A promoter specifically expressed in root cells under low phosphorus induction and its application

Abstract

This invention discloses a promoter specifically induced by low phosphorus in root cells and its application, relating to the field of genetic engineering technology. The key technical points are: the promoter has the nucleotide sequence shown in SEQ ID NO: 1. This invention also provides a vector containing the above promoter and its application in expressing the reporter gene GUS or the acid phosphatase gene OsPAP16 in rice. The beneficial effects of this invention are: the Pro-PRE provided by this invention is induced by low phosphorus and specifically expressed in root cells, driving the expression of exogenous genes, such as acid phosphatase or organic acid secretion proteins, under low phosphorus conditions, thereby improving the crop's tolerance to low phosphorus and providing important guidance for breeding low phosphorus-tolerant varieties.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a promoter that is specifically expressed in root cells under low phosphorus induction and its application. Background Technology

[0002] Phosphorus is one of the essential macronutrients for plant growth and development, participating in a series of physiological and biochemical processes within plants, such as photosynthesis and respiration. It is also a crucial structural component of many biological macromolecules, including DNA, RNA, and phospholipids. While soil contains relatively abundant phosphorus, the concentration of inorganic phosphorus available for plant absorption is generally only 1-10 μM, far below the requirements for normal plant growth and development. To address soil phosphorus deficiency, large amounts of phosphate fertilizer are applied in agricultural production to increase crop yields. Phosphorus, along with nitrogen and potassium, is collectively known as the "three essential fertilizer elements." However, compared to nitrogen and potassium fertilizers, phosphate fertilizer is more easily converted into fixed phosphates, which are difficult for crops to absorb, resulting in a utilization rate of only 15%–25% in the current season. Unutilized phosphate fertilizer not only increases agricultural production costs but also leads to eutrophication of nearby water bodies through soil erosion, causing serious environmental and ecological damage. Therefore, how to improve the ability of crops to activate and utilize soil phosphorus pools, and make soil phosphorus pools a substitute for phosphate rock resources, is a major scientific problem that urgently needs to be solved. It is of great significance to ecological protection and improving agricultural production efficiency, and is also an inevitable requirement for sustainable agricultural development.

[0003] Soil phosphorus pools contain only a small amount of water-soluble inorganic phosphorus available for direct plant absorption, but the content is often low and insufficient to meet the normal growth and development needs of plants; most phosphorus is fixed in the soil. Phosphorus in the soil that cannot be directly utilized by plants includes fixed inorganic phosphorus and organic phosphorus. To utilize these forms of phosphorus, plants have evolved a series of physiological and biochemical mechanisms to enhance their ability to absorb phosphorus from the soil. Phosphorus deficiency induces plant roots to secrete different types of acid phosphatases, which can degrade organic phosphorus in the soil into inorganic phosphorus for plant absorption. In addition, plant roots can also secrete hydrogen ions and organic acids, activating fixed inorganic phosphorus in the soil for root absorption. Overexpression of plant secretory acid phosphatases using constitutive promoters can effectively improve the plant's ability to utilize organic phosphorus in the soil. Similarly, overexpression of organic acid secretion proteins can enhance the organic acid secretion capacity of plant roots, promoting the root's ability to absorb fixed inorganic phosphorus from the soil.

[0004] However, using constitutive promoters to overexpress secretory acid phosphatases or organic acid secretory proteins leads to the target gene being overexpressed not only in the roots but also in the aboveground tissues, resulting in energy waste and inhibiting plant growth. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems and provide a promoter that is specifically expressed in root cells under low phosphorus induction and its application.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a promoter specifically expressed in root cells under low phosphorus induction, named Pro-PRE, whose nucleotide sequence is shown in SEQ ID NO: 1.

[0007] Furthermore, a recombinant expression vector is provided, wherein a reporter gene or a target gene is linked downstream of the promoter. A simplified diagram of the vector structure is shown below. Figure 2 As shown.

[0008] The application of a promoter that is specifically induced to be expressed in root cells by low phosphorus is described, in which the recombinant expression vector is transformed into rice callus, and then the transformed callus is regenerated into a complete transgenic plant through a genetic transformation system.

[0009] Furthermore, the reporter gene GUS or the gene encoding acid phosphatase OsPAP16 was expressed in rice using the Pro-PRE promoter. Under low phosphorus conditions, the Pro-PRE promoter induced specific expression of GUS in phosphorus-deficient rice root cells.

[0010] Compared with the prior art, the beneficial effects of this solution are as follows: The Pro-PRE provided by this invention is induced by low phosphorus and is specifically expressed in root cells. Under low phosphorus conditions, it drives the expression of exogenous genes, such as acid phosphatase or organic acid secretion proteins, thereby improving the crop's tolerance to low phosphorus. This has important guiding significance for the breeding of low phosphorus tolerant varieties. Attached Figure Description

[0011] Figure 1 This is an agarose gel electrophoresis image of Pro-PRE in an embodiment of the present invention;

[0012] Figure 2 These are maps of the expression of downstream genes GUS or OsPAP16 initiated by Pro-PRE in embodiments of the present invention, wherein (a) is a map of GUS gene expression and (b) is a map of OsPAP16 gene expression.

[0013] Figure 3 These are GUS staining images of roots and leaves of Pro-PRE::GUS-transferred rice cultured under normal and phosphorus-deficient conditions in an embodiment of the present invention. (a) shows GUS staining of leaves under normal phosphorus conditions, (b) shows GUS staining of leaves under phosphorus-deficient conditions, (c) shows GUS staining of roots under normal phosphorus conditions, and (d) shows GUS staining of roots under phosphorus-deficient conditions.

[0014] Figure 4This refers to the GUS activity of roots and leaves of Pro-PRE::GUS-transferred rice cultured under normal and phosphorus-deficient conditions in this embodiment of the invention.

[0015] Figure 5 The images show the phenotypic diagrams, dry weights, and root surface acid phosphatase activities of rice cultured under normal and phosphorus-deficient conditions in this embodiment of the invention. (a) shows the phenotypic diagrams of rice under normal and phosphorus-deficient conditions, (b) shows the aboveground dry weight, (c) shows the root dry weight, and (d) shows the root surface acid phosphatase activity. Detailed Implementation

[0016] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0018] Example

[0019] Construction of Pro-PRE::GUS or Pro-PRE::OsPAP16 vectors

[0020] Searching for PR through NCBI (National Center for Biotechnology Information) E The promoter sequence (SEQ ID NO: 1), with PR E The 2005 bp upstream of the 5' end of the start codon of the gene was designated as the promoter sequence, named Pro-PRE. Primers for amplifying the promoter fragment were designed based on the vector's restriction enzyme sites. Both the forward primer PF1 and the reverse primer PR1 had an 18 bp sequence overlapping with the vector at their 5' ends. The primer sequences were: forward primer PF1: 5'ctgcaggtcgacggatccAAATTTTCTCCAAACTTGCATG 3', reverse primer PR1: 5'acataagggactgaccacGGTGACTGTGAGCTAGCCGCAC 3'.

[0021] Using genomic DNA from the rice variety Nipponbare as a template, the Pro-PRE sequence was amplified using a standard PCR procedure. The PCR reaction mixture consisted of: 1 μL DNA, 1 μL forward primer PF1 (10 μM), 1 μL reverse primer PR1 (10 μM), 25 μL 2x Tag DNA polymerase, and 2 μL H2O. The PCR amplification program was as follows: 94℃ pre-denaturation for 2 minutes; 94℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 2 minutes, for 32 cycles; and a final extension at 72℃ for 2 minutes. After agarose gel electrophoresis, the fragment was excised and recovered; the amplified fragment size was 2005 bp.

[0022] The vector DX2181b was digested with the restriction endonuclease XmaⅠ, and the linearized vector was recovered. Using a seamless cloning kit provided by Wuhan Aibote Biotechnology Co., Ltd., the amplified promoter product was ligated into the digested DX2181b vector, and the vector was transformed into *E. coli* DH5α strain using the heat shock method. Positive clones were selected for PCR verification, and the verified positive clones were sequenced. The plasmid that was correctly aligned was transformed into *Agrobacterium* EHA105 for later use. The vector was named Pro-PRE::GUS (a simplified diagram of the vector structure is shown below). Figure 2 (as shown in a).

[0023] The Pro-PRE-OsPAP16 vector was constructed using the same seamless cloning kit. The primers for amplifying the promoter were:

[0024] Forward primer PF2:

[0025] 5'atgaccatgattacgccaAAATTTTCTCCAAACTTGCATG3',

[0026] Reverse primer PR2:

[0027] 5'gcgacgccagcaccgcatGGTGACTGTGAGCTAGCCGCAC3'.

[0028] The CDS primers for amplifying OsPAP16 are:

[0029] Forward primer PF3: 5'ggctagctcacagtcaccATGCGGTGCTGGCGTC3',

[0030] Reverse primer PR3: 5'cttctcctttgcccatatCTAGGAGCTCAAGC3'.

[0031] The CDS sequence of OsPAP16 was amplified by PCR using cDNA from the rice variety Nipponbare as a template. The PCR reaction mixture consisted of 1 μL cDNA, 1 μL forward primer PF3 (10 μM), 1 μL reverse primer PR3 (10 μM), 25 μL 2x Tag DNA polymerase, and 2 μL H2O. The PCR amplification program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 58℃ annealing for 20 s, 72℃ extension for 35 s, for 34 cycles; and a final extension at 72℃ for 5 min. After agarose gel electrophoresis, the fragment was excised and recovered, with a size of 1149 bp. The vector PBI121 was digested with restriction endonucleases HindIII and XbaI, and the linearized vector was recovered. Using the seamless cloning kit provided by Wuhan Aiboteke Biotechnology Co., Ltd., the amplified promoter product and the CDS product of OsPAP16 were ligated into the enzyme-digested PBI121 vector, and transformed into *E. coli* DH5α strain using the heat shock method. Positive clones were selected for PCR verification, and the verified positive clones were sequenced. The plasmid that was correctly aligned was transformed into *Agrobacterium* EHA105 for later use. The vector was named Pro-PRE::OsPAP16 (a simplified diagram of the vector structure is shown below). Figure 2 (as shown in b).

[0032] Obtain Pro-PRE::GUS or Pro-PRE::OsPAP16 transgenic rice materials

[0033] (1) Experimental material: wild-type (i.e. non-GMO) rice variety Nipponbare.

[0034] (2) Solution preparation

[0035] 1. MS stock solution (10x)

[0036] NH4NO3 16.5g

[0037] KH2PO4 1.7g

[0038] KNO3 19.0g

[0039] MgSO4·7H2O 3.7g

[0040] 3.32g CaCl2 or 4.4g CaCl2·2H2O

[0041] Bring the volume to 1L and store at 4℃ for extended periods.

[0042] 2. MS micro-stock solution (100x)

[0043] MnSO4·4H2O 2.23g

[0044] ZnSO4·7H2O 0.86g

[0045] KI 0.083g

[0046] H3BO3 0.62g

[0047] Na₂MoO₄·2H₂O 0.025g

[0048] CoCl2·6H2O 0.0025g

[0049] CuSO4·5H2O 0.0025g

[0050] Bring the volume to 1L and store at 4℃ for extended periods.

[0051] 3. N6 large stock solution (10x)

[0052] KNO3 28.3g

[0053] (NH4)2SO4 4.63g

[0054] KH2PO4 4.0g

[0055] MgSO4·7H2O 1.85g

[0056] 1.25g CaCl2 or 1.66g CaCl2·2H2O

[0057] Bring the volume to 1L and store at 4℃ for extended periods.

[0058] 4. B5 micro-stock solution (100x)

[0059] KI 0.08g

[0060] H3BO3 0.16g

[0061] ZnSO4·7H2O 0.15g

[0062] Add 0.44g of MnSO4·4H2O or 0.3335g of MnSO4·H2O to a final volume of 1L and store at 4℃ for long-term storage.

[0063] 5. Fe 2+ -EDTA (ethylenediaminetetraacetic acid) stock solution (100x)

[0064] Add 800mL of H2O to a beaker and bring to a boil. Then add 3.73g of Na2EDTA·2H2O. After cooling slightly, add 200mL of H2O to another beaker and dissolve 2.78g of FeSO4·7H2O. While stirring, slowly pour the solution into the Na2EDTA·2H2O mixture. Stir and keep warm at 70℃ for 2 hours. After cooling, bring the volume to 1L and store at 4℃ away from light.

[0065] 6. Organic reagent stock solution (100x)

[0066] Nicotinic acid 0.1g

[0067] Thiamine HCl (VB1) 0.1g

[0068] Pyridoxine HCl (VB6) 0.1g

[0069] Inositol 10g

[0070] Glycine 0.2g

[0071] Bring the volume to 1L and store at 4℃.

[0072] 7. KT (kinetin) stock solution (1 mg / mL)

[0073] Dissolve 100 mg of KT in 1 mL of 1 M KOH, shake well, add water to dissolve and bring the volume to 100 mL. Store at room temperature. (KT may precipitate during long-term storage; it is recommended to prepare only 10 mL at a time.)

[0074] 8. 2,4-D (2,4-dichlorophenoxyacetic acid) stock solution (1 mg / mL)

[0075] Add 100 mg of 2,4-D to 1 mL of 1 M KOH, shake well, dissolve in water and bring the volume to 100 mL, and store at room temperature.

[0076] 9. 200mMAS (acetylsyleugenone) stock solution

[0077] Dissolve 0.4g AS in 10mL DMSO (dimethyl sulfoxide), filter and sterilize, aliquot into sterile 1.5mL centrifuge tubes, and store at -20℃.

[0078] 10. NAA (Naphthaleneacetic Acid) stock solution (1 mg / mL)

[0079] Add 100 mg NAA to 1 mL of 1 M KOH, shake well, add water to dissolve and bring the volume to 100 mL, and store at room temperature.

[0080] (3) Culture medium formulation (prepare before use)

[0081] Table 1 Induction Culture Medium

[0082]

[0083] Table 2 Subculture Culture Media

[0084]

[0085] Table 3 Suspension Culture Medium

[0086]

[0087] Table 4 Co-culture medium

[0088]

[0089] Table 5. Selected Culture Media

[0090]

[0091] Add after sterilization:

[0092]

[0093]

[0094] Table 6 Differentiation Culture Media

[0095]

[0096] Table 7 Rooting Culture Medium

[0097]

[0098] (4) Genetically modified operation steps

[0099] All the following operations shall be performed in a laminar flow hood, with sterilization in the laminar flow hood lasting at least 30 minutes and air blowing for 20 minutes.

[0100] 1. Inducing callus formation

[0101] After the mature rice seeds are hulled, select plump, smooth, and sterile seeds and place them in centrifuge tubes. Disinfect with 75% ethanol for 2 minutes. Discard the ethanol and add 30% (v / v) NaClO solution (or 84 disinfectant: sterile water (v:v) = 1:1) for 30 minutes, inverting and mixing several times during this process. Discard the NaClO solution and rinse five times with sterile water, finally soaking in sterile water for 30 minutes, inverting and mixing several times during this process. Discard the sterile water and blot the seeds dry on sterile filter paper (place absorbent paper at the bottom of the petri dish and filter paper on top). Use tweezers to transfer the seeds into the induction medium, placing 10-12 seeds per bottle (embryo facing upwards). Incubate in the dark at 28°C for one month.

[0102] 2. Succession

[0103] From the induced callus, select pale yellow, granular, dry, and viable callus tissues and transfer them to subculture medium for dark culture for 20 days; (it is best to perform infection after one subculture, and subculture a maximum of two times, otherwise the callus transformation efficiency will be very low. When subculturing for the first time, be careful to remove other tissues such as endosperm and buds attached to the callus tissue).

[0104] 3. Infection and Co-cultivation

[0105] Two days before the experiment, the preserved Agrobacterium strain stock solution was streaked in YEP medium (with the corresponding antibiotic added, the resistance of the strain determined by the strain) at 28°C for activation. In a sterile 250mL Erlenmeyer flask, 100mL of suspension medium (with 50μl 200mMAS and 1mL 50% glucose) was poured in. The streaked Agrobacterium was scraped into a pea-sized portion of the suspension medium and incubated at 28°C and 200rpm for 30 minutes, until slightly turbid. Rice callus particles that had grown to a certain size were picked out and soaked in the Agrobacterium suspension for 30 minutes. The callus tissue was removed, placed on sterile filter paper to drain for 2 hours, and then evenly placed on a co-culture medium (with a layer of sterile filter paper on top to prevent overgrowth of Agrobacterium) and incubated in the dark at 19°C for 3 days.

[0106] 4. Screening for resistant callus

[0107] After 3 days of co-culturing, callus was collected in 250 mL blue-capped bottles and rinsed with sterile water until the water in the bottles was clear, indicating that the Agrobacterium was thoroughly cleaned. Finally, sterile water containing 1000 mg / L carbenicillin sodium was added and the callus was soaked for 30 minutes. The sterile water was then discarded, and the callus was spread on filter paper and air-dried for 3 hours. The dried callus was then transferred to a selection medium containing 400 mg / L carbenicillin sodium and 50 mg / L hygromycin or G418 for the first round of selection, incubated in the dark at 28°C for 14 days. The initially grown callus was then transferred to a selection medium containing 300 mg / L carbenicillin sodium and 80 mg / L hygromycin or G418 for the second round of selection, incubated in the dark at 28°C until granular resistant callus tissue grew. If no resistant callus grew after four weeks, the callus was transferred to a selection medium with the same composition for a third round of selection.

[0108] 5. Differentiation and seedling formation of resistant callus

[0109] Select 2-3 resistant calluses from the same callus and place them on differentiation medium. Incubate at 26°C under light [14h / 10h (day / light) photocycle, light intensity 2000lx]. After 30-50 days of differentiation culture, the callus tissue will differentiate into seedlings. When the green shoots grow to about 3-5cm, remove the young roots with scissors and transfer them to rooting medium. Incubate at 26°C under light.

[0110] 6. Transplantation and molecular identification of transgenic seedlings

[0111] After 10-15 days of rooting culture, select seedlings with well-differentiated roots and stems, remove the sealing film, add an appropriate amount of distilled or sterile water, and harden them off in a culture room for 5-7 days. Then, wash off the agar and transfer them to rice nutrient solution for 2 weeks of cultivation. Use screening marker genes to select resistant transgenic materials. Finally, transplant the obtained transgenic positive seedlings into the field or pots for seed harvesting.

[0112] Pro-PRE initiates GUS gene expression.

[0113] Pro-PRE::GUS transgenic seeds were soaked in 1% nitric acid for 14 hours, rinsed several times with distilled water, and then germinated at 37℃. After the seeds showed signs of germination, they were cultured in rice nutrient solution (formulation as shown in Table 8). The greenhouse light cycle was 12 hours of light / 12 hours of darkness, with a light intensity of 3000 lux. The daytime culture temperature was 30℃, and the nighttime culture temperature was 22℃. Seedlings cultured normally for 10 days were treated with normal phosphorus and phosphorus-deficient (without NaH2PO4·2H2O in the nutrient solution) treatments for 10 days. Some plants were used for GUS staining, and others were used to determine GUS activity.

[0114] Root tips and leaves were taken separately, placed in GUS staining solution, and incubated at 37°C for 12 hours. After removing chlorophyll with 70% ethanol, they were embedded in 2.7% agar, transversely sectioned using a vibratory microtome, and observed and photographed under a microscope. Results are as follows: Figure 3 As shown, under normal phosphorus conditions, some roots showed a light blue color, while under phosphorus-deficient conditions, the roots were stained a distinct blue, and the leaves under both normal and phosphorus-deficient treatments did not show any color.

[0115] Total protein was extracted from leaves and roots, and the GUS activity of the plants was quantitatively determined based on the principle that GUS reacts with the substrate MUG (4-methylumbelliferone-β-glucuronide) to produce the fluorescent substance MU (4-methylumbelliferone). Results are as follows: Figure 4 As shown, leaf GUS activity did not differ significantly under different phosphorus treatments, but root GUS activity under phosphorus deficiency was 5 times that under normal phosphorus conditions. The results of GUS staining and quantitative determination of activity fully demonstrate that this promoter is induced by phosphorus deficiency and is specifically expressed in roots.

[0116] Materials that promote OsPAP16 expression via Pro-PRE enhance rice growth.

[0117] Seeds of the Pro-PRE::OsPAP16 transgenic material were soaked in 1% nitric acid for 14 hours, rinsed several times with distilled water, and then germinated at 37℃. Once the seeds showed signs of germination, they were cultured in rice nutrient solution (formulation as shown in Table 8). The greenhouse light cycle was 12 hours of light / 12 hours of darkness, with a light intensity of 3000 lux. The daytime culture temperature was 30℃, and the nighttime culture temperature was 22℃. Seedlings cultured normally for 10 days were then treated with normal phosphorus and phosphorus-deficient (without NaH2PO4·2H2O in the nutrient solution) treatments for 10 days. Afterward, photographs were taken, and the aboveground parts and roots were placed separately in paper bags, dried at 70℃, and their dry weight was recorded. Results are as follows: Figure 4 As shown in ac, the aboveground and root dry weights of transgenic material (OX) were higher than those of wild type (WT) under both normal phosphorus and phosphorus-deficient conditions.

[0118] Rice plants treated with normal phosphorus and phosphorus deficiency were used to determine the root surface acid phosphatase activity. The roots were quickly rinsed with distilled water, and then the seedlings were transferred to 30 mL of rice culture medium (pH 5.5) containing 10 mM pNPP (disodium 4-nitrophenyl phosphate). A blank sample was prepared without plant samples. The reaction was carried out at 30℃ for 30 min. 370 μL of the reaction solution was then added to 1.66 mL of 1 M NaOH to terminate the reaction, and the OD410 absorbance was measured. After the measurement, the root weight was recorded. The root surface acid phosphatase activity was calculated based on the pNP standard curve. The results are as follows: Figure 4 As shown in d, the root surface acid phosphatase activity of transgenic material (OX) was higher than that of wild type (WT) under both normal phosphorus and phosphorus-deficient conditions.

[0119] Table 8 Rice Nutrient Solution Formula

[0120]

[0121] Note: Adjust the pH of the nutrient solution to 5.0-5.5 with 1M hydrochloric acid.

[0122] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A promoter specifically expressed in root cells under low phosphorus induction, characterized in that, The promoter shown was named Pro-PRE. Using the genomic DNA of the rice variety Nipponbare as a template, the Pro-PRE sequence was amplified using a standard PCR procedure, resulting in a promoter fragment size of 2005 bp. Primers for amplifying the promoter fragment were designed based on the vector restriction sites. Both the forward primer PF1 and the reverse primer PR1 have an 18 bp sequence overlapping with the vector at their 5' ends. The primer sequences for amplifying the promoter are as follows: Forward primer PF1: 5'ctgcaggtcgacggatccAAATTTTCTCCAAACTTGCATG 3', Reverse primer PR1: 5'acataagggactgaccacGGTGACTGTGAGCTAGCCGCAC 3'.

2. A recombinant expression vector comprising the promoter of claim 1, which is specifically expressed in root cells under low phosphorus induction, characterized in that, The recombinant expression vector expresses the reporter gene or target gene connected downstream of the promoter.

3. The application of the promoter specifically expressed in root cells under low phosphorus induction as described in claim 1, characterized in that, The recombinant expression vector containing the promoter Pro-PRE as described in claim 1 is transformed into rice callus, and the transformed callus is then regenerated into a complete transgenic plant through a genetic transformation system, wherein the plant is rice.

4. The application as described in claim 3, characterized in that, The reporter gene GUS or the acid phosphatase gene OsPAP16 is expressed in rice using the promoter Pro-PRE described in claim 1. Under low phosphorus conditions, the promoter Pro-PRE described in claim 1 induces specific expression of GUS in phosphorus-deficient rice root cells.

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