and electron transporter within the respiratory chain, while FAD is primarily involved in the oxidative dehydrogenation of organic compounds, including fatty acids. Glucose-6-phosphate dehydrogenase catalyzes the conversion of glucose-6-phosphate and NADP+ into 6-phosphogluconic acid and NADP. Following a 5-min preincubation at 30 °C, geraniol (at a final concentration of 2.5 mmol/L) was introduced to initiate the reaction. The mixture was incubated for 3 h at 30 °C, after which methanol was added to terminate the reaction. Subsequently, the reaction mixture was centrifuged at 12,000 rpm for 5 min, and then analyzed using HPLC.

Analysis of the LjG10H gene silencing expression in Lonicera japonica

The pCMV201-2b_N81 plasmid was double-digested with the restriction enzymes QuickCut Kpn I and QuickCut Xba I (TaKaRa, Beijing, China). The reaction system consisted of pCMV201-2b_N81 plasmid 1 μg, QuickCut Kpn I 1 μL, QuickCut Xba I 1 μL, 10× QuickCut Buffer 5 μL, and ddH₂O supplementation to 20 μL, and the enzyme was digested at 37 C for 30 min.

In order to construct the pCMV201-2b_N81-LjG10H vector, the specific fragment of LjG10H, which had Kpn I and Xba I restriction sites, was amplified and primers LjG10H F and LjG10H R (Table 1) were designed using Snapgene. The PCR product was then linked to the digested pCMV201-2b_N81 plasmid using T4 ligase (TaKaRa, Beijing, China), and transformed into E. coli (DH5). Recombinant plasmid pCMV201-2b_N81-LjG10H with correct sequencing was taken to transform Agrobacterium receptive C58C1. The Agrobacterium tumefaciens C58C1 strain, harboring the plasmids pCMV101, pCMV201-2bN81, pCMV201-2b_N81-LjG10H, and pCMV301, was cultured in LB medium supplemented with kanamycin at a concentration of 50 μg/mL and rifampicin at a concentration of 25 μg/mL. The culture conditions were maintained at 28 °C with shaking at 180 rpm for a duration of 48 h. The shaken bacterial solution at a 1:100 dilution was transferred into the 50 mL LB medium, to achieve an A₆₀₀ value of Agrobacterium tumefaciens between 0.6 and 1.0. Following centrifugation at 4,000 rpm for 10 min, the bacterial cells were harvested, and the supernatant was discarded. An infection buffer was prepared by adding 10 mmol/L MgCl (Tianjin Dingshengxin Chemical Co., Ltd., Tianjin, China), 10 mmol/L MES (Shanghai Maclin Biochemical Technology Co., Ltd., Shanghai, China), and 100 μmol/L acetyl butyryl (AS, Shanghai Maclin Biochemical Technology Co., Ltd., Shanghai, China) to ddH₂O to a final volume of 100 mL. The absorbance of the washed and resuspended bacterial cells was adjusted to A₆₀₀ = 1.0, followed by incubation for a minimum of 3 h to induce protein expression. Subsequently, the bacteria harboring pCMV101, pCMV201-2bN81-LjLjG10H, and pCMV301 were mixed at a volume ratio of 1:1:1.

The healthy and robust Lonicera japonica seedlings were injected with a syringe. Using a needle to make a slight scar on the back of the cotyledon, a 1 mL sterile syringe without the needle was then used to align the wound and inject the mixed bacteria solution. The infected Lonicera japonica seedlings were first dark-treated for 48 h, and then cultured in a culture chamber (light 16 h/dark 8 h, 23 C, 60%ofhumidity). A total of 15 days later, new leaves were sampled to determine the relative expression level of LjG10H and iridoid content.

Determination of iridoid content

To quantify the iridoid content, the concentrations of loganic acid, morroniside, swertiamarin, loganin, and dehydrogenation loganin were detected in the sample. The leaves of transgenic Lonicera japonica were subjected to freezing and drying for a duration of 48 h, after which they were ground into a fine powder and passed through a sieve with a pore size of 4. A precise weight of 0.5 g was measured and placed into a 100 mL stoppered conical flask, followed by the addition of 50% methanol to achieve a final volume of 50 mL. The weight of the flask was recorded, and the mixture underwent ultrasound treatment for 1 h. Subsequently, the solution was allowed to stand at room temperature, and the weight was measured again to account for any loss, which was compensated by the addition of 50% methanol. The solution was then thoroughly mixed and centrifuged at 4,000 rpm for 10 min. Finally, the supernatant was filtered using a 0.22 μm organic filter membrane. The solution obtained was subjected to analysis via HPLC. The chromatographic conditions were established as follows: the mobile phase comprised 0.5% phosphoric acid (A) -acetonitrile (B), gradient elution: 0–5 min, 92% A; 5–35 min, 92–87% A; 35–43 min, 87–79% A; 43–48 min, 79% A; 48–60 min, 79–74%. The column temperature was 30 °C, the flow rate was set at 1 mL/min, and the sample volume was 10 μL. The detection wavelength was established at 240 nm.

Data processing

The relative expression level of the LjG10H gene in the leaves of Lonicera japonica was determined utilizing the 2^−∆∆Ct method. Significant differences in gene expression were assessed using SPSS software, and a graphical representation was generated employing GraphPad Prism software.

Cloning and sequence analysis of the LjG10H gene in Lonicera japonica

The LjG10H gene was successfully cloned from Lonicera japonica, and subsequent sequence analysis confirmed that the amplified full-length cDNA of LjG10H is 1,497 bp, encoding a protein of 498 amino acids. The LjG10H protein exhibited a homology range of 72.93–83.90% with seven other G10H amino acid sequences derived from various sources. The alignment of the protein sequences revealed that LjG10H possesses a conserved domain structure characteristic of most plant P450s, which includes a proline-rich region (PPGPxPLP) located at positions 33–40, a central helix (AGTDTT) at positions 302–307, and a heme-binding domain (PFGxGRRxCxG) at positions 432–442, consistent with the known characteristics of G10H in plants (Fig. 2B). Furthermore, a phylogenetic tree was constructed based on the alignment of 21 amino acid sequences, including LjG10H, which indicated that the LjG10H protein from Lonicera japonica is closely clustered with proteins from Valeriana jatamansi, suggesting a close genetic relationship (Fig. 2A).

Expression analysis of the LjG10H gene

The findings indicated that the expression of the LjG10H gene in flowers was significantly higher than in other tissues, with leaves exhibiting the next highest expression levels, while the expression in fruits was the lowest (Fig. 2C). This result aligns closely with previous research. Additionally, the concentration of iridoids was markedly greater in flowers compared to stems and leaves, where they were predominantly expressed. For example, the concentration of secoxyloganin in flowers was 34.4 times and 4.77 times greater than that in leaves and stems, respectively (Wang et al., 2023).

Prokaryotic expression of the LjG10H gene

Following induction with IPTG, a prominent band was observed at an approximate molecular mass of 72 kDa in both the supernatant and precipitate of the cells (Fig. 3, lane 5 and 6). This observation suggests effective expression, as the detected mass exceeded the expected value of 55.45 kDa, likely due to the presence of the thioredoxin (TRX)-tag. The protein was successfully purified through elution with 5% imidazole (Fig. 3, lane 7). Conversely, no bands were detected in Lane 2 when 30% imidazole was employed to elute the cleaved supernatant, which may be attributed to the high concentration of imidazole.

Detection of the LjG10H enzyme activity

In the enzyme activity detection system, geraniol served as the substrate for HPLC analysis. The retention times for the standard 10-hydroxygeraniol and geraniol were recorded at 5.0 and 25.2 min, respectively (Figs. 4A and 4B). Within the reaction system designed for the purification of LjG10H protein, the retention time of the target product was observed at 5.1 min (Fig. 4C). The intensity of the target peak increased upon the introduction of 10-hydroxy geraniol standard into the same system, confirming its identification as 10-hydroxygeraniol (Fig. 4D). Conversely, in the reaction that did not include LjG10H protein, no peak was detected at this retention time (Fig. 4E). These results suggest that LjG10H exhibits catalytic activity in the conversion of geraniol to 10-hydroxygeraniol in vitro.

Silencing plant screening and content determination

To investigate the specific role of LjG10H in Lonicera japonica, a silencing expression system was established, and five silenced expression Lonicera japonica seedlings were selected for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of their newly developed leaves. The results indicated that, in comparison to normally growing Lonicera japonica seedlings, the relative expression level of the LjG10H gene was significantly diminished (Fig. 5A). The expression levels for Line-1, Line-2, Line-3, Line-4, Line-5, and Line-6 were recorded as 0.26, 0.43, 0.50, 0.42, 0.13, and 0.32 times that of the wild type (WT), respectively. This finding suggests that the silenced expression plants were successfully generated, allowing for progression to subsequent experiments. Plants exhibiting a lower degree of gene silencing were subsequently selected for further validation to enhance the reliability of the gene silencing results. Among these, Lines 2, 3, and 4 demonstrated suboptimal levels of gene silencing efficacy and were therefore chosen for the determination of iridoid contents. The concentration of iridoid compounds in the newly developed leaves was analyzed using high-performance liquid chromatography (HPLC). In comparison to normally growing Lonicera japonica seedlings, the iridoid compound content in the silenced plants was significantly reduced to 0.58, 0.64, and 0.57 times that of the control group (Fig. 5B). These results indicate a positive correlation between the relative expression level of the LjG10H gene and the variation in iridoid compound content. The observed reduction in iridoid content in Lonicera japonica with a lower degree of silencing further corroborates that a more complete silencing leads to a more substantial decrease in iridoid content.

Correlation analysis

According to the Pearson correlation analysis, the correlation coefficient between iridoid content and the LjG10H gene was found to be 0.506. This correlation was statistically significant at the 0.01 level, indicating a strong and significant positive relationship between iridoid content and the LjG10H gene (see Table 2).