Root-specific high-expression promoter pPYKS as well as recombinant expression vector and application thereof

Abstract

The invention discloses a root-specific high-expression promoter pPYKS and a recombinant expression vector and application thereof, an over-integration genome, transcriptome and single cell group data optimization promoter, and also discloses a multifunctional plant expression vector construction method based on the promoter. Through cross-species transformation (tobacco and arabidopsis thaliana), the root specificity high expression characteristic and the potential in metabolic pathway precise regulation and control and complex character intelligent design are verified, and an efficient regulation and control tool is provided for plant metabolic engineering, synthetic biology and stress-resistant breeding; and core element support is provided for constructing an environment response type intelligent breeding decision-making system.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, specifically to a root-specific highly expressed promoter pPYKS, and also to a recombinant expression vector containing the promoter and its applications. Background Art

[0002] As a core element in gene expression regulation, the tissue specificity and expression efficiency of promoters directly determine the application effects of genetic engineering. Their design requires in-depth association with the genotype-phenotype regulatory network. Traditional promoter screening relies on experimental trial and error, lacking the integration of multi-omics data and the support of intelligent algorithms, resulting in low efficiency of element design and poor environmental adaptability. Especially in the field of crop intelligent breeding, it is urgent to realize the rational design and optimization of gene elements through the standardized analysis of phenome, epigenome and single-cell group data.

[0003] Although the currently widely used constitutive promoters (such as CaMV 35S) can drive gene expression in various plant tissues, they lack spatiotemporal specificity, easily leading to energy redundancy, metabolic imbalance and side effects in non-target tissues. Especially in the field of plant secondary metabolite synthesis (such as tropane alkaloids, medicinal terpenoids), there is an urgent need for promoters with strong tissue specificity and high expression efficiency to precisely regulate metabolic pathways.

[0004] As a key species for tropane alkaloid synthesis, the root metabolic network of Atropa belladonna is regulated at multiple levels. However, the current research on Atropa belladonna root-specific promoters is still blank, and the existing promoters cannot meet the dual requirements of high expression efficiency and broad applicability in metabolic engineering. The present invention successfully cloned the Atropa belladonna root-specific highly expressed promoter pPYKS by integrating single-cell transcriptome, chromatin open region (ATAC-seq) and methylation data of Atropa belladonna root tissues. Further cross-species verification (tobacco, Arabidopsis thaliana) confirmed its broad applicability, providing a standardized tool for dynamic reprogramming of metabolic pathways and design of high-yield and stress-tolerant traits. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a root-specific highly expressed promoter pPYKS; the second objective of the present invention is to provide a recombinant expression vector containing the root-specific highly expressed promoter pPYKS; the third objective of the present invention is to provide a cell containing the root-specific highly expressed promoter pPYKS or the recombinant expression vector; the fourth objective of the present invention is to provide the application of the root-specific highly expressed promoter pPYKS in regulating the root-specific and highly efficient expression of a target gene.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] 1. The root-specific highly expressed promoter pPYKS, and the nucleotide sequence of the promoter pPYKS is as shown in SEQ ID NO.3.

[0008] 2. A recombinant expression vector containing the root-specific highly expressed promoter pPYKS.

[0009] Preferably in the present invention, the recombinant expression vector is obtained by ligating the sequence shown in SEQ ID NO.3 into the 5' end of the GUS gene of the plant expression vector.

[0010] 3. A cell containing the root-specific highly expressed promoter pPYKS or the recombinant expression vector.

[0011] Preferably in the present invention, the cell is a plant cell.

[0012] Preferably in the present invention, the plant cell is a belladonna cell, a tobacco cell or an arabidopsis cell.

[0013] 4. Application of the root-specific highly expressed promoter pPYKS in regulating the root-specific and highly efficient expression of a target gene.

[0014] The beneficial effects of the present invention are as follows: The present invention discloses the root-specific highly expressed promoter pPYKS, its recombinant expression vector and application. The promoter is constructed on a plant expression vector, and its root-specific highly expressed characteristics are verified by cross-species transformation (tobacco, arabidopsis), providing an efficient regulation tool for plant metabolic engineering, synthetic biology and stress-resistant breeding, and providing core element support for constructing an environment-responsive intelligent breeding decision-making system. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the following drawings are provided for illustration:

[0016] Figure 1 Results of qPCR analysis and transcriptome analysis (A: qPCR analysis; B: transcriptome analysis);

[0017] Figure 2 Paraffin section diagram of lateral roots of transgenic belladonna plants stained with GUS;

[0018] Figure 3 Quantitative detection of GUS reporter gene in transgenic belladonna;

[0019] Figure 4 Quantitative detection of GUS reporter gene in transgenic tobacco;

[0020] Figure 5 Quantitative detection of GUS reporter gene in transgenic arabidopsis. Detailed implementation manners

[0021] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0022] Example 1: Cloning of the highly root-specific expression promoter pPYKS of Atropa belladonna and construction of a plant expression vector

[0023] (1) Integration of multi-omics data

[0024] Based on the single-cell transcriptome and epigenome data of Atropa belladonna roots, the PlantRegMap tool was used to predict the cis-regulatory elements of root-specifically expressed genes, and a 1.3 kb region upstream of the AbPYKS gene was screened out as a candidate promoter.

[0025] (2) Cloning of the promoter pPYKS

[0026] Take the fibrous root tissue of Atropa belladonna (Atropa belladonn), grind it in liquid nitrogen, and extract genomic DNA by the CTAB method. The integrity of the DNA was detected by 1% agarose gel electrophoresis, and the concentration was measured by a spectrophotometer (A260 / A280 = 1.8 - 2.0).

[0027] According to the Atropa belladonna genome database, the Softberry and Plantcare software were used to predict the promoter region of the AbPYKS gene (including the TATA box and the transcription start site), and specific primers were designed:

[0028] pAbPYKS-HindⅢ-F: 5'-cgcaagctttttacttactttttaggctaggtt-3' (SEQ ID NO.1);

[0029] pAbPYKS-BglⅡ-R: 5'-cgcagatcttttccctttctttgtttggtttaa-3' (SEQ ID NO.2);

[0030] Using the Atropa belladonna genomic DNA as a template, PCR amplification was carried out using the HyPerFusion Plus DNA polymerase. After the amplification product was verified by 1% agarose gel electrophoresis, the gel was cut and recovered and purified (AxyPrep TM PCR Cleanup Kit), and sequencing confirmed that a pPYKS promoter fragment with a length of about 1.3 kb was obtained. The specific sequence is shown in SEQ ID NO.3, and sequencing confirmed that its sequence was consistent with the genome database.

[0031] Through qPCR analysis and transcriptome analysis, the primer sequences for qPCR are as follows:

[0032] qPCR-pAbPYKS-F: 5'-cttcaggagatgggctggac-3' (SEQ ID NO.4)

[0033] qPCR-pAbPYKS-R: 5'-aagcatcacagaagggcagg-3' (SEQ ID NO.5)

[0034] The results are as Figure 1 shown. It was found that PYKS had a relatively high expression level in the roots of plants, especially in the fibrous roots.

[0035] (3) Construction of plant expression vector

[0036] pPYKS::GUS vector: The vector is the pCAMBIA1305.1-GUS-EPSPS vector modified in this laboratory. pCAMBIA1305.1 is a plant binary vector containing the GUS reporter gene (β-glucuronidase) and the hygromycin resistance marker (Hygromycin). Then, EPSPS (5-enolpyruvylshikimate-3-phosphate synthase, glyphosate resistance gene) was inserted as a selectable marker. The specific method was to double-digest pCAMBIA1305.1 with the restriction enzyme XhoI to generate sticky ends, mix the EPSPS digested with the same enzyme with the vector backbone in an equimolar ratio, and ligate using T4 DNA ligase (see Qiaozhuo Zhang. Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna. Metabolic Engineering 72 (2022) 237–246). The ligation product was transformed into Escherichia coli, and the LB plate containing the appropriate antibiotic was spread. The single colonies grown were detected for positivity, and the plasmid was extracted and sequenced to ensure that the EPSPS sequence had no mutations and was in the correct direction.

[0037] The pCAMBIA1305.1-GUS-EPSPS vector and the promoter fragment were double-digested with the restriction enzymes HindⅢ and BglⅡ, and ligated using T4 Ligase (Thermo Scientific) to obtain the vector pPYKS::GUS. The ligation product was transformed into Escherichia coli DH5α and spread on the LB solid medium supplemented with kanamycin, and cultured overnight at 37°C.

[0038] Perform positive detection on the grown single colonies, and the primers are as follows:

[0039] pAbPYKS-check-F: 5'-TCATTGAAATTATTGAGGTGGGCT-3' (SEQ ID NO.6);

[0040] GUS-check-R: 5'-ATCGGGTACAGACTAGTTCGTC-3' (SEQ ID NO.7);

[0041] Transfer the positive strain to a 10 mL LB liquid medium containing kanamycin for enlarged culture to extract the plasmid (refer to the reagent kit instruction manual for plasmid extraction). After the plasmid is correctly sequenced, it is used for subsequent experiments.

[0042] (2) p35S::GUS control vector

[0043] Insert the cauliflower mosaic virus 35S promoter (p35S) into the same site of pCAMBIA1301 to construct the p35S::GUS vector. Verify the correctness of the vector by restriction enzyme digestion and sequencing.

[0044] Restriction enzyme digestion and sequencing verification show that the pPYKS::GUS and p35S::GUS vectors are successfully constructed.

[0045] (3) Preparation of engineering bacteria

[0046] Preparation of competent cells: Select the strain (EHA105), and culture it in an LB liquid medium containing antibiotics with shaking until OD600 = 0.5 - 0.85. After ice-bathing for 30 minutes, centrifuge to collect the bacteria, resuspend them with pre-cooled 20 mM CaCl2 solution, aliquot and store at -80 °C for later use.

[0047] Transform the constructed vector into Agrobacterium tumefaciens EHA105. In this experiment, the freeze-thaw method is used. Add the plasmid to the thawed Agrobacterium tumefaciens competent cells, ice-bathe for 5 minutes, quickly freeze in liquid nitrogen for 8 minutes, heat-shock at 37 °C for 5 minutes, and finally resuscitate with LB medium for 3 - 5 hours. Coat the LB plate containing antibiotics, screen for positive clones and verify them by PCR or restriction enzyme digestion. Obtain the engineering strains EHA105-pPYKS::GUS and EHA105-p35S::GUS.

[0048] Example 2, Transformation of Atropa belladonna plants and result analysis

[0049] (1) Activation of engineering bacteria

[0050] Inoculate the engineered EHA105 strains (EHA105-pPYKS::GUS and EHA105-p35S::GUS) by streaking on YEP solid medium containing Rif and Kan antibiotics, and incubate them upside down in an incubator at 28 °C for about 48 hours to obtain monoclonal colonies.

[0051] Pick monoclonal EHA105 Agrobacterium and inoculate it into 10 mL of YEP liquid medium containing Rif and Kan antibiotics, and culture it on a shaker at 28 °C for about 24 hours. After positive detection of the activated engineered strains, they are used for subsequent genetic transformation of Atropa belladonna.

[0052] (2) Induction of transgenic Atropa belladonna plants

[0053] Cut the cotyledons of aseptic Atropa belladonna seedlings that have grown for about 15 days, and inoculate the explants into the resuspended bacterial solution for 5 minutes. Then transfer the explants to sterile absorbent paper to blot dry the surface bacterial solution, and finally place the explants on a co-culture solid medium covered with sterile filter paper and co-culture them in the dark at 25 °C for 4 days. After the co-culture is completed, transfer the explants to the regeneration medium and culture them at 25 °C under a 16-hour photoperiod, and subculture by changing the medium every 3 weeks. After 6 weeks of screening culture, transfer the explants with multiple buds to jars containing fresh screening medium. After 3 - 4 weeks, cut 1 - 3 cm long buds from each regenerated explant and transfer them to MS rooting medium (added with hygromycin for screening, concentration 20 mg / L) for rooting culture.

[0054] (3) Acclimatization and transplantation of regenerated aseptic Atropa belladonna seedlings

[0055] Acclimatize and transplant the rooted pG2 transgenic plants and wild-type plants into an artificial climate chamber for cultivation and use in subsequent experiments. Wash the medium on the roots with running water to avoid root rot caused by moldy residual medium. After air-drying the surface moisture of the seedling roots, transplant them into the sterilized mixed substrate (vermiculite:humus:perlite = 6:3:1). To avoid water loss and death of Atropa belladonna plants, remove the excess leaves of the seedlings, seal the seedlings with a transparent cover to reduce water loss. After culturing in the artificial climate chamber for 7 - 10 days, remove the transparent cover for subsequent experiments.

[0056] (4) GUS histochemical staining and activity detection

[0057] GUS staining: Take transgenic Atropa belladonna root, stem, and leaf tissues, immerse them in GUS staining solution (1 mM X-Gluc, 0.1% Triton X-100, 50 mM phosphate buffer pH 7.0), incubate them in the dark at 37 °C for 12 hours, and observe the blue precipitate after decolorization with 70% ethanol.

[0058] GUS fluorescence quantitative analysis: The GUS activity was determined using the 4-MUG (4-methylumbelliferyl-β-D-glucuronide) method. 100 mg of tissue was homogenized, and the extraction buffer (50 mM NaPO4 pH 7.0, 10 mM β-mercaptoethanol) was added. After centrifugation, the supernatant was reacted with 4-MUG, and the activity was detected by a fluorescence spectrophotometer (excitation 365 nm, emission 455 nm).

[0059] The β-glucuronidase (GUS) reporter gene system has been used to characterize and localize gene expression. When the promoter is cloned upstream of the GUS reporter gene, the promoter drives the expression of the GUS reporter gene, and the activation expression of the promoter can be visualized by detecting the cleavage products of β-glucuronidase. To further determine the tissue localization of pAbPYKS, in this study, pAbPYKS was constructed on the pCAMBIA1305.1 (EPSPS)-GUS vector, and transgenic Atropa belladonna plants were successfully constructed through Agrobacterium tumefaciens EHA105-mediated transformation. Paraffin sections of the lateral roots of the transgenic plants stained with GUS were observed, and the results are as Figure 2 shown. The results showed that almost all tissue cells in the GUS sections of pAbPYKS had GUS blue signals, indicating that pAbPYKS was ubiquitously expressed in the roots. To further determine the activity of the promoter, in this study, a GUS reporter gene quantitative detection kit (product number SL7161) from Coolaber Technology Co., Ltd. in Beijing was used for the quantitative detection of the GUS reporter gene. First, 100 mg of the root, stem, and leaf tissues of fresh tobacco were taken, and the materials were rapidly frozen with liquid nitrogen, and then the tissues were ground in a mortar by liquid nitrogen grinding. The ground and broken tissues were transferred to an EP tube, and 1 mL of the extraction solution was immediately added and mixed well. Centrifuge at 12,000 rpm at 4 °C for 10 min. Transfer the supernatant to another clean EP tube and centrifuge at 12,000 rpm at 4 °C for 10 min. The obtained supernatant was the protein extract and was placed on ice for use. The protein concentration was determined using the Bradford method, and the GUS enzyme activity was determined according to the GUS enzyme activity assay kit. The results showed that the GUS activity driven by pAbPYKS was higher in the roots than that driven by 35S.

[0060] Example 3. Transformation of tobacco plants and analysis

[0061] Activation of engineering bacteria (EHA105-pPYKS::GUS and EHA105-p35S::GUS) was carried out according to the method of Example 2.

[0062] Tobacco genetic transformation: The activated engineering bacteria were centrifuged at 5000 rpm for 10 min to collect the bacterial cells. In the ultra-clean bench, the supernatant was poured out, and the bacterial cells were resuspended with the tobacco transformation solution to make the OD600 of the bacterial solution between 0.1 and 0.3, and kept for standby. Select tobacco seedlings with good growth conditions, cut off the healthy leaves, and cut the leaves into 1 cm2 The left and right leaf segments were immersed in the Agrobacterium suspension resuspended with transformation solution for 10 min. The leaf segments were clamped with sterile paper to absorb the bacterial solution on the leaf surface, and then laid flat on the co-culture Petri dish covered with filter paper. After co-culturing for 3 days at 25 °C in the dark, they were transferred to the bud differentiation medium and induced under the light condition at 25 °C. The medium was changed about two weeks later. The explants were screened and cultured until adventitious buds about 1 cm long grew out. The buds were cut and transferred to the rooting medium (hygromycin was added for screening, with a concentration of 20 mg / L) to induce rooting.

[0063] Soil culture of sterile tobacco seedlings: The sterile tobacco seedlings with roots about 8 cm long were transferred to the artificial climate chamber. After 24 h, the sterile seedlings in the culture tank were taken out, the root medium was rinsed clean, and then transplanted into the pre-moistened nutrient soil. A transparent cover was covered to prevent the seedlings from losing water excessively. After 2 days, the transparent cover was removed and the seedlings were continuously cultured for about 30 days for subsequent experiments.

[0064] In this study, pAbPYKS was constructed on the pCAMBIA1305.1 (EPSPS)-GUS vector, and transgenic tobacco plants were successfully constructed through Agrobacterium-mediated transformation by EHA105. To determine the activity of the promoter, quantitative detection of the GUS reporter gene activity in the roots, stems and leaves of transgenic plants was carried out in this study. The method was the same as that in Example 2, and the results were as Figure 4 shown. The results showed that the GUS activity driven by pAbPYKS was higher in roots than that driven by the 35S promoter.

[0065] Example 4. Transformation of Arabidopsis thaliana plants and analysis

[0066] Activation of engineering bacteria (EHA105-pPYKS::GUS and EHA105-p35S::GUS) was carried out according to the method in Example 2.

[0067] Agrobacterium-mediated floral dip transformation of Arabidopsis thaliana: Healthy-growing Arabidopsis thaliana (Columbia ecotype) was selected. At the early flowering stage (the main inflorescence bolted 4 - 5 cm), the top was cut off to promote the growth of lateral branches, and transformation was carried out 4 - 5 days later. Before transformation, the soil needed to be kept moist and the influence of pests should be avoided. The verified Agrobacterium suspension was centrifuged and resuspended with 10 mM MgCl 2+ 5% sucrose solution to OD600 = 0.7 - 0.8, and 0.02% surfactant (such as Silwet L-77) was added to enhance permeability.

[0068] Flower dipping operation: It is optimal when the plant has 20 - 30 inflorescences. Cut off the mature pods to avoid interference. Dipping method: Invert the plant and fully immerse the inflorescence in the Agrobacterium suspension for 10 seconds (the immersion time for Ler-0 ecotype needs to be extended to 10 minutes). After dipping, wrap it with plastic film to maintain a dark and high humidity environment (16 - 24 hours), and then resume normal light culture. To improve the transformation efficiency, repeat the dipping 2 - 3 times at an interval of 7 days. Collect the dry T0 generation seeds about 4 - 6 weeks after transformation.

[0069] Resistance screening: Sow on MS medium containing antibiotics (hygromycin is added for screening, with a concentration of 15 mg / L). The surviving seedlings are candidate transgenic plants of the T1 generation. Quantitatively detect the GUS activity of the reporter gene, and the method is the same as in Example 2.

[0070] It can be seen from the GUS activity detection results ( Figure 5 ), the GUS activity driven by pAbPYKS is higher than that driven by the 35S promoter in the root, indicating that pAbPYKS is a novel promoter with high specific expression in the root. The GUS activity analysis shows that pPYKS has a high driving ability in the root, providing an important regulatory element for subsequent metabolic engineering research.

[0071] The above-mentioned embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. The root-specific highly expressed promoter pPYKS, characterized in that: The nucleotide sequence of the promoter pPYKS is shown in SEQ ID NO.

3.

2. A recombinant expression vector containing the root-specific highly-expressed promoter pPYKS as claimed in claim 3.

3. The recombinant expression vector according to claim 2, characterized in that: The said recombinant expression vector is obtained by ligating the sequence shown in SEQ ID NO. 3 to the 5'-end of the GUS gene of a plant expression vector.

4. A cell containing the root-specific highly-expressed promoter pPYKS as claimed in claim 1 or the recombinant expression vector as claimed in claim 2.

5. The cell according to claim 4, wherein: The said cell is a plant cell.

6. The cell according to claim 5, wherein: The said plant cell is a belladonna cell, a tobacco cell or an arabidopsis cell.

7. Use of the root-specific highly-expressed promoter pPYKS as claimed in claim 1 in regulating the root-specific and highly-efficient expression of a target gene.

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