741 poplar PtoWRKY24 gene promoter responding to multiple stress and application of 741 poplar PtoWRKY24 gene promoter
By introducing the PtoWRKY24 gene promoter into poplar, the problem of the lack of poplar stress-resistant gene promoters that respond to multiple stresses in the existing technology was solved, the stress resistance of poplar under multiple stresses was enhanced, and a theoretical basis for molecular breeding was provided.
Patent Information
- Application Number
- CN202510998860.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-21
- Publication Date
- 2025-10-28
AI Technical Summary
The existing technology lacks poplar stress resistance gene promoters that can specifically respond to multiple biotic/abiotic stresses, which limits the progress of molecular breeding for improving poplar stress resistance.
The PtoWRKY24 gene promoter and its recombinant vector are provided. They are introduced into plant cells by Agrobacterium transformation, and their stress response activity is analyzed in combination with the reporter gene GUS to verify their specific response function under multiple stresses.
The study verified that the PtoWRKY24 promoter has specific responsiveness to salt stress, drought, hormone stress, and pest stress, providing a theoretical basis for molecular breeding to improve the stress resistance of poplar trees and enhancing the stress response of plants.
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Figure CN120843518A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular breeding technology, and in particular to the 741 Yang PtoWRKY24 gene promoter in response to multiple stresses and its applications. Background Technology
[0002] WRKY transcription factors are plant-specific zinc finger transcription regulators that regulate plant signal transduction and physiological and biochemical processes, playing a crucial role in plant disease resistance and immunity. The important role of WRKY family transcription factors in plant physiological regulation not only affects the plant growth cycle and sugar metabolism but also plays a vital regulatory role in key life activities such as leaf senescence, secondary metabolite production, and stress responses. As core components of the plant signal transduction system, their functional mechanisms are of great value for understanding plant adaptability. WRKY has been identified in various traditional Chinese medicinal herbs, including *Artemisia annua*, *Catharanthus roseus*, *Salvia miltiorrhiza*, *Ophiorrhiza pumila*, *Taxus chinensis*, and *Rosabanksiae*, in regulating the synthesis of artemisinin, terpenoid indole alkaloids, tanshinone, camptothecin, paclitaxel, and sesquiterpenes. Many WRKY genes are expressed during various stages of plant life, including seed germination, root development, seedling growth, flowering, and fruit ripening, and they also regulate plant senescence. When plants are infected by pathogens, WRKY transcription factors can be activated, binding to the W-box of defense-related gene promoters to activate or inhibit gene expression, thereby enhancing plant disease resistance. Under drought and salt stress, the expression levels of some WRKY genes change, participating in plant stress responses. WRKY transcription factors themselves can form dimers or multimers, and different WRKY proteins can interact, affecting their DNA-binding activity and transcriptional regulatory functions.
[0003] Promoter classification is complex, currently mainly based on different transcription patterns into constitutive promoters, inducible promoters, and tissue-specific promoters. Constitutive promoters are not limited by time and space, nor are they affected by the external environment. Their expression intensity is usually stable, and they can efficiently and non-specifically promote the expression of exogenous genes in most plant tissues, with similar expression levels across different tissues. As the earliest type of promoter studied, it is widely used in the field of plant genetic engineering.
[0004] Poplar is an important economic tree species. Studying its stress resistance gene regulation mechanism can provide molecular targets for breeding new salt-tolerant and drought-resistant varieties. However, there is currently a lack of poplar stress resistance gene promoters that can specifically respond to multiple biotic / abiotic stresses. Summary of the Invention
[0005] The purpose of this invention is to provide a promoter for the 741 poplar PtoWRKY24 gene that responds to multiple stresses and its application, providing a precise regulatory element for molecular breeding of poplar stress resistance.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] This invention provides a PtoWRKY24 gene promoter, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0008] The present invention also provides a recombinant vector comprising the above-mentioned PtoWRKY24 gene promoter and an operatively ligable downstream reporter gene or target gene.
[0009] The present invention also provides a host cell comprising the above-described recombinant vector.
[0010] Preferably, the host cell is Agrobacterium or Escherichia coli.
[0011] The present invention also provides a transgenic plant cell or transgenic plant tissue, wherein the genome of the transgenic plant cell or transgenic plant tissue is integrated with the above-mentioned recombinant vector.
[0012] The present invention also provides a transgenic plant in which the above-mentioned recombinant vector is integrated into the genome of the transgenic plant.
[0013] This invention also provides the application of the above-mentioned PtoWRKY24 gene promoter, recombinant vector, or host cell in enhancing plant stress response.
[0014] Preferably, the stress is salt stress, drought stress, hormone stress, or pest stress.
[0015] Preferably, the plant is poplar or tobacco.
[0016] The present invention also provides a method for enhancing the stress response of plants, comprising the following steps:
[0017] The PtoWRKY24 gene promoter was introduced into plant cells using Agrobacterium-mediated transformation.
[0018] The beneficial effects of this invention are:
[0019] This invention analyzes the activity changes of the PtoWRKY24 promoter in the model plant tobacco, combined with the reporter gene GUS, under stress conditions such as high salt, drought, bollworm, and hormones, to verify whether it possesses a specific stress response function. This invention provides new clues to understanding the stress resistance function and molecular mechanism of the PtoWRKY24 gene, lays a theoretical foundation for improving the stress resistance and adaptability of poplar through molecular breeding, and is of great significance for promoting molecular breeding for plant stress resistance and ecological restoration. Attached Figure Description
[0020] Figure 1 The results of cloning and PCR identification of the PtoWRKY24 promoter are shown, including: (a) amplification of the target gene; (b) E. coli smear; (c) single colony PCR identification, M: BM 2000 DNA Maker (from top to bottom: 2000, 1000, 750, 500, 250, 100bp).
[0021] Figure 2 This is a diagram showing the distribution of cis-acting elements of the promoter.
[0022] Figure 3 Electrophoresis diagrams were constructed for the vector, including: (a) plasmid detection; (b) single colony PCR identification, SM: SuperDNAMaker (10000, 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100bp) M: BM2000 DNAMaker (2000, 1000, 750, 500, 250, 100bp);
[0023] Figure 4 The image shows the GUS staining results 12 hours after stress, where: CK1: blank control; CK2: transformation control;
[0024] Figure 5 The image shows the GUS staining results after 36 hours of stress, where: CK1: blank control; CK2: transformation control. Detailed Implementation
[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0026] The >PtoWRKY24 promoter sequence in this invention is shown in SEQ ID NO.1:
[0027]
[0028] Example
[0029] Cloning of the PtoWRKY24 promoter
[0030] Genomic DNA was extracted from leaves of Populus 741 using the CTAB method. Based on the Populus 741 genome information, the promoter sequence of the PtoWRKY24 gene was obtained. W24Pro-F / R primers were designed, and the target gene promoter sequence was amplified by PCR using the obtained DNA template and KOD high-fidelity enzyme. The reaction system was KOD One. TM PCR MasterMix 10 μL, DNA 2 μL, W24Pro-F 0.6 μL, W24Pro-R 0.6 μL, ddH2O to a final volume of 20 μL. The reaction program was: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 5 min. PCR products were detected by electrophoresis using a BM2000 marker at 220V for approximately 15 min. Fragment sizes were analyzed to ensure they matched expectations. If they did, photographs were taken, and the target bands were excised and recovered.
[0031] promoter cloning vector construction
[0032] Using the pTOPO-TA / Blunt Simple Universal Cloning Kit, the recombinant product was recovered from the gel and constructed into a T vector. The recombinant vector was transformed into *Escherichia coli* DH5α competent cells and plated on culture plates containing ampicillin. Single colonies were picked for culture PCR using KOD One. TM PCR MasterMix 10 μL, bacterial culture 2 μL, W24Pro-F 0.6 μL, W24Pro-R 0.6 μL, ddH2O to a final volume of 20 μL. The reaction program was: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 5 min. Positive clones were sent to the sequencing facility. An equal volume of sterile 70% glycerol was added to the correctly sequenced bacterial culture, and the mixture was stored at -80℃. Additionally, the PtoWRKY24 promoter sequence was submitted to the PlantCARE online website to predict the cis-acting elements of the promoter region (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).
[0033] Construction of promoter expression vector
[0034] Plasmids were extracted using the Plasmid Miniprep Kit (BW-PD1211) according to the manufacturer's instructions, followed by agarose gel electrophoresis to obtain a positive plasmid containing the PtoWRKY24 promoter in the T vector. Using this positive plasmid as a template, PCR amplification was performed using pBI121-W24pro-F / R primers containing the adapter, and the promoter fragment was recovered from the gel. The pBI121 expression vector plasmid was extracted and digested with HindIII and BamHI restriction enzymes, and the digestion products were recovered from the gel. Novizan was used... Using the Entry One Step Cloning Kit (C114), the promoter was ligated to the pBI121 vector via seamless cloning according to the instructions. The ligation product was transformed into competent DH5α *E. coli* cells, and resistant colonies were obtained through Kans PCR selection. Colony identification was performed using W24proJC-F / R primers. Simultaneously, positive clones of the recombinant strain were sent to a sequencing company for sequencing to further confirm the accuracy of the amplified target fragment and recombinant plasmid. The positive vector was named ProWRKY24:GUS expression vector and transformed into competent GV3101 *Agrobacterium tumefaciens* cells.
[0035] Instantaneous transformation of tobacco
[0036] Positive clones were added to LB liquid medium at a ratio of 1:100 and cultured at 28°C until OD reached. 600 The bacterial cell concentration was set to 0.6-0.8. After centrifugation, the cells were collected. The cells were then treated with a resuspension solution (10 mM MgCl2, 10 mM MES pH 5.7, and 0.2 mM acetylsylcholine), using the resuspension solution as a control, and adjusted to an OD value of 0.6-0.8. 600 The value is approximately 0.5-1.0. Wild-type tobacco plants that have grown for about 4 weeks are thoroughly watered and placed under an incandescent lamp for 1 hour before injection to open their stomata. Select healthy plants, and choose the second or third leaf from the top of each plant. Draw 1 mL of the suspension into a syringe without a needle and inject it into the back of the tobacco leaf. Mark the injection area, and incubate in the dark for 36 hours before placing it under normal light.
[0037] Promoter stress response and GUS staining
[0038] After the dark treatment, the plants were cultured under normal light conditions for 12 hours until the stomata were fully opened. They were then irrigated with 100 mL of 100 mM and 150 mM NaCl aqueous solutions and 100 mM PEG6000 aqueous solution, respectively; sprayed with 50 mL of 0.1 mM abscisic acid aqueous solution and 0.1 mM methyl jasmonate aqueous solution; and treated with 15 first-instar cotton bollworms to create 6 transiently transformed plants as experimental groups. Control groups were also set up: one was a blank control (wild-type tobacco without any treatment); the other was a transformation control (tobacco plants infected with Agrobacterium (containing the target promoter) but not subjected to stress treatment).
[0039] After 12 and 36 hours of treatment, 20 circular leaves (5 cm in diameter) were taken from each plant using a perforator and transferred to GUS staining solution. Staining was performed in the dark at 38°C for 12 hours, followed by destaining in 75% ethanol solution, with the ethanol solution changed periodically until the leaves were completely destaining. The leaves were then observed and photographed under a dissecting microscope. Based on the obtained images, the response characteristics of transiently transformed tobacco to different stresses were analyzed.
[0040] (1) Cloning of the PtoWRKY24 promoter
[0041] Using genomic DNA from Yang 741 as a template, the promoter fragment of the target gene was obtained by PCR amplification, and the results are as follows. Figure 1 As shown in Figure a, the size is as expected. The target band was recovered from the gel and constructed into a cloning vector. The recombinant vector was transformed into competent *E. coli* cells. The transformation product was evenly spread onto LB solid culture dishes containing ampicillin and incubated upside down in a 37°C incubator. The results are as follows. Figure 1 As shown in b. Single colonies were picked for PCR detection, and the results are as follows. Figure 1 As shown in c. The full-length sequence of the target gene, totaling 2039 bp, was obtained through sequencing.
[0042] (2) Bioinformatics Analysis
[0043] PlantCARE analysis revealed that the PtoWRKY24 promoter region contains multiple cis-acting elements (Table 1). These elements include several cis-acting elements or motifs related to plant stress resistance, such as the abscisic acid-responsive element ABRE, the methyl jasmonate-responsive elements TGACG-motif and CGTCA-motif, and the loss-responsive element WUN-motif. The distribution of these cis-acting elements in the promoter region is shown in the table below. Figure 2 As shown.
[0044] Table 1. Cis-Action Elements of PtoWRKY24 Promoter
[0045]
[0046]
[0047] (3) Construction of promoter expression vector
[0048] The positive clone plasmid containing the PtoWRKY24 promoter was extracted from (1), and the results are as follows: Figure 3 As shown in Figure a, the ProWRKY24:GUS expression vector was successfully constructed using the positive clone plasmid and transformed into Agrobacterium (GV3101) competent cells. Two single colonies were picked for PCR, and the results are shown in Figure a. Figure 3 As shown in b, the target fragment was amplified from the colony, and the positive clone strain was preserved.
[0049] (4) Initiating sub-stress response
[0050] Tobacco leaves from the control and treatment groups were collected 12 h and 36 h after stress treatment, respectively. GUS staining revealed that no blue area appeared in the blank control; only a slight blue tinge was observed in the transformation control; while the leaves of the transgenic tobacco lines treated with different stresses all showed varying degrees of blue areas, with the blue being deepest at the veins. Figure 4 and Figure 5 This indicates that the PtoWRKY24 promoter sequence has initiation activity and can drive the expression of the GUS gene.
[0051] After 12 hours of stress treatment, the PtoWRKY24 promoter responded to salt, drought, abscisic acid, methyl jasmonate, and bollworm larval feeding stress. The staining results were most pronounced in tobacco leaves treated with first-instar bollworms, followed by abscisic acid, drought, methyl jasmonate, and salt. The results are as follows: Figure 4 As shown.
[0052] After 36 hours of stress treatment, the PtoWRKY24 promoter responded to salt, drought, abscisic acid, methyl jasmonate, and bollworm larvae feeding stress. The staining results in tobacco leaves treated with abscisic acid were the most pronounced, followed by drought, methyl jasmonate, salt, and first-instar bollworms. The results are as follows: Figure 5 As shown. However, the overall color at 36 hours of stress was lighter than that at 12 hours of treatment.
[0053] The above conclusions indicate that the PtoWRKY24 promoter can respond to abscisic acid, methyl jasmonate, salt, drought, and bollworm feeding stress, further suggesting that the PtoWRKY24 gene may enhance plant stress resistance by responding to external biotic and abiotic stresses.
[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A PtoWRKY24 gene promoter, characterized in that, The nucleotide sequence of the PtoWRKY24 gene promoter is shown in SEQ ID NO.
1.
2. A recombinant vector, characterized in that, It includes the PtoWRKY24 gene promoter as described in claim 1 and an operable downstream reporter gene or target gene.
3. A host cell, characterized in that, It includes the recombinant vector as described in claim 2.
4. The host cell according to claim 3, characterized in that, The host cell is Agrobacterium or Escherichia coli.
5. A transgenic plant cell or transgenic plant tissue, characterized in that, The recombinant vector of claim 2 is integrated into the genome of the transgenic plant cell or transgenic plant tissue.
6. A transgenic plant, characterized in that, The recombinant vector of claim 2 is integrated into the genome of the transgenic plant.
7. The application of the PtoWRKY24 gene promoter of claim 1, the recombinant vector of claim 2, or the host cell of claim 3 or 4 in enhancing plant stress response.
8. The application according to claim 7, characterized in that, The stresses are salt stress, drought stress, hormone stress, or pest stress.
9. The application according to claim 7, characterized in that, The plant in question is either poplar or tobacco.
10. A method for enhancing plant stress response, characterized in that, Includes the following steps: The PtoWRKY24 gene promoter described in claim 1 was introduced into plant cells using Agrobacterium-mediated transformation.