ChABCB1 gene for transporting hancine from extracellular to intracellular in cephahotaxus hainanensis and application thereof

Abstract

The application discloses a ChABCB1 gene for transporting harringtonine from outside cells to inside cells in Cephalotaxus hainanensis and application thereof, a nucleotide sequence of the ChABCB1 gene is shown as SEQ ID NO:1, and an amino acid sequence of a protein coded by the ChABCB1 gene is shown as SEQ ID NO:2; an expression cassette containing the gene, a carrier, an engineering bacterium or a transgenic cell line are also disclosed, and application of the ChABCB1 gene, the protein coded by the ChABCB1 gene, the expression cassette containing the ChABCB1 gene, the carrier, the engineering bacterium or the transgenic cell line in transporting harringtonine from outside cells to inside cells is disclosed.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the ChABCB1 gene in Hainan Cephalotaxus fortunei that transports cephalotaxus sinensis from the extracellular to the intracellular space and its application, particularly the ChABCB1 gene, an ATP-binding cassette transporter (ABC transporter) cloned during the transcellular transport of cephalotaxus sinensis in Hainan Cephalotaxus fortunei, and its encoded protein and its application. Background Technology

[0002] Hainan corn nut ( Cephalotaxus hainanensis *Cephalotaxus* is a rare medicinal plant containing the natural anticancer drugs holoharponine and homoharponine. Holoharponine and homoharponine are ester alkaloids with holoharponine as their core structure. Their pharmaceutical preparations can be used clinically to treat non-lymphocytic leukemia and various malignant tumors such as breast cancer, choriocarcinoma, and cervical cancer, making them among the few natural anticancer drugs with significant efficacy and a clear pharmacological mechanism. However, due to the scarcity and slow growth of *Cephalotaxus* species, the production cost of these drugs remains high. Furthermore, *Cephalotaxus* species are listed as a Class II National Key Protected Endangered Plant, prohibiting the acquisition of raw materials through logging. Although holoharponine alkaloids can be produced artificially, their high synthesis difficulty and cost have prevented commercial production. Therefore, using genetic engineering or cell engineering techniques to solve the raw material source problem of holoharponine alkaloids has become a current research focus, aiming to reduce production costs and improve their clinical application value.

[0003] ATP-binding cassette (ABC) transporters are a class of membrane transporters widely found in organisms, functioning to transport various substrate molecules. In plants, ABC transporters participate in many important physiological processes, including the transport of plant hormones, the accumulation of secondary metabolites, and the excretion of exogenous toxic substances. For example, ABC transporters in some plants have been found to be closely related to drug resistance, disease resistance, and the transport of secondary metabolites. *Cephalotaxus fortunei* is an important medicinal plant, containing anticancer alkaloids in its roots, stems, and leaves. Cephalotaxus sinensis is the parent nucleus of the anticancer compounds cephalotaxus sinensis and homocephalotaxus sinensis in *Cephalotaxus fortunei*, and its synthesis and transport directly affect the content of cephalotaxus sinensis and homocephalotaxus sinensis. However, the transport mechanism of cephalotaxus sinensis within *Cephalotaxus fortunei* remains unclear. Summary of the Invention

[0004] The purpose of this invention is to provide a ChABCB1 gene and its encoded protein that transports cephalotaxine from the extracellular to the intracellular environment in Hainan Torreya grandis.

[0005] The present invention also aims to provide an expression cassette, vector, engineered bacteria or transgenic cell line containing the ChABCB1 gene, which transports cephalotaxine from the extracellular to the intracellular environment in the above-mentioned Hainan Cephalotaxus fortunei.

[0006] The final objective of this invention is to provide the application of the ChABCB1 gene, the protein encoded by the ChABCB1 gene, the expression cassette containing the ChABCB1 gene, vectors, engineered bacteria or transgenic cell lines, etc., in the extracellular to intracellular transport of cephalotaxine.

[0007] The first objective of the present invention can be achieved by the following technical solution: a ChABCB1 gene in Hainan Torreya grandis that transports cephalotaxine from the extracellular to the intracellular space, wherein the nucleotide sequence of the ChABCB1 gene is shown in SEQ ID NO:1.

[0008] This invention analyzes the full-length transcriptome sequencing results of Cephalotaxus fortunei in Hainan, designs a pair of specific primers, and performs PCR amplification on cDNA from Cephalotaxus fortunei leaf samples to obtain the CDS sequence of the ChABCB1 gene that transports cephalotaxine. The specific CDS sequence of the ChABCB1 gene is shown in SEQ ID NO:1.

[0009] The present invention also provides a protein encoded by the ChABCB1 gene described above, the amino acid sequence of which is shown in SEQ ID NO: 2, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids to the sequence.

[0010] The second objective of the present invention can be achieved by the following technical solution: an expression cassette, vector, engineered bacteria or transgenic cell line containing the ChABCB1 gene, which transports cephalotaxine from the extracellular to the intracellular space in the Hainan Cephalotaxus fortunei.

[0011] As a preferred embodiment of the present invention, the engineered bacteria provided by the present invention constructs the ChABCB1 gene into the vector pDR196, transforms Saccharomyces cerevisiae with the obtained recombinant vector, and screens positive strains to construct engineered yeast bacteria.

[0012] This invention, through initial screening and comparative transcriptome analysis of *Cephalotaxus fortunei* from Hainan, identified several candidate transporter genes whose levels significantly differed before and after feeding with cephalotaxus sinensis. Among them, the expression level of ChABCB1, a transporter gene belonging to the ABC family and ABCB subfamily, was significantly increased, suggesting that it may play an important role in the transmembrane transport of cephalotaxus sinensis. *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) Saccharomyces cerevisiaeChABCB1 is a model organism widely used for the expression and functional study of exogenous genes. The full-length sequence of this gene was cloned from *Torreya grandis* hainanensis and inserted into a *Saccharomyces cerevisiae* expression vector for functional verification. By expressing ChABCB1 in *Saccharomyces cerevisiae*, this study provides an important tool for exploring its function in alkaloid metabolism and transport.

[0013] As another preferred embodiment of the present invention, the transgenic cell line provided by the present invention constructs the ChABCB1 gene into the vector pK7FGW2, transforms tobacco BY2 cells with the obtained recombinant vector, and screens for positive transgenic cell lines.

[0014] The final objective of this invention can be achieved through the following technical solution: the application of the above-mentioned ChABCB1 gene, the protein encoded by the ChABCB1 gene, the expression cassette containing the ChABCB1 gene, the vector, engineered bacteria or transgenic cell lines, etc., in the transport of holotaxine, especially in the transport of holotaxine from the extracellular to the intracellular environment.

[0015] It can also be further applied in biosynthetic technologies such as genetic engineering and cell engineering.

[0016] Furthermore, genetically engineered plants (with increased cephalotaxine content) can be utilized in the food, health product, and biopharmaceutical industries.

[0017] Compared with existing technologies, this invention has the following advantages: Addressing the current weakness in functional gene research in *Cephalotaxus fortunei*, this invention is the first to clone the transporter protein ChABCB1, which transports cephalotaxine, from *Cephalotaxus fortunei*. Furthermore, this invention employs genetic engineering methods to transform the ChABCB1 gene into target plants or suspension cells, which can promote the transport of cephalotaxine. This provides an important theoretical basis for obtaining drugs or foods with anticancer activity using genetic engineering or cell engineering techniques in the future, and has broad application prospects and significant economic value. Attached Figure Description

[0018] Figure 1 The results of the phylogenetic tree analysis of the ABCB transporter protein system of *Torreya grandis* from Hainan in Example 1;

[0019] Figure 2 The relative expression levels of the ChABCB1 gene in different tissues of *Cephalotaxus fortunei* in Example 1;

[0020] Figure 3The images shown are: PCR verification diagram (A) and SDS-PAGE identification diagram (B) of competent yeast cells in Example 1. Note: M: DL 8000bp; 1-5: 5 different single clones; M': protein marker; 1': ChABCB1; 2': pDR196.

[0021] Figure 4 The accumulation of holoharbitis alkaloid in pDR196-ChABCB1 yeast cells and negative control at different incubation times in Example 2. Note: The blank column corresponding to ND indicates the peak area of ​​the peak time corresponding to the peak of holoharbitis alkaloid that was not detected.

[0022] Figure 5 The cephalotaxine tolerance test was performed on the pDR196 and pDR196-ChABCB1 yeast transformants in Example 3.

[0023] Figure 6 This is an experiment in Example 4 on the transport of different types of alkaloids by pDR196-ChABCB1 yeast cells;

[0024] Figure 7 The effect of ATP inhibitors on the internal uptake of cephalotaxine in pDR196-ChABCB1 yeast cells in Example 5. Note: Verapamil (Vm), 100 μM; Glibenclamide (Gb), 100 μM; Baforomycin A1 (Baf A1), 0.1 μM;

[0025] Figure 8 The image shows the GFP green fluorescence signal and subcellular localization of the ChABCB1 gene after being transformed into BY2 tobacco cells, as observed by laser confocal microscopy in Example 6. The successfully transformed BY2 tobacco cells can clearly express GFP green fluorescence. EGFP represents the green fluorescence signal of the fusion protein containing ChABCB1::EGFP at 488nm. DIC represents the imaging of differential interference contrast under transmitted white light. Merge represents the overlay of DIC and the fluorescence field.

[0026] Figure 9 The figures represent the accumulation of cephalotaxine in ChABCB1 transgenic tobacco cell lines and wild-type cells at different incubation times in Example 7. Note: Purple circle: The accumulation of cephalotaxine in 1 mL of wild-type tobacco cell line (WT) within 24 h after providing exogenous cephalotaxine; Orange square: The accumulation of cephalotaxine in 1 mL of ChABCB1 transgenic cell line (ChABCB1) within 24 h. Detailed Implementation

[0027] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, all examples were conducted under conventional experimental conditions, such as those described in Sam Brook et al.'s Molecular Cloning: A Laboratory Manual (Sam Brook J & Russell DW, 2001), or as recommended by the manufacturer's instructions. Unless otherwise specified, all reagents and raw materials used were commercially available. For example, pDR196 was recommended, but not limited to, purchased from Miaoling Biotechnology, as a Saccharomyces cerevisiae expression vector; pK7FGW2 was recommended, but not limited to, purchased from Thermo Fisher Scientific, as a plant expression vector; tobacco BY2 cells were recommended, but not limited to, a gift from Associate Professor Guan Pingyin of China Agricultural University; cephalotaxine, holoharponene, homoharponene, sanguisorbin, hyoscyamine, nicotine, etc., were recommended, but not limited to, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and ATP inhibitors verapamil, glibenclamide, bafloxacin A1, etc., were recommended, but not limited to, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0028] Example 1: Cloning of the ChABCB1 gene

[0029] Based on previous analysis of the full-length transcriptome sequencing results of *Cephalotaxus fortunei* hainanensis, a pair of specific primers were designed (forward primer ChABCB1-F: 5'-atggttcttggtactattggttctg, as shown in SEQ ID NO: 3 and reverse primer ChABCB1-R: 5'-tgaagaggccttctgcaa, as shown in SEQ ID NO: 4). Total RNA was extracted from *Cephalotaxus fortunei* leaves using the Tiangen RNAprep Pure Kit, and cDNA was synthesized via reverse transcription. The CDS sequence of the ChABCB1 gene, which transports cephalotaxine in *Cephalotaxus fortunei* hainanensis, as shown in SEQ ID NO: 1, was amplified from the cDNA obtained using the aforementioned primers ChABCB1-F and ChABCB1-R. The full-length CDS sequence of the ChABCB1 gene is 3936 bp (as shown in SEQ ID NO: 1, including the stop codon tag). The ChABCB1 protein encodes 1311 amino acids (as shown in SEQ ID NO: 4). NO: 2), molecular mass (kDa) 136.88, molecular formula C 6108 H 9841 N 1653 The isoelectric point is 8.04.

[0030] Various transport proteins play different roles in plants, and different transport proteins have different transport substrates. To further explore the function of ChABCB1 in plants, the amino acid sequence encoded by ChABCB1 was homologously aligned using NCBI, identifying the corresponding amino acid sequences encoded by transport protein genes in other species. Based on this, a phylogenetic tree was constructed using MEGA11. The phylogenetic analysis results of the ChABCB1 ABCB transport protein in *Cephalotaxus fortunei* are as follows: Figure 1 As shown, from Figure 1 As can be seen, ChABCB1 is most closely related to TcMDR found in dwarf yew, and is on the same evolutionary branch as CjMDR1 and AtMDR4, which have been identified as inbound transporters; while it is far removed from the outbound transporters AtMDR1 and AtMDR19.

[0031] The specific steps for cloning the ChABCB1 gene are as follows:

[0032] (1) Cool and grind fresh Hainan Torreya leaves with liquid nitrogen, accurately weigh 100mg of powder and add it to a 2mL centrifuge tube pre-cooled with liquid nitrogen, add Tiangen RNAprep Pure Kit total RNA lysis buffer, vortex mix and let stand for 10min.

[0033] (2) Total RNA was extracted from the leaves of *Torreya grandis* Hainane according to the instructions of the Tiangen RNAprep Pure Kit.

[0034] (3) The total RNA extracted from the leaves of Hainan Torreya grandis was used as a template and the first strand of cDNA was synthesized by reverse transcription using the PrimeScript™ RT Master Mix reverse transcriptase kit (purchased from Takara Bio Engineering Dalian Co., Ltd.). The reaction conditions were carried out according to the kit instructions.

[0035] (4) The CDS sequence of the ChABCB1 gene that transports cephalotaxine in Hainan Torreya grandis was amplified from the cDNA obtained by reverse transcription of RNA using the above primers ChABCB1-F and ChABCB1-R.

[0036] (5) Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2.5 min, 35 cycles; 72℃ extension for 10 min; The PCR products obtained by amplification were seamlessly cloned into expression vectors pDR196 (Miaoling Biotechnology, Saccharomyces cerevisiae expression vector) and pK7FGW2 (Thermo Fisher Scientific, plant expression vector), transformed into Escherichia coli competent cells, positive clones were screened and sequenced to obtain the required full-length gene; Plasmids carrying the ChABCB1 gene sequence were extracted from the positive clones and named pDR196-ChABCB1 plasmid and pK7FGW2-ChABCB1 plasmid.

[0037] Tissue-specific expression analysis: Total RNA was extracted from the stems and leaves of *Cephalotaxus fortunei* and reverse transcribed into first-strand cDNA as templates for qPCR identification. The expression pattern of the ChABCB1 gene in the two tissues of *Cephalotaxus fortunei* was analyzed based on the qPCR results. Using the gene expression level in the stem as a control, ChABCB1 expression was lower in the stem and higher in the leaf. Figure 2 Furthermore, previous studies have shown that the accumulation of cephalotaxine in leaves is higher than in stems. The experimental results indicate that the tissue expression pattern of the ChABCB1 gene is the same as the accumulation site of cephalotaxine in *Torreya grandis* hainanensis, suggesting that ChABCB1 is highly likely involved in the transport process of cephalotaxine.

[0038] qPCR identification was performed using the ChamQ Universal SYBR qPCR Master Mix kit (Novizan, China) and a real-time PCR instrument (ABI / QuantStudio 6 Flex). The housekeeping gene was Ch18S. Primers were designed using BeaconDesigner 7 software, and the primers used are as follows:

[0039] ChABCB1-qF: gtcaagtcctctgtcaat, as shown in SEQ ID NO: 5;

[0040] ChABCB1-qR: ctcaatgtcaccaatcac, as shown in SEQ ID NO: 6;

[0041] Ch18S-qF: caatggcaatgacaatgg, as shown in SEQ ID NO: 7;

[0042] Ch18S-qR: cagagaacaatccgaact, as shown in SEQ ID NO: 8.

[0043] Construction of the pDR196-ChABCB1 expression vector: Based on the gene sequence and the pDR196 vector sequence, a gene fragment containing the pDR196 homologous linker was obtained using Hainan Cephalotaxus cDNA as an amplification template. Homologous recombination was then performed using seamless cloning to construct the pDR196-ChABCB1 yeast system expression vector, specifically including:

[0044] Using the pEASY®-Uni Seamless Cloning and Assembly Kit (Beijing), the pDR196 *Saccharomyces cerevisiae* expression vector fragment and the ChABCB1 fragment with identical homologous arms were ligated according to the instructions. The PCR reaction program was 50℃, 30 min; 4℃, ∞. The ligation product was transformed into *E. coli* DH5α competent cells. After screening and identification of pDR196-ChABCB1 positive clones, the pDR196-ChABCB1 circular plasmid was extracted and stored at -20℃ for later use. SDS-PAGE identification of ChABCB1 protein: Following the instructions of the classic yeast transformation kit (Coolaber), yeast AD12345678 competent cells were prepared and transformed. The pDR196-ChABCB1 recombinant plasmid was transformed into defective yeast competent cells AD12345678, which is often used to study the function of transport proteins. After incubation at 30℃, PCR verification was performed. Gel electrophoresis showed that the ChABCB1 gene was present in yeast cells. Figure 3 Figure A shows that the pDR196-ChABCB1 expression vector was successfully transformed into competent yeast cells. After expansion culture, expression was induced in half-strength medium, and the supernatant was collected by sonication for subsequent protein detection. The recombinant protein was identified using SDS-PAGE, with the pDR196 expression vector serving as a negative control. SDS-PAGE protein gel analysis showed successful expression of ChABCB1 protein in yeast cells. The target protein, with a molecular weight of approximately 136.9 kDa, was obtained from the lysed supernatant. However, no band of the same size was found at a similar location in the pDR196 lane. Figure 3 (Figure B in the middle)

[0045] Example 2: HPLC detection of the transport capacity of ChABCB1 for the substrate cephalotaxine.

[0046] To analyze the gene function of ChABCB1, this experiment conducted a substrate transport assay of ChABCB1 protein in Saccharomyces cerevisiae. Recombinant yeast was induced to express the protein, and then Saccharomyces cerevisiae cells containing pK7FWG2 and pDR196 were incubated in a medium containing cephalotaxine (50 µM). Samples were taken at planned time points (0 h, 2 h, 4 h, 6 h, 12 h, and 24 h). The substrate was determined by high performance liquid chromatography, and the content of cephalotaxine was calculated based on the peak area, thus verifying its function of transporting cephalotaxine.

[0047] The specific steps are as follows:

[0048] (1) Using the transformed pDR196 Saccharomyces cerevisiae expression vector yeast as a negative control, ChABCB1 protein was induced to be expressed. Three replicates were set up. In the last step, the precipitate was resuspended with 2.5 mL 1 / 2 SD-U sterile medium and 50 µM of cephalotaxine was added. The culture was carried out at 30℃ and 200 rpm for 2 h, 4 h, 6 h, 12 h and 24 h.

[0049] (2) Centrifuge at 4000 rpm at room temperature for 15 min, and collect the supernatant and precipitate at the same time;

[0050] (3) Resuspend the precipitate with 10 mL ddH2O, centrifuge at 4000 rpm for 15 min at room temperature, and discard the supernatant;

[0051] (4) Resuspend the precipitate with 1 mL ddH2O, transfer it to a 2 mL centrifuge tube, centrifuge at 4000 rpm for 15 min at room temperature to completely remove the supernatant;

[0052] (5) After weighing, add 100 μL of 100% methanol to the tube, vortex to mix, extract by sonication for 40 min, and centrifuge at 13000 rpm at room temperature for 10 min.

[0053] (6) Filter the supernatant through a 0.22 μm hydrophobic filter membrane and store at 4 °C for HPLC detection;

[0054] (7) HPLC was used for detection. Agilent 1260 Infinity II column (Plus C18, 250×4.6mm, 5 μm), column temperature 30℃, flow rate 1mL / min, sample loading volume 20μL, detection wavelength of cephalotaxine 290nm, the mobile phase ratio changes as follows: 0.1% formic acid water (A), acetonitrile (B).

[0055] Table 1. Cephalotaxine elution procedure

[0056] .

[0057] Using 5-fold serial dilutions of cephalotaxine solution as standards, HPLC analysis was performed, and regression curves were plotted. The regression equation was y = 121.33x + 7.384 (R²). 2 =0.9965); calculate the content of cephalotaxine in the sample, and the results are as follows. Figure 4 As shown, compared with the negative control yeast strain containing the expression vector plasmid, the pDR196-ChABCB1 yeast strain had a higher content of cephalotaxine; the experimental results indicate that the ChABCB1 protein is involved in the transport of cephalotaxine.

[0058] Further investigation revealed the relationship between the accumulation of cephalotaxine in yeast cells and culture time. The cephalotaxine accumulation in the pDR196-ChABCB1 transformed yeast strain was consistently higher than that in the negative control group at every time point. It was found that from 0h to 12h, the cephalotaxine content in both transformed yeast cells continuously increased with increasing time (e.g., ...). Figure 3 This indicates that when yeast cells are in a culture medium containing 50 µM cephalotaxine, the yeast cells continuously absorb cephalotaxine from the culture medium without exceeding their maximum capacity or absorption capacity. Analysis of the detection results at 12 h and 24 h showed that the content of cephalotaxine in the two types of transformed yeast cells showed a decreasing trend opposite to that from 0 h to 12 h. Therefore, it is believed that during the period of 12 h to 24 h, the yeast cells transformed by pDR196 and pDR196-ChABCB1 gradually stopped transporting cephalotaxine inward, and it is possible that other transport proteins were used to expel cephalotaxine from the yeast cells to maintain the normal physiological activity and stability of the yeast cells.

[0059] Example 3 Spot tolerance test of Saccharomyces cerevisiae

[0060] To further confirm whether the ChABCB1 protein has the ability to internalize cephalotaxine from the culture medium into transgenic yeast cells, the transgenic yeast cells were incubated with half-strength culture media containing different concentrations of cephalotaxine. The results are as follows: Figure 5 .

[0061] from Figure 5 The results show that different concentrations of cephalotaxine were used to evaluate the uptake and tolerance capacity of transgenic yeast cells for cephalotaxine. When no cephalotaxine was added to the culture medium, both pDR196 and pDR196-ChABCB1 yeast cells grew normally. However, as the concentration of cephalotaxine increased, the growth inhibition of transgenic yeast cells became more significant (due to the action of transport proteins, transgenic yeast cells absorbed cephalotaxine faster and in greater quantities, resulting in significant inhibition). At the same cephalotaxine concentration, the growth of pDR196 yeast cells was superior to that of transgenic yeast cells. When the concentration of cephalotaxine was increased to 1 mM, the growth of both pDR196 and pDR196-ChABCB1 yeast cells was poor. Therefore, overexpression of ChABCB1 in yeast cells enhances the uptake of cephalotaxine, and when the uptake of cephalotaxine reaches its peak, it exerts toxicity on the cells, inhibiting cell growth.

[0062] Example 4: Substrate-specific analysis of transporters

[0063] Saccharomyces cerevisiae cells containing pDR196-ChABCB1 and pDR196 plasmids were incubated in a medium containing different types of alkaloids at the same concentration (0.16 µg / μL), and samples were taken at 12 h. After extraction with 100% methanol, the contents of each type of alkaloid were determined by high-performance liquid chromatography (HPLC). The content of each type of alkaloid was calculated based on peak area. Each treatment time point was repeated three times. The HPLC system was an Agilent 1260 Infinity II with a column (Plus C18, 250 × 4.6 mm, 5 μm). The HPLC detection methods for different alkaloids differ, and the detection methods are as follows:

[0064] Homoharringtonine: Flow rate 1 mL / min, sample volume 20 μL, detection wavelength of homoharringtonine 290 nm, mobile phase ratio changes as follows: 0.1% formic acid water (A), acetonitrile (B);

[0065] Homoharringtonine: flow rate 0.8 mL / min, sample volume 20 μL, detection wavelength of homoharringtonine 290 nm, mobile phase ratio changes as follows: 0.1% formic acid water (A), acetonitrile (B);

[0066] Sanguisorbine: column temperature 35℃, flow rate 0.8mL / min, sample loading volume 20μL, detection wavelength of sanguisorbine 284nm, mobile phase ratio changes as follows: 0.1% phosphoric acid water (A), acetonitrile (B);

[0067] Hyoscyamine: Column temperature 35℃, flow rate 1mL / min, sample loading volume 20μL, detection wavelength of hyoscyamine 210nm, mobile phase ratio changes as follows: 0.1% phosphoric acid water (A), acetonitrile (B);

[0068] Nicotine: Column temperature 30℃, flow rate 1mL / min, sample loading volume 20μL, nicotine detection wavelength 260nm, mobile phase ratio changes as follows: 0.02 mol / L Na2HPO4, pH=6 (A), methanol (B).

[0069] Transgenic yeast cells were incubated with other types of alkaloids, with the same yeast strain transformed from pDR196 plasmid used as a negative control. The specific uptake of harvestinoline by ChABCB1 was investigated. Sensitivity to various alkaloids was determined by comparing the uptake levels of isoquinoline alkaloids (sanguisorbin), pyrrolidine alkaloids (hyoscyamine), pyridine alkaloids (nicotine), and two types of ester alkaloids from 1-phenylethylisoquinoline (harvestinoline and homoharvestinoline are derivatives of harvestinoline and different from harvestinoline) in transgenic yeast cells and pDR196 yeast cells. This study aimed to investigate substrate specificity. The results are as follows: Figure 6 As shown.

[0070] from Figure 6 The results show that when fed nicotine, the absorption of nicotine by the negative control and transgenic yeast cells was basically the same. Under the same conditions, the systemic absorption of other types of alkaloids by the negative control group was higher than that by the transgenic yeast cells. Therefore, the pDR196-ChABCB1 yeast cells did not play a role in the incubation of the four types of alkaloids. In addition, although the active absorption capacity for each type of alkaloid was different, ChABCB1 was more sensitive to the absorption of cephalotaxine. In summary, no active transport function of different types of alkaloids was found in the transgenic yeast cells, indicating that ChABCB1 has relatively strong transport substrate specificity.

[0071] Therefore, the yeast cells of pDR196-ChABCB1 in this invention can only transport cephalotaxine and cannot transport other compounds, including cephalotaxine and homoharringtonine. Cephalotaxine and homoharringtonine are derivatives of cephalotaxine, which are based on the molecular structure of cephalotaxine with the addition of other functional groups. Therefore, the above experiments have verified the substrate specificity of ChABCB1.

[0072] Example 5: ATP-dependent analysis of substrate uptake

[0073] To determine whether the uptake of cephalotaxine by yeast cells depends on ChABCB1, the effects of ATP inhibitors and the presence of ATP in yeast cells were analyzed. Compared to the control group without inhibitors, verapamil (100 μM) acted as an ATP inhibitor of plant ABC transporters and Ca2+. 2+ Channel blockers inhibit the inward absorption of cephalotaxine by ChABCB1. Figure 7 Bafloxacin A1 (0.1 μM), a specific type V ATPase inhibitor, and NH4Cl (5 mM) disrupt the transmembrane pH environment. Similar to verapamil, bafloxacin A1 and NH4Cl inhibit the inward absorption of cephalosporin by ChABCB1. Glifenamide (100 μM) is another inhibitor of sulfonylurea receptors and plant ABC transporters, and is also a K... + Channel inhibitors; compared to the other two inhibitors, the uptake of cephalosporin in transgenic yeast cells treated with glibenclamide was slightly higher than that in pDR196 yeast cells. Meanwhile, the cephalosporin content in the glibenclamide-treated transgenic yeast cells was significantly lower than that in the control group (the control group was the no-inhibitor group). Glibenclamide did not significantly inhibit the inward uptake of cephalosporin. In conclusion, ChABCB1 functions as a cephalosporin influx transporter and requires ATP for energy.

[0074] Example 6 Construction and transformation of the ChABCB1 gene expression vector into tobacco BY2 suspension cells

[0075] To understand the physiological function of ChABCB1 in plants, its subcellular localization was determined in stably transformed tobacco BY2 cells. First, a binary expression vector pK7FWG2-ChABCB1::EGFP was constructed according to Gateway (Invitrogen, USA), and green fluorescent protein (GFP) was fused to the C-terminus of ChABCB1. After transformation into Agrobacterium, ChABCB1 was stably integrated into the genome of tobacco BY2 cells according to the methods of Buschman and Gao. Wild-type tobacco cells were cultured and subcultured according to Rajabi's method; cells in the late exponential growth stage were observed under a microscope. Compared to wild-type tobacco cell lines, green fluorescent signals were clearly visible distributed in the cytoplasmic membrane and around the nucleus of the transgenic cells. Figure 8 In summary, the transgenic cell line was successfully transformed, and ChABCB1 was localized on the cell membrane.

[0076] Specifically, to better analyze the function and subcellular localization of the protein encoded by the ChABCB1 gene, the gene was further constructed into a GFP-labeled vector and then transfected into tobacco BY2 suspension cells to achieve recombinant expression. Subcellular localization was observed using laser confocal microscopy. The specific steps are as follows:

[0077] (1) The pK7FGW2-ChABCB1 plasmid obtained in Example 1 was transformed into Agrobacterium by heat shock transformation. The vector carries a GFP green fluorescent tag, and the transformation result can be verified by observing the green fluorescence through laser confocal microscopy.

[0078] (2) Select a single Agrobacterium clone and inoculate it into 5 mL of LB medium and incubate overnight at 28°C;

[0079] (3) Take 1.5 mL from the overnight cultured Agrobacterium and transfer it to 50 mL of fresh LB medium. Incubate at 28°C with shaking for 3-5 h until OD600 = 0.8-1.0;

[0080] (4) Collect Agrobacterium bacterial suspension and centrifuge, discard the supernatant, and resuspend the bacterial cells in 180 μL of cotransfer medium;

[0081] (5) Select tobacco BY2 suspension cells cultured for 3-4 days, wash and filter them with 700-1000 mL of co-transfer medium, and then take 5 mL of BY2 suspension cells into Agrobacterium cells and shake and culture for 15 min.

[0082] (6) Transfer the BY2 suspension cells after shaking culture to a co-transfer medium plate and co-culture for 3-4 days;

[0083] (7) Transfer the co-cultured BY2 suspension cells to a selection medium plate to screen for positive cells and then transfer them to a liquid selection medium for suspension culture;

[0084] (8) Observation using laser confocal microscopy showed that BY2 tobacco cells successfully transfected with ChABCB1 protein clearly expressed GFP green fluorescence, and the protein was located on the cell membrane. ChABCB1 may act as a cephalotaxine uptake protein on the cell membrane. See details. Figure 7 .

[0085] Example 7: Substrate Analysis of ChABCB1 Overexpressing Transgenic Tobacco Cell Lines

[0086] To directly demonstrate that ChABCB1 possesses the ability to systemically transport cephalotaxine, the coding sequence of ChABCB1 was transformed and inserted into the tobacco genome, with expression driven by a strong 35S promoter. Transgenic cell lines were fed cephalotaxine, with wild-type cells serving as a negative control. Cells were harvested at 2h, 4h, 6h, 8h, 12h, and 24h for HPLC analysis. The results showed that (…). Figure 9 The transgenic tobacco cell lines exhibited higher alkaloid uptake and accumulation from the outset. The content of cephalotaxine in transgenic cells gradually increased between 2 and 4 hours, remained unchanged between 4 and 12 hours, and then showed an upward trend between 12 and 24 hours. Furthermore, when the intracellular cephalotaxine content was relatively stable, the cephalotaxine content in transgenic cells was nine times that of wild-type cells. In contrast, wild-type tobacco cell lines showed lower levels of alkaloid accumulation, maintaining approximately the same content between 2 and 12 hours. However, the cephalotaxine content in wild-type tobacco cells increased between 12 and 24 hours, consistent with the behavior in transgenic cells. Therefore, ChABCB1 expression can increase intracellular accumulation of cephalotaxine by uptake of extracellular cephalotaxine. However, when tobacco cells were co-cultured with cephalotaxine for an extended period, the cephalotaxine content in both wild-type and transgenic cells increased.

[0087] Therefore, this invention screened a highly expressed transporter candidate gene by analyzing comparative transcriptome data of *Cephalotaxus fortunei* in Hainan. This candidate gene belongs to the ABC family and is named ChABCB1. Green fluorescence signals were observed on the cell membrane of the ChABCB1 transgenic cell line. ChABCB1 may be involved in the uptake of cephalotaxine into the cell on the cell membrane. The ChABCB1 was characterized and its function was verified: (1) Substrate transport assay in yeast cells verified the function of ChABCB1 in transporting cephalotaxine. The substrate diversity and ATP dependence of the transporter protein of ChABCB1 were characterized. ChABCB1 specifically transports cephalotaxine. After inhibiting the energy source of the transporter protein, the content of cephalotaxine in the transgenic cells was lower than that in pDR196 cells. ChABCB1, as an inflow pump located on the cell membrane, relies on the energy of ATP hydrolysis to play a role on the cell membrane, specifically transporting cephalotaxine into the cytoplasm, thereby accelerating the metabolic flow; (2) ChABCB1 transgenic tobacco cells have the ability to transport cephalotaxine, and the content of cephalotaxine in their cells is much higher than that of wild type. It has been confirmed in plant cells that ChABCB1 takes up cephalotaxine internally; (3) The expression of ChABCB1 in Hainan Torreya grandis is tissue-specific: it is expressed at a high level in leaves and at a very low level in stems.

[0088] Although the invention has been described in conjunction with preferred embodiments, it should be understood that the scope of protection of the invention is not limited to the embodiments described herein. Other embodiments of the invention will be readily conceived and understood by those skilled in the art in light of the description and practice of the invention disclosed herein. The description and embodiments are to be considered exemplary only, and the true scope and spirit of the invention are defined by the claims.

Claims

1. A ChABCB1 gene in *Torreya grandis* that transports cephalotaxine from the extracellular to the intracellular space, characterized in that... The nucleotide sequence of the ChABCB1 gene is shown in SEQ ID NO:

1.

2. The protein encoded by the ChABCB1 gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. An expression cassette containing the ChABCB1 gene of claim 1.

4. A vector containing the ChABCB1 gene of claim 1.

5. An engineered bacterium or transgenic cell line containing the ChABCB1 gene of claim 1, the expression cassette of claim 3, and the vector of claim 4.

6. The application of the ChABCB1 gene of claim 1, the protein encoded by the ChABCB1 gene of claim 2, the expression cassette of claim 3, the vector of claim 4, and the engineered bacteria or transgenic cell line of claim 5 in the extracellular to intracellular transport of cephalotaxine in yeast cells or tobacco.

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