Promoter mdpar7 driving specific expression in root hair epidermal cells of plants and use thereof

Abstract

The application discloses a plant root hair epidermis cell-specific expression promoter MdPAR7 and application thereof, and belongs to the technical field of genetic engineering and molecular breeding. The application provides the plant root hair epidermis cell-specific expression promoter MdPAR7, and the promoter can promote expression of downstream genes in plant root hair epidermis cells, including expression of a root hair development positive regulation gene and a root hair development negative regulation gene. The inducible recombinant expression vector, the construct or the expression cassette provided by the application can play a synergistic effect through tissue specificity of the MdPAR7 promoter and inducibility of an estrogen response element, so that double regulation of root hair specificity and estrogen induction is realized. The MdPAR7 promoter provided by the application can drive target functional genes to express in root hairs, meets functional requirements of plant root hairs under different environmental stresses, has a wider application scene, and has promotional value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically relating to a promoter MdPAR7 that drives the specific expression of plant root hair epidermal cells and its applications. Background Technology

[0002] Root hairs are tubular structures formed by specialized epidermal cells of plant roots. They are key organs for absorbing water and mineral nutrients such as nitrogen and phosphorus, and their development level directly affects a plant's tolerance to low fertilizer levels and drought resistance. As a perennial woody fruit tree, apples in major producing areas are often subjected to low-phosphorus soils and seasonal drought stress. Poor root hair development leads to decreased nutrient absorption efficiency, weakened stress resistance, and seriously affects yield stability.

[0003] Root hair-specific promoters are crucial elements for precise regulation of root genes, driving the specific expression of target genes in root hairs and reducing redundant expression in non-target tissues. Several root hair-specific promoters have been identified in model plants such as Arabidopsis thaliana, rice, and barley, as well as in gramineous crops. Some of these promoters contain conserved RHE motifs and are universally applicable across related species. They can improve root function by regulating genes related to root hair development, stress resistance, and nutrient absorption.

[0004] The mechanisms of core root hair development regulatory genes such as RSL4, RHD6, and GL2 in Arabidopsis thaliana are relatively well understood. However, the root hair development cycle, regulatory network, and environmental response patterns of woody plants differ significantly from those of model plants, making it difficult to directly apply model plant genes and promoters for precise regulation in woody plants. Current woody plant genetic engineering primarily utilizes CaMV 35S constitutive promoters, which suffer from poor tissue specificity, energy redundancy, and interference with normal plant growth. Furthermore, the discovery and application of endogenous root hair-specific promoters in woody plants remain largely unexplored, hindering the efficiency of molecular breeding for root hair development. Summary of the Invention

[0005] Based on the above-mentioned technological needs, the purpose of this invention is to provide a promoter, MdPAR7, that drives specific expression in plant root hair epidermal cells and its applications. This invention, through the exploration of plant root hair-specific promoters, obtains the promoter MdPAR7, which can induce specific expression in plant root hair epidermal cells. The MdPAR7 promoter is suitable for molecular breeding scenarios that improve plant root hair morphology and stress resistance and nutrient absorption characteristics by directionally regulating the expression of key genes in root hair development. It solves the problems of lack of plant root hair-specific expression tools, low precision of constitutive promoter regulation, and difficulty in flexibly controlling the timing and intensity of gene expression in existing technologies. It enables the directional and on-demand expression of exogenous genes in plant root hair epidermal cells, achieving precise improvement of fruit tree root hair morphology. The promoter MdPAR7 provided by this invention, and the root hair-specific and inducible gene expression system constructed using it, have significant practical implications for improving root function in dicotyledonous plants.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution: The present invention provides a promoter MdPAR7 that drives specific expression in plant root hair epidermal cells, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides an amplification primer pair for the promoter MdPAR7, the amplification primer pair comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO.3.

[0008] Furthermore, the promoter MdPAR7 has a specific initiation function for plant root hair epidermal cells.

[0009] Furthermore, the plant root hair epidermal cell-specific activation function means that the MdPAR7 promoter has a activation function or corresponding expression level in root hair epidermal cells that is much greater than the activation function or corresponding expression level in other plant tissues, or that it has an activation function only in plant root hair epidermal cells.

[0010] The present invention also provides an inducible recombinant expression system, construct or expression cassette containing the promoter MdPAR7.

[0011] Furthermore, the inducible recombinant expression system includes an inducible recombinant expression vector and a supporting induction regulation system. The inducible recombinant expression vector adopts a T-DNA binary vector architecture, with the following elements connected in series from left to right: RB boundary → MdPAR7 promoter → LexA-VP16-hER-TE9 response element → Ter terminator → OlexA-mini35S promoter → target functional gene → GFP marker gene → LB boundary. The supporting induction regulation system includes an inducer regulation module and an environmental temperature co-regulation module.

[0012] Furthermore, the inducer regulation module includes a core inducer, a synergistic inducer, and an alternative inducer; the core inducer is estradiol, with a concentration gradient of 5.0-50.0 μmol / L, including a low concentration of 5.0-15.0 μmol / L, a medium concentration of 20.0-30.0 μmol / L, and a high concentration of 40.0-50.0 μmol / L. The synergistic inducer is ethanol, with a concentration gradient of 1%-10% (v / v), including a low concentration of 1%-3%, a medium concentration of 4%-6%, and a high concentration of 7%-10%. The alternative inducer is dexamethasone, with a concentration gradient of 0.5-8.0 μmol / L, including a low concentration of 0.5-2.0 μmol / L, a medium concentration of 3.0-5.0 μmol / L, and a high concentration of 6.0-8.0 μmol / L; the target functional gene is selected from genes related to root hair development regulation, including silent expression fragments of positive or negative root hair development regulatory genes.

[0013] Furthermore, the LexA-VP16-hER-TE9 in the inducible recombinant expression vector is an estradiol-responsive element that can specifically sense exogenous estradiol signals and activate downstream transcription processes through conformational changes, thereby achieving induction switch control of gene expression.

[0014] Furthermore, the GFP marker gene in the inducible recombinant expression vector is a green fluorescent protein reporter gene, used to track vector transformation efficiency, expression localization, and expression intensity, providing a visual basis for vector function verification.

[0015] Furthermore, the host cell containing the inducible recombinant expression vector is selected from prokaryotic cells or higher eukaryotic cells, wherein the prokaryotic cells include Escherichia coli and Agrobacterium, and the higher eukaryotic cells include plant cells.

[0016] Furthermore, the plant cells are preferably those from apple, Arabidopsis thaliana, or tobacco.

[0017] Furthermore, the construct contains a foreign gene and an MdPAR7 promoter element operatively linked to the foreign gene.

[0018] Furthermore, the exogenous genes include resistance genes, selection marker genes, antigen protein genes, RNAi genes, root hair development regulation genes, stress resistance-related genes, nutrient absorption-related genes, biological agent genes, and plant quality-related genes.

[0019] Furthermore, the exogenous gene is expressed only in the root hair epidermal cells of the plant.

[0020] Furthermore, the screening marker gene or reporter gene is selected from the β-glucuronidase gene or the green fluorescent protein gene.

[0021] Furthermore, the stress-resistance-related genes are selected from low-phosphorus tolerance genes or drought-resistance genes.

[0022] Furthermore, the expression cassette, from 5' to 3', sequentially comprises the following elements: the MdPAR7 promoter element with root hair epidermal cell-specific initiation function, the gene ORF sequence, and the terminator; or from 5' to 3', sequentially comprises the following elements: the MdPAR7 promoter, the LexA-VP16-hER-TE9 response element, the Ter terminator, the OlexA-mini35S promoter, the gene ORF sequence, the GFP marker gene, and the terminator; the expression cassette may also include elements selected from poly(A) elements and / or enhancers.

[0023] The present invention also provides a recombinant vector containing the promoter MdPAR7, wherein the recombinant vector contains the MdPAR7 promoter element, construct or expression cassette; the vector is selected from bacterial plasmids, plant binary vectors or shuttle vectors.

[0024] The plant cells containing the inducible recombinant expression vector, or the chromosomes thereof having the core expression elements of the vector integrated therein, the core expression elements including the MdPAR7 promoter, the LexA-VP16-hER-TE9 estradiol response element, the OlexA-mini35S promoter, the target functional gene, and the GFP marker gene.

[0025] The present invention also provides the application of the aforementioned promoter MdPAR7 in driving gene expression in plant root hair epidermal cells.

[0026] Furthermore, the plants include dicotyledonous plants, including apple, Arabidopsis thaliana, tobacco, and tomato.

[0027] Furthermore, the application specifically includes the following steps: (1) Using genetic transformation methods, an inducible recombinant expression vector, construct or expression cassette containing the promoter MdPAR7 is introduced into plant explants to obtain transformed plant cells; (2) The plant cells regenerate into a plant, resulting in a transgenic plant; (3) Add an inducer to the transgenic plant and induce it at an ambient temperature of 18-28℃ to induce the specific expression of the target functional gene in the root hair epidermal cells of the plant.

[0028] Furthermore, the genetic transformation method is an Agrobacterium-mediated transformation method, wherein the Agrobacterium includes, but is not limited to, strains K599, EHA105, and GV3101 (commercially available strains purchased from Sangon Biotech (Shanghai) Co., Ltd.).

[0029] Furthermore, the inducer includes one or more of estradiol, ethanol, and dexamethasone.

[0030] Furthermore, the molar concentration of estradiol is 5.0-50.0 μmol / L, the volume concentration of ethanol is 1%-10% v / v, and the molar concentration of dexamethasone is 0.5-8.0 μmol / L.

[0031] Furthermore, the target gene includes a silent expression fragment of a gene that positively regulates plant root hair development or a gene that negatively regulates plant root hair development.

[0032] Furthermore, the target functional gene is selected from genes related to root hair development regulation, including positive regulatory genes for root hair development. MdASL , MdAHD Or, genes that negatively regulate root hair development MdAL2 RNAi silencing fragments.

[0033] Furthermore, the plant explants include plant leaves, stem segments, callus tissue, somatic embryos, or root tip tissue.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The MdPAR7 promoter provided by this invention has extremely high expression specificity, driving gene expression only in plant root hair epidermal cells. The constructed inducible recombinant expression vector can achieve dual regulation of "root hair specificity + estradiol induction" through the tissue specificity of the MdPAR7 promoter and the inducible synergistic effect of the LexA-VP16-hER-TE9 estradiol response element. This avoids redundant expression of the target gene in non-target tissues and allows for flexible control of gene expression timing and intensity, reducing interference with plant growth. The MdPAR7 promoter has high regulatory precision, driving the expression of any functional gene in root hairs, including genes related to root hair development, stress resistance substance synthesis, and rhizosphere exudates, to achieve functions such as regulating root hair density and length, enhancing stress resistance, and attracting beneficial soil microorganisms.

[0035] 2. The MdPAR7 promoter provided by this invention has a significant effect on root hair-specific expression in plants. Experiments have confirmed that the MdPAR7 promoter can drive the expression of MdASL / MdAHD, which can increase the length of plant root hair by more than 30%, improve phosphorus absorption efficiency by 20%-30% under low phosphorus conditions, enhance the ability of apples to capture soil moisture after optimizing root hair morphology, increase the relative water content of leaves by 15%-20% under drought stress, significantly improve yield stability, and fill the gap in root hair-specific promoters. The supporting technical system provides a standardized tool for the study of root hair development mechanism and helps to promote the analysis of root hair regulatory network in woody plants.

[0036] Compared to traditional constitutive expression, this system adds an estradiol induction switch, which can flexibly control the expression timing of the target gene, avoiding unnecessary expression during the seedling stage from interfering with root hair development, and is more suitable for the needs of gene function verification and precision breeding. At the same time, the expression is highly controllable. The expression level of the target gene can be adjusted by adjusting the concentration of inducers such as estradiol, ethanol, and dexamethasone, as well as the ambient temperature. It can adapt to the root hair functional needs under different environmental stresses such as mild drought and severe drought, and has a wider range of applications.

[0037] 3. The promoter MdPAR7 provided by this invention can be used to specifically and inducibly regulate the expression of exogenous genes in plant root hair epidermal cells, or for molecular breeding to improve plant root hair morphology, enhance stress resistance, and optimize nutrient absorption efficiency, or for research on root hair development mechanisms; the regulation includes precise control of the timing, intensity, and duration of exogenous gene expression by adjusting the concentration of inducer or the ambient temperature. Attached Figure Description

[0038] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0039] Figure 1 This is a single-cell sequencing map.

[0040] Figure 2 This is a schematic diagram of the structure of the pMdPAR7-N7GFP vector.

[0041] Figure 3 This is a diagram showing the connection of elements in an inducible expression vector.

[0042] Figure 4 The growth of the transgenic adventitious roots mediated by Agrobacterium in this invention.

[0043] Figure 5 This shows the luminescence of the transgenic adventitious roots.

[0044] Figure 6 The degree of root hair growth of the transgenic lines is represented by A, where A is MdPAR7::MdASL; B is MdPAR7::MdAHD; and C is MdPAR7::MdAL2.

[0045] Figure 7 The degree of root hair growth is represented by the wild-type strain, where A is WT-1; B is WT-2; and C is WT-3.

[0046] Figure 8 This is GFP-specific fluorescence of transgenic adventitious roots.

[0047] Figure 9The data represents the root hair length statistics of transgenic lines after the addition of inducing agents. Among them, A represents the root hair length statistics of transgenic lines in the estradiol single-induction group; B represents the root hair length statistics of transgenic lines in the estradiol + ethanol synergistic induction group; and C represents the root hair length statistics of transgenic lines in the dexamethasone substitution induction group. Detailed Implementation

[0048] The technical solutions and beneficial effects of the present invention will be described in detail below with reference to embodiments, so as to fully understand the purpose and features of the present invention. It should be noted that the embodiments described here are merely illustrative and are not intended to limit the entirety of the present invention. Other embodiments obtained by those skilled in the art based on the content of the present invention without creative effort are all within the protection scope of the present invention.

[0049] Example 1: Acquisition of the MdPAR7 promoter Using Gala apple genomic DNA as a template, specific primers were designed based on the regulatory region sequence of MdPAR7. Genomic DNA was extracted from the apple using liquid nitrogen grinding and amplified using conventional PCR methods to obtain a 3000 bp DNA fragment upstream of the ATG start codon of the apple MdPAR7 gene. Sequencing verification of the amplified product showed that it matched the wild-type nucleotide sequence of apple.

[0050] The promoter sequence was subsequently optimized. The advantages of the optimized sequence include: eliminating excessively long single-base repetitive sequences, reducing the probability of mismatches and deletions during PCR amplification, and improving the integrity and uniformity of the amplification products; adjusting the base distribution of non-functional regions without altering the core regulatory elements, which aligns with the transcriptional preferences of plant cells and helps improve the expression efficiency of downstream genes; and reducing base combinations that easily form secondary structures, making the promoter sequence more stable during in vitro cloning and in vivo transcription, and reducing the risk of sequence degradation or mutation. Figure 1 This is a single-cell sequencing map. The optimized full-length MdPAR7 promoter sequence is shown in SEQ ID NO.1. The primer pairs for amplifying the optimized MdPAR7 promoter are shown below: Forward primer: PAR7-F:CAAGCTTGCATGCCTGCAGGagtagcgctgcgagctatactatg (SEQ ID NO. 2); Reverse primer: PAR7-R:GGTACCCTCGAGGGATCCgtcaggtgacgcaagatccgctcg (SEQ ID NO. 3).

[0051] The promoter MdPAR7 provided by this invention was precisely screened using single-cell sequencing technology. Single-cell transcriptome sequencing analysis of cell populations related to root hair development successfully identified candidate promoter fragments with root hair-specific expression characteristics. Subsequent experiments verified that the screened MdPAR7 promoter can specifically drive the overexpression of downstream genes in plant root hair epidermal cells. Its expression activity is strictly limited to root hair epidermal cells and does not affect gene expression in other tissues and organs such as stems, leaves, and non-root hair roots, demonstrating extremely strong tissue specificity.

[0052] Example 2: Construction of expression vector This embodiment provides the construction of an expression vector, specifically including the following steps: (1) Fragments of apple root hair regulatory genes MdASL, MdAHD and MdAL2 were cloned respectively. The accession number of MdASL is: MD05G1243700 (SEQ ID NO.6); the accession number of MdAHD is: MD16G1266300 (SEQ ID NO.7); the accession number of MdAL2 is: MD17G1278000 (SEQ ID NO.8).

[0053] (2) The optimized MdPAR7 promoter obtained in Example 1 and the above-mentioned target gene were inserted into the pROK2-N7GFP vector (sequence as shown in SEQ ID NO.9, purchased from Wuhan Miaoling Biotechnology Co., Ltd.). The original CaMV 35S promoter in the vector was replaced with the MdPAR7 promoter to construct three recombinant expression vectors: pMdPAR7::MdASL-GFP (SEQ ID NO.10), pMdPAR7::MdAHD-GFP (SEQ ID NO.11), and pMdPAR7::MdAL2-GFP (SEQ ID NO.12). These vectors were then transformed into Escherichia coli TOP10 strains and preserved.

[0054] (3) The recombinant vector was transformed into Agrobacterium K599 strain (purchased from Sangon Biotech (Shanghai) Co., Ltd.) using the heat shock transformation method, and the positive Agrobacterium was verified by colony PCR. The structural diagram of the vector pMdPAR7-GFP is shown below. Figure 2 As shown.

[0055] (4) Construction of inducible expression vectors: The inducible expression vector uses a T-DNA binary vector (SEQ ID NO.13). The modified T-DNA arrangement from left to right is: RB boundary → MdPAR7 promoter → LexA-VP16-hER-TE9 estradiol response element → Ter terminator → OlexA-mini35S promoter → target functional gene (MdASL, MdAHD, or MdAL2) → GFP marker gene → LB boundary, achieving dual regulation of "root hair specificity + estradiol induction". The expression vector is constructed using the pER10-35S-EGFP plasmid (sequence as shown in SEQ ID NO.14, purchased from Wuhan Miaoling Biotechnology Co., Ltd.) as the backbone. The T-DNA region of this plasmid naturally carries the RB boundary, LexA-VP16-hER, EGFP, LB boundary, and kanamycin selection element.

[0056] The MdPAR7 promoter (SEQ ID NO.1) was first amplified, and the TE9-Ter fragment (SEQ ID NO.4) and OlexA-mini35S fragment (SEQ ID NO.5) were synthesized. At the same time, the target fragments MdASL, MdAHD, and MdAL2 were amplified.

[0057] The sequence of synthetic TE9-Ter fragment is as SEQ ID Shown in NO.4: GATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATG ACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATC.

[0058] The sequence of OlexA-mini35S fragment is as SEQ ID Shown in NO.5: TGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTACTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTTCGCCGACAGTGGTCCCA AAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCAACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATAAGGAAGTTCATTTCATTTGGAGAGAACA.

[0059] Homologous recombination was used. The original promoter driving LexA in the original vector was removed, and the MdPAR7 promoter was inserted. TE9 and a terminator were then linked downstream of the LexA-VP16-hER vector. The original 35S promoter upstream of EGFP was then removed, and OlexA-mini35S and the target gene were inserted sequentially, linking the target gene to EGFP in cis. After verification by PCR, enzyme digestion, and sequencing, three recombinant vectors were constructed and transformed into Agrobacterium for subsequent experiments. The target gene was not expressed in the transgenic material without estradiol treatment; estradiol application induced expression only in the root hairs. The structure of the inducible expression element is shown below. Figure 3 As shown.

[0060] Example 3: Experiment on transformation and root induction of Gala apple leaves Based on the Agrobacterium tumefaciens introduced with the vector pMdPAR7::MdASL-GFP obtained in Example 2, this example provides Agrobacterium tumefaciens-mediated transformation and root induction of apple Gala leaves, specifically including the following steps: (1) Activation of bacterial strain: Take 200 μL of positive Agrobacterium tumefaciens strain and add it to 2 mL of LB medium (containing SPE antibiotic) for initial activation; take 500 μL of initially activated bacterial strain and add it to 20 mL of LB medium (containing SPE antibiotic) for secondary activation; take 1 mL of secondary activated bacterial strain and add it to 20 mL of LB medium (containing SPE antibiotic) for tertiary activation. Add AS 1.5 h after shaking, with a final concentration of 50-100 μM / L, and shake until the OD600 is about 0.5.

[0061] (2) Preparation of infection solution: Centrifuge the activated bacterial solution at 5000 rpm for 5 min and discard the supernatant; add sterile water to resuspend and centrifuge again at 5000 rpm for 5 min and discard the supernatant; add infection solution containing AS (1 / 2 MS + 15 g / L sucrose, pH 5.2) and adjust OD600 to 0.2-0.3.

[0062] (3) Explant treatment: Take Gala apple tissue culture seedlings that have been subcultured for 20-30 days, cut off the leaves at the top of the plant, then cut off two-thirds of the leaves and remove the leaf tips, immerse them in the infection solution for 1 minute, and then use sterile filter paper to absorb the excess infection solution.

[0063] (4) Co-culture: Transfer the leaves to co-culture medium (1 / 2MS + 15g / L sucrose + 7g / L agar, pH 5.2) and incubate in the dark at 22℃ for 3 days.

[0064] (5) Rooting induction: The co-cultured leaves were transferred to rooting medium (1 / 2 MS + 15 g / L sucrose + 7 g / L agar + 300 mg / L termethin, pH 5.8) and cultured at 25°C under light (16 h light / 8 h dark). The medium was replaced every 10 days, and transgenic adventitious roots were obtained after 3 weeks.

[0065] Example 4: Promoter Specificity Verification Based on the transgenic adventitious roots prepared in Example 3, this example provides the preparation of root tip sections and verification of promoter specificity of transgenic adventitious roots, specifically including the following steps: (1) Preliminary specificity verification: The luminescence of the transgenic adventitious roots was initially verified using a handheld laser spotlight, and the luminescent roots were marked for subsequent material collection.

[0066] (2) Preparation of fixative: Dilute 4% PFA solution to 2.5% with 1×PBS solution and pre-cool on ice.

[0067] (3) Material fixation: Take the root tip of the transgenic plant (about 1 cm in length), immerse it in a pre-cooled 2.5% PFA solution, and then place it in a 4℃ refrigerator overnight for fixation.

[0068] (4) Sucrose gradient washing: Dilute 4% PFA solution to 1% with 1×PBS solution, and prepare 10%, 20% and 30% sucrose gradient washing solutions with 1% PFA. Wash the fixed root tips in sequence, each step for 20 minutes, to ensure that the material is fully dehydrated.

[0069] (5) Embedding: Prepare a 5% low melting point agarose solution (4g agarose + 80mL water), heat until completely dissolved, cool to 45-55℃, pour the agarose solution into a square container, take out the root tip from the sucrose solution, use filter paper to absorb the excess liquid from the root tip, then embed it in the agarose solution and place it on ice to solidify quickly.

[0070] (6) Sectioning: Wrap the solidified agarose block with plastic wrap and store at 4°C (use within one week). Prepare 30μm thick root tip longitudinal sections using a shaker. Collect the sections into 1×PBS solution containing DAPI staining solution for nuclear staining.

[0071] (7) Specificity verification: The slide was placed on a glass slide and observed by ultra-high resolution laser confocal microscopy (excitation wavelength 360 nm, emission wavelength 460 nm). GFP-specific fluorescence was detected only in the root hair epidermis, confirming the root hair epidermal specific expression of the MdPAR7 promoter. Figure 8 ).

[0072] Example 5: Phenotypic Analysis of Transgenic Adventitious Roots Based on Examples 3 and 4, this example further analyzes and verifies the phenotype and function of transgenic adventitious roots, specifically including the following steps: (1) Phenotypic measurement: The root hair length (n>100) of transgenic and wild-type plants was measured using ImageJ software. The results are shown (see Figure 4 , 5 (6, 7): The root hair length of MdPAR7::MdASL and MdPAR7::MdAHD transgenic lines increased by 32%-45% compared with the wild type; the root hair length of MdPAR7::MdAL2 transgenic lines increased by 25%-30% compared with the wild type.

[0073] (2) Low phosphorus stress treatment: Transgenic plants and wild-type plants were planted in soil with an available phosphorus content of 5 mg / kg. After 4 weeks of cultivation, the phosphorus content of the plants was measured. The results showed that the phosphorus absorption efficiency of the transgenic lines was 20%-30% higher than that of the wild type.

[0074] (3) Drought stress treatment: PEG6000 was used to simulate drought (mass fraction 20%). After 7 days of treatment, the relative water content of leaves was measured. The relative water content of leaves of transgenic lines was 15%-20% higher than that of wild type, and the drought resistance was significantly enhanced.

[0075] Example 6: Functional Verification of Inducible Vector This embodiment provides functional verification of the inducible vector, specifically including the following parts: Agrobacterium infection and transgenic adventitious root induction were performed according to the procedure in Example 3. The obtained transgenic adventitious roots were divided into the following experimental groups, with three biological replicates in each group. Wild-type apple roots and transgenic adventitious roots without inducing agents were used as controls: Estradiol-induced group: Different concentrations of estradiol were added to the rooting medium, with final concentrations of 5.0 μmol / L (low concentration), 20.0 μmol / L (medium concentration), and 40.0 μmol / L (high concentration).

[0076] Estradiol + ethanol synergistic induction group: 1%, 4%, and 7% (v / v) ethanol were added to a culture medium containing 20.0 μmol / L estradiol.

[0077] Dexamethasone substitution induction group: Different concentrations of dexamethasone were added to the rooting medium, with final concentrations of 0.5 μmol / L (low concentration), 3.0 μmol / L (medium concentration), and 6.0 μmol / L (high concentration).

[0078] Temperature-coordinated control group: Based on 20.0 μmol / L estradiol induction, the culture temperatures were set at 18℃, 25℃, and 28℃, and the photoperiod was maintained at 16h light / 8h dark.

[0079] All experimental groups underwent follow-up testing 10 days after induction treatment. The experimental results are as follows: Figure 9 As shown.

[0080] Results analysis: In the precise regulatory mechanism of the estradiol-induced system, the hER domain in the LexA-VP16-hER-TE9 element can specifically bind to estradiol. In the absence of estradiol, this element is inactive, the OlexA-mini35S promoter is silenced, and the target gene is not expressed. After the addition of estradiol, the binding of the hER domain to estradiol triggers a conformational change, activates the LexA-VP16 transcription activator, and then initiates the expression of the target gene. Precise regulation of expression intensity can be achieved by adjusting the estradiol concentration. The basic effective range is 5.0-10.0 μmol / L. Low concentration (5.0-15.0 μmol / L) is suitable for mild regulation under mild stress (such as mild low phosphorus and mild drought), and root hair length is moderately increased by 15%-20%. Medium concentration (20.0-30.0 μmol / L) is suitable for moderate stress, and root hair length is increased by 25%-35%. High concentration (40.0-50.0 μmol / L) is suitable for severe stress (such as severe low phosphorus and severe drought), and root hair length can be increased by 35%-45%, thus achieving efficient expression of the target gene.

[0081] Ethanol, as a co-inducer of estradiol, optimizes induction efficiency and expression intensity by adjusting ethanol concentration. Low concentrations of ethanol (1%-3%, v / v) enhance the permeability of estradiol in plant cells and increase the binding efficiency of the hER domain to estradiol, resulting in a 10%-15% increase in target gene expression at the same estradiol concentration. Medium concentrations of ethanol (4%-6%, v / v) further enhance the induction effect, increasing expression by 20%-25%. High concentrations of ethanol (7%-10%, v / v) moderately inhibit expression, suitable for scenarios requiring weakened target gene expression, enabling fine-tuning of expression intensity to meet the regulatory needs of different stages of root hair development.

[0082] When dexamethasone is used as an alternative inducer, it cross-binds with the hER domain in the LexA-VP16-hER-TE9 element, initiating induced expression. At concentrations of 0.5–2.0 μmol / L, the target gene is expressed at low levels, suitable for mild regulation in the early stages of root hair development; at concentrations of 3.0–5.0 μmol / L, expression levels significantly increase, suitable for enhanced regulation in the mid-stage; and at concentrations of 6.0–8.0 μmol / L, expression levels reach their peak, suitable for highly efficient regulation under severe stress. Furthermore, dexamethasone induction is characterized by rapid action and stable duration, meeting the needs for short-term rapid regulation.

[0083] Based on the above-mentioned inducer regulation, further optimization was achieved by adjusting the ambient temperature. 22-25℃ is the optimal base temperature, at which the inducer binds most efficiently to the hER domain, resulting in the best expression regulation stability. A low temperature environment of 18-21℃ can reduce the induction reaction rate, decreasing the expression level of the target gene by 15%-20% and prolonging the expression duration. A high temperature environment of 26-28℃ can accelerate the induction reaction rate, allowing the target gene to quickly reach peak expression, increasing the expression level by 10%-15%, but shortening the duration, thus adapting to the time-specific regulatory needs under different growth cycles and environmental stresses.

[0084] Using Gala apple genomic DNA as a template, the complete MdPAR7 promoter sequence was obtained by PCR amplification and sequenced for verification. Three MdPAR7 promoter-driven recombinant expression vectors were constructed: MdPAR7::MdASL, MdPAR7::MdAHD, and MdPAR7::MdAL2. All vectors contain a GFP green fluorescent marker gene, allowing for real-time tracking of vector transformation efficiency, expression localization, and expression intensity. The fluorescence signal intensity visually reflects the induction and regulation effect, providing a visual basis for precise optimization of regulatory parameters.

[0085] This invention utilizes single-cell sequencing to screen for the MdPAR7 promoter, which exhibits extremely high root hair epidermal cell specificity. Based on this promoter, three GFP-labeled recombinant expression vectors and a T-DNA binary inducible recombinant expression vector were constructed, forming a multidimensional, precise regulatory system of "root hair specificity + multiple inducible systems + temperature synergy." Estradiol serves as the core inducer, adapting to different stress intensities through concentration gradients; ethanol as a co-inducer and dexamethasone as an alternative inducer both allow for fine-tuning of expression intensity based on concentration; environmental temperature further optimizes the precision of regulation and the persistence of expression, achieving directional, quantitative, and scenario-specific adaptation for root hair development.

[0086] Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A promoter MdPAR7 that drives specific expression in plant root hair epidermal cells, characterized in that, The nucleotide sequence of the promoter MdPAR7 is shown in SEQ ID NO.

1.

2. The amplification primer pair for promoter MdPAR7 as described in claim 1, characterized in that, The amplification primer pair includes a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3.

3. An inducible recombinant expression system, construct, or expression cassette containing the promoter MdPAR7 as described in claim 1.

4. The application of the promoter MdPAR7 as described in claim 1 in driving gene expression in plant root hair epidermal cells.

5. The application according to claim 4, characterized in that, The plants include dicotyledonous plants, which include apple, Arabidopsis thaliana, tobacco and tomato.

6. The application according to claim 4, characterized in that, The application specifically includes the following steps: (1) Using genetic transformation methods, an inducible recombinant expression system, construct or expression cassette containing the promoter MdPAR7 is introduced into plant explants to obtain transformed plant cells; (2) The transformed plant cells regenerate into plants, resulting in transgenic plants; (3) Apply an inducer to the transgenic plant and induce it at an ambient temperature of 18-28℃ to induce the specific expression of the target functional gene in the root hair epidermal cells of the plant.

7. The application according to claim 6, characterized in that, The inducer includes one or more of estradiol, ethanol, and dexamethasone.

8. The application according to claim 7, characterized in that, The molar concentration of estradiol is 5.0-50.0 μmol / L, the volume concentration of ethanol is 1%-10% v / v, and the molar concentration of dexamethasone is 0.5-8.0 μmol / L.

9. The application according to claim 6, characterized in that, The target functional genes include the silenced expression fragments of positive and negative regulatory genes for plant root hair development.

10. The application according to claim 4, characterized in that, The plant explants include plant leaves, stem segments, callus tissue, somatic embryos, or root tip tissue.

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