TcuJAZ3 gene of taxus cuspidata and application of TcuJAZ3 gene in increasing yield of paclitaxel

Abstract

The invention discloses a taxus cuspidata TcuJAZ3 gene and application of the taxus cuspidata TcuJAZ3 gene in increasing the yield of paclitaxel, and belongs to the field of gene engineering. The low-temperature response gene TcuJAZ3 is cloned from taxus cuspidata, expression of enzyme genes in almost all paclitaxel biosynthetic pathways is remarkably activated by overexpression of the TcuJAZ3, the TcuJAZ3 is a key regulatory factor of the paclitaxel biosynthetic pathways, and the paclitaxel content in transgenic leaves is remarkably increased. The TcuJAZ3 gene can be used as a key target for regulating and controlling the paclitaxel content, and a new way is provided for culturing a new variety of taxus chinensis with high paclitaxel content and obtaining paclitaxel by utilizing gene engineering and cell engineering.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a Taxus cuspidata TcuJAZ3 gene and application thereof in improving paclitaxel yield. BACKGROUND

[0002] Taxus spp. is a common name of Taxus plants, which has extremely high medicinal value. Paclitaxel isolated from Taxus plants is a natural anti-tumor active diterpene alkaloid compound, which can inhibit mitosis of cancer cells and effectively prevent proliferation of cancer cells. With the increasing number of cancer patients year by year, the market demand for paclitaxel is also increasing year by year. However, Taxus plants grow extremely slowly in natural conditions, have poor regeneration ability, have a long cultivation cycle and have extremely low paclitaxel content. In addition, the wild resources of Taxus plants are severely damaged due to the predatory exploitation of human beings. In order to alleviate the contradiction between the demand for paclitaxel and the protection of Taxus resources, researchers have developed various paclitaxel production methods, such as chemical total synthesis method, cell culture method and microbial fermentation method. Unfortunately, due to the complex structure of paclitaxel, the synthesis path involves multiple reaction steps, so the above methods still have problems such as high cost, complicated synthesis steps and difficult control of reaction conditions, which makes it difficult to efficiently obtain paclitaxel.

[0003] It is known that low temperature is one of the main abiotic stresses faced by plant growth and development, but low temperature can also promote the accumulation of plant secondary metabolites. Jasmonic acid (JA) as a key stress signal molecule plays an important role in transmitting low temperature signals and regulating the accumulation of secondary metabolites. JA signal is proved to be able to significantly promote the synthesis of paclitaxel.

[0004] Taxus cuspidata Siebold & Zucc. is a plant of Taxaceae and Taxus, which is an internationally recognized endangered and rare anticancer plant. The species has a history of 2.5 million years on earth and belongs to a plant fossil. In 1996, it was listed as a rare and endangered plant in the world by the United Nations Educational, Scientific and Cultural Organization, and in 1999, it was included in the list of first-class rare and endangered wild plant protection in China. SUMMARY

[0005] The technical problem to be solved by the present application is how to develop a method for improving paclitaxel yield of Taxus plants.

[0006] The technical solution of the present application is a Taxus cuspidata TcuJAZ3 gene, wherein the TcuJAZ3 gene is a nucleotide fragment encoding a protein shown in SEQ ID No. 2.

[0007] Further, the sequence of the nucleotide fragment is shown in SEQ ID No. 1.

[0008] The expression vector containing the TcuJAZ3 gene.

[0009] The application of the TcuJAZ3 gene or the overexpression vector in improving the paclitaxel production of Taxus cells.

[0010] Further, the application is overexpressing the TcuJAZ3 gene in Taxus to improve the paclitaxel production of Taxus.

[0011] A method for cultivating a Taxus cell line with high paclitaxel production, the overexpression vector containing the TcuJAZ3 gene is genetically transformed into Taxus cells, and positive cells are screened to obtain a Taxus cell line with high paclitaxel production.

[0012] A method for cultivating a Taxus strain with high paclitaxel content, the overexpression vector containing the TcuJAZ3 gene is genetically transformed into Taxus tissues, and positive plants are screened after tissue culture to obtain a Taxus strain with high paclitaxel content.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] The present application clones a low-temperature response gene TcuJAZ3 from Taxus cuspidata, and the overexpression of TcuJAZ3 significantly activates the expression of almost all enzyme genes in the paclitaxel biosynthesis pathway, is a key regulatory factor of the paclitaxel synthesis pathway, and the paclitaxel content in the transgenic leaves is significantly improved. TcuJAZ3 can be used as a key target for regulating the paclitaxel content, and provides a new way for preparing Taxus cells with high paclitaxel production. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The paclitaxel and taxane content changes in the TcuJAZ3 overexpressed Taxus leaves.

[0016] Figure 2 The expression level changes of target genes and paclitaxel biosynthesis pathway enzyme genes in the TcuJAZ3 overexpressed Taxus leaves. DETAILED DESCRIPTION

[0017] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the experimental materials used are purchased from commercial channels unless otherwise specified.

[0018] The plant material used in the present application, six-year-old Taxus cuspidata cuttings and two-year-old Taxus cuspidata seedlings, are all planted in the research greenhouse of the Chinese Academy of Forestry.

[0019] Example 1 Cloning of Taxus cuspidata TcuJAZ3 Gene

[0020] 1. Extraction of Total RNA from Taxus cuspidata

[0021] Total RNA was extracted from Taxus cuspidata using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) according to the kit instructions. The quality of the extracted total RNA was determined by agarose gel electrophoresis and NanoDrop One / OneC Ultra-micro UV Spectrophotometer. TM The concentration and integrity of the RNA were determined by the NanoDrop One / OneC Ultra-micro UV Spectrophotometer. The concentrations of the four replicate samples were 595.6 ng / mL, 635.6 ng / mL, 627.7 ng / mL, and 978.9 ng / mL, respectively. All of the RNA had clear and complete 28S and 18S bands, indicating that the extracted total RNA was intact and could be used for subsequent gene cloning experiments.

[0022] 2. Synthesis of the First Strand of cDNA

[0023] The RNA was reverse transcribed into cDNA using the FastKing cDNA First-Strand Synthesis Kit (De-Gene) according to the kit instructions. The obtained cDNA was diluted 5-fold and stored at -20°C.

[0024] 3. Cloning of Taxus cuspidata TcuJAZ3 Gene

[0025] (1) Design of Cloning Primers

[0026] Based on the expression data of the JAZ gene family in the Taxus cuspidata transcriptome after low-temperature treatment, a highly expressed JAZ gene was selected and named TcuJAZ3. According to the sequence information of TcuJAZ3, specific primers TcuJAZ3-F: 5'-TATTCTGTGTATCCATCCCG-3' and TcuJAZ3-R: 5'-AGTGACGCCACCTTGTCTAG-3' were designed using the Primer Premier 5 software.

[0027] (2) PCR Amplification of the Full-Length CDS of TcuJAZ3

[0028] The TcuJAZ3 gene was cloned using specific primers and Taxus cuspidata cDNA as the template. The amplification system is shown in Table 1, and the amplification program is set as shown in Table 2.

[0029] Table 1 KOD One TM PCR Master Mix Enzyme Reaction System 1

[0030]

[0031] Table 2 KOD One TM PCR Master Mix Enzyme Reaction Program 1

[0032]

[0033] (3) Gel recovery of PCR products

[0034] 50 μL of PCR product was separated using a 1.2% agarose gel at 150 V for 20 min. The target gene fragment was then recovered using the M5 Hiper Gel Extraction Kit (MF029-01), following the kit's instructions. Finally, NanoDrop gel was used to extract the fragment. TM The concentration of the recovered gel product was determined using a One / OneC ultra-micro UV spectrophotometer.

[0035] (4) Target gene ligation into pTOPO-Blunt vector

[0036] The corresponding reaction system was established according to the instructions in the M5 HiPer pTOPO-Blunt Cloning Kit (MF021). The gel-purified product was ligated into the pTOPO-Blunt vector to obtain a T vector containing the target gene.

[0037] (5) Transformation of E. coli competent cells and identification of positive monoclonal cells

[0038] Escherichia coli competent cells were purchased from Tiangen Biotech (Beijing) Co., Ltd., and the transformation procedure was performed according to the manufacturer's instructions. After overnight culture, single colonies were picked and cultured in LB liquid medium containing 50 μg / mL Amp. A PCR verification experiment was performed in the culture. The primers were universal primers M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3'. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4. After PCR, 10 μL of the PCR product was subjected to agarose gel electrophoresis for detection, and positive bacterial cultures were sent to Sino-American Taihe Biotechnology (Beijing) Co., Ltd. for sequencing.

[0039] Table 3. 2X M5 HiPer plus Taq HiFi PCR mix enzyme reaction system

[0040]

[0041] Table 4. Enzyme Reaction Procedure for 2X M5 HiPer plus Taq HiFi PCR mix

[0042]

[0043] TcuJAZ3 was successfully cloned from the Northeast yew. The full length of TcuJAZ3 is 549bp (SEQ ID No.1) and it encodes 182 aa (SEQ ID No.2).

[0044] (6) Plasmid extraction

[0045] use Plasmids were extracted using the Plasmid MiniPrep Kit (EM101-02). Detailed instructions are provided in the kit's manual. NanoDrop was used for plasmid extraction. TM The concentration of the extracted plasmid was detected using a One / OneC ultra-micro UV spectrophotometer and stored at -20℃.

[0046] Example 2: Construction of TcuJAZ3 transgenic leaves from Taxus chinensis

[0047] The TcuJAZ3 gene cloned in Example 1 was constructed into the pBI121-GUS plasmid, transformed into Escherichia coli competent cells and positive plasmids were extracted. The positive plasmids were then transformed into Agrobacterium tumefaciens competent cells GV3101. Single colonies were picked for bacterial PCR verification. The correctly sequenced Agrobacterium tumefaciens bacterial solutions were added to sterilized glycerol (final concentration to 25%) and stored at -80℃.

[0048] (1) Disinfection pretreatment of yew leaves

[0049] Take fresh leaves from the current year of the Northeast yew tree, wash off the surface dirt, and rinse with running water for 2 hours. In a clean bench, disinfect the leaves by soaking them in a 2% sodium hypochlorite solution for 20 minutes, then rinse them carefully with sterile water 3-5 times. Use sterile filter paper to absorb the moisture from the leaf surface, and then use a blade to make even cuts on the leaves.

[0050] (2) Preparation of Agrobacterium infection solution

[0051] ① Take out the Agrobacterium glycerol mixture stored at -80℃, streak it on an LB agar plate containing 50 μg / mL Kana and 20 μg / mL Rif for activation, and incubate upside down at 28℃ for 48–72 h. Pick positive single colonies and place them in 1 mL of LB liquid medium containing 50 μg / mL Kana and 20 μg / mL Rif, and incubate at 28℃ with shaking at 200 rpm for 10–12 h.

[0052] ② Add the turbid bacterial culture to 80 mL of LB liquid medium containing 50 μg / mL Kana and 20 μg / mL Rif, and incubate at 28°C and 200 rpm with shaking until the bacterial culture reaches OD.600 The value is approximately 0.8.

[0053] ③ Collect bacterial cells by centrifugation at 5000 rpm for 15 min, resuspend the bacterial cells in resuspending buffer (1 / 2 MS + 100 μM AS), and adjust the OD of the infection solution. 600 The value was reduced to around 0.8, and the mixture was left to stand in the dark at room temperature for 2 hours.

[0054] (3) Infection of Taxus chinensis leaves

[0055] The pretreated leaves were placed in Agrobacterium infection solution, with Agrobacterium infection solution containing empty vector plasmid as a control. Each group of experiments was set up with 3 biological replicates, each biological replicate containing leaves from 2 Taxus chinensis plants. The leaves were cultured in the dark at 28℃ and 200rpm for 16h.

[0056] The infected leaves were removed, and the Agrobacterium infection solution on the leaf surface was washed off with sterile water. The leaves were then dried with sterile filter paper and inoculated onto a co-culture solid medium (1 / 2 MS + 30 g / L sucrose + 5.0 g / L agar + 100 μM AS) and incubated in the dark for 24 h.

[0057] (4) Collection and preservation of transgenic leaf samples

[0058] First, rinse the yew leaves 3–5 times with sterile water containing 300 mg / L termethin, then rinse them 3–5 times with sterile water. Finally, blot the surface moisture with sterile filter paper. A portion of the leaves were flash-frozen in liquid nitrogen and stored at -80°C for subsequent experiments. The remaining leaves were placed in sterile water and shaken at 120 rpm for 3 days, then dried at 60°C and ground into powder for the determination of paclitaxel and its metabolites.

[0059] Example 3: Detection of paclitaxel and other taxanes in transgenic leaves

[0060] (1) Preparation of mixed reference solution

[0061] Accurately weigh 6.0 mg of each of the following reference standards: Baccatin III, 10-Deacetylbaccatin III (10-DAB), Paclitaxel, Cephalomannine, 10-Deacetylpaclitaxel (10-DAT), and 7-EpiPaclitaxel. Prepare a reference standard stock solution with a mass concentration of 0.60 mg / mL using methanol. Take 1.0 mL of each stock solution, mix them, and then dilute 100 times to prepare a mixed reference standard stock solution with a mass concentration of 0.001 mg / mL.

[0062] (2) Preparation of test samples

[0063] Accurately weigh 0.1 g of the transgenic sample powder, add 5 mL of 80% ethanol, mix thoroughly, and extract by sonication at 50°C for 1 h. After cooling to room temperature, briefly centrifuge, collect the supernatant, and centrifuge to dry in a nitrogen evaporator. Redissolve the sample with 1 mL of deionized water, and after complete dissolution, add 1 mL of ethyl acetate for extraction. Collect the organic phase solution and centrifuge to dry in a nitrogen evaporator. Before detection, dissolve the sample thoroughly with 1 mL of methanol, filter twice using a 0.22 μm filter membrane, and collect the final filtrate to obtain the test sample.

[0064] (3) Chromatographic-mass spectrometry conditions

[0065] Chromatographic conditions: ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm), mobile phase: acetonitrile-water. Elution gradient: 0–0.5 min, 5% acetonitrile; 0.5–12 min, 5%–95% acetonitrile; 12–12.1 min, 95%–5% acetonitrile; 12.1–15 min, 5% acetonitrile; flow rate: 0.3 mL / min; column temperature: 30 °C; injection volume: 10 μL.

[0066] Mass spectrometry conditions: electrospray ionization source; positive ion detection; multiple reaction detection scanning mode; ion source temperature 320℃; ionization voltage 4200V; sheath gas flow rate 10L / min; scanning range 150~2000m / z.

[0067] (4) Plotting the standard curve

[0068] Take the stock solution of the mixed reference standard and dilute it by 5, 10, 20, 50, 100, 200, 500, and 1000 times, respectively. Take 10 μL of each concentration of the mixed reference standard and perform analysis. Finally, plot a standard curve with peak area as the ordinate (Y) and the mass concentration of each reference standard as the abscissa (X). Analyze the linear regression equation and correlation coefficient (R²) for each reference standard. 2 See Table 5.

[0069] Table 5. Linear equations and correlation coefficients for six taxane compounds.

[0070]

[0071]

[0072] (5) The effects of TcuJAZ3 overexpression on taxane metabolism were systematically analyzed by determining the contents of paclitaxel and taxane compounds in transgenic leaves. Data showed ( Figure 1Overexpression of TcuJAZ3 significantly increased the content of 10-DAB, baccatin III, paclitaxel and 7-epitaphthol in leaves (P<0.01). Among them, the content of 7-epitaphthol, which increased the least, was nearly 1.6 times that of the control CK, and the content of paclitaxel was about 1.7 times that of the control CK, while the content of cephalotaxine decreased significantly by 14%.

[0073] Example 4: Expression analysis of target genes and paclitaxel synthesis pathway enzyme genes in transgenic leaves

[0074] The results of TcuJAZ3 gene overexpression analysis showed that ( Figure 2 Compared with the control group (CK), the transcription level of TcuJAZ3 in the TcuJAZ3 overexpression group (TcuJAZ3-OE) was significantly increased (P<0.01), indicating that the transient gene overexpression system was successfully constructed. Overexpression of TcuJAZ3 significantly activated the expression of enzyme genes in almost all taxane biosynthesis pathways. Among them, the expression level of TASY, the core enzyme that determines taxane backbone synthesis, was 1.63 times that of the CK group in the TcuJAZ3-OE group (P<0.01). The expression levels of other key enzyme genes that modify the taxane backbone, such as T13αH, T10βH, T7βH, T14βH, and T2αH, were all significantly increased (P<0.01), with T7βH increasing to nearly 12 times that of the control and T13αH increasing to nearly 6 times that of the control. The expression levels of acylationases TAT, TBT, DBAT, BAPT, and DBTNBT were all significantly increased by multiple times (P<0.01), and the expression levels of side chain synthesis-related enzymes PAM and PCL were also significantly increased to 3 to 5 times that of the control (P<0.05).

Claims

1. The TcuJAZ3 gene of Taxus chinensis, wherein the TcuJAZ3 gene is a nucleotide fragment encoding the protein shown in SEQ ID No.

2.

2. The TcuJAZ3 gene according to claim 1, characterized in that, The nucleotide sequence of the nucleotide fragment is shown in SEQ ID No.

1.

3. An overexpression vector containing the TcuJAZ3 gene as described in claim 1 or 2.

4. The application of the TcuJAZ3 gene as described in claim 1 or 2 or the overexpression vector as described in claim 3 in increasing the yield of paclitaxel from Taxus chinensis.

5. The application according to claim 4, characterized in that, The application is to overexpress the TcuJAZ3 gene as described in claim 1 or 2 in yew, thereby increasing the yield of paclitaxel in yew.

6. A method for preparing a yew cell line with high paclitaxel production, characterized in that, Transform yew cells with an overexpression vector containing the TcuJAZ3 gene as described in claim 1 or 2, screen for positive cells, and obtain a yew cell line that produces high levels of paclitaxel.

7. A method for cultivating a yew strain with high paclitaxel content, characterized in that, Transform yew tissue with an overexpression vector containing the TcuJAZ3 gene as described in claim 1 or 2, screen positive plants after tissue culture, and obtain yew lines with high paclitaxel content.

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