Application of alfalfa MsWRKY49 gene in regulating and controlling elongation of pollen tube under low boron stress
By overexpressing the MsWRKY49 gene in alfalfa and regulating the ROS level in the pollen tube, the problem of pollen tube elongation under low boron stress was solved, crop improvement through genetic engineering was achieved, the use of boron fertilizer and the risk of pollution were reduced, a molecular regulatory mechanism was provided, and a new strategy was offered for crop breeding.
Patent Information
- Application Number
- CN202510954042.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-09-26
AI Technical Summary
In the existing technology, the application effect of boron fertilizer is unstable and may cause environmental pollution. There is a lack of effective gene regulation methods to solve the problem of pollen tube elongation under low boron stress.
By overexpressing the MsWRKY49 gene in alfalfa, genetic engineering technology was used to regulate the level of reactive oxygen species (ROS) in pollen tubes and enhance the elongation ability of pollen tubes under low boron stress.
It reduces the use of boron fertilizers, lowers the risk of agricultural pollution, provides gene regulation technology to replace traditional boron fertilizer application, achieves precise improvement of crop traits, reveals the ROS-mediated molecular regulation mechanism, and provides a theoretical basis and gene manipulation targets for plant breeding for boron stress tolerance.
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Figure CN120700031A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to an application of the MsWRKY49 gene in regulating pollen tube elongation under low boron stress. Background Art
[0002] Boron is one of the key trace elements necessary for plant growth and development, and plays a vital role in plant cell wall synthesis, cell membrane stability, and various metabolic processes. In particular, the role of boron is particularly significant in pollen development. Boron interacts with rhamnogalacturonan-II (RG-II) in the cell wall to form borate esters, thereby enhancing the porosity and elasticity of the cell wall. This interaction is particularly important for rapidly growing areas such as root tips, buds, and flowers, which are extremely susceptible to boron deficiency. The invention shows that boron deficiency can lead to a significant decrease in seed yield in a variety of crops such as rice, corn, wheat, and alfalfa, seriously affecting the benefits of agricultural production.
[0003] The traditional solution to the problem of boron deficiency is to supplement the boron element in the soil through the application of boron fertilizer. However, this solution has many shortcomings. First, the effect of boron fertilizer is often unstable and is affected by various factors such as soil type, climatic conditions, and fertilization technology, making it difficult to accurately control the supply of boron. Second, excessive application of boron fertilizer may also cause environmental pollution and pose a potential threat to the ecosystem. In addition, although boron fertilizer can promote the reproductive development of crops and increase seed yield to a certain extent, its physiological mechanism of action on flower and seed development is not fully understood, and it cannot fundamentally solve the problem of boron deficiency.
[0004] While scientists have recognized the crucial effects of boron on pollen germination and pollen tube elongation, the specific molecular mechanisms of boron deficiency remain elusive. In particular, little is known about the role of WRKY transcription factors in mediating boron-regulated pollen tube elongation. As an important class of regulatory factors, WRKY transcription factors play a key role in plant stress responses and growth and development, but their specific function in regulating pollen tube elongation under low boron stress has not been fully explored.
[0005] With the continuous development of genetic engineering technology, its application in crop improvement is becoming increasingly widespread. Through genetic engineering, crop genetic characteristics can be precisely manipulated, achieving targeted improvements in crop traits. However, relatively few genetic discoveries have been made regarding pollen tube elongation under low-boron stress. While some discoveries have focused on genes related to pollen development and elongation, the specific functions and regulatory mechanisms of these genes under low-boron stress remain unclear.
[0006] The shortcomings of the existing technology mainly lie in the limitations of boron fertilizer application and the lack of effective gene regulation methods. The application of boron fertilizer is unstable and may cause environmental pollution, while existing genetic inventions have failed to fully reveal the molecular regulatory mechanism of pollen tube elongation under low-boron stress. Therefore, the development of an effective gene regulation method to improve plant pollen viability under low-boron conditions by regulating the expression of specific genes has become an urgent need for current inventions. To address the above problems, the applicant proposes the use of the alfalfa MsWRKY49 gene to regulate pollen tube elongation under low-boron stress. Summary of the Invention
[0007] The purpose of the present invention is to provide an application of an alfalfa MsWRKY49 gene in regulating pollen tube elongation under low boron stress, so as to solve the problems in the prior art.
[0008] To achieve the above object, the present invention provides the following technical solution: an application of the MsWRKY49 gene in alfalfa to regulate pollen tube elongation under low boron stress, comprising the following steps:
[0009] The MsWRKY49 gene was overexpressed in alfalfa through genetic transformation to create a MsWRKY49 overexpression strain.
[0010] The MsWRKY49 overexpressing strain and wild-type alfalfa were cultured under low-boron conditions, and their pollen tube lengths were measured and compared;
[0011] It was verified that the MsWRKY49 gene enhances the elongation ability of pollen tubes under low boron stress by regulating the level of reactive oxygen species (ROS) in pollen tubes.
[0012] Optionally, the genetic transformation method is: inserting the full-length coding sequence of the MsWRKY49 gene into a plant expression vector by homologous recombination technology, and introducing the vector into the alfalfa genome by Agrobacterium-mediated method to obtain a stably inherited MsWRKY49 overexpression strain.
[0013] Optionally, the low-boron condition is: the boric acid concentration in the pollen culture medium is 0 mM to 0.03235 mM.
[0014] Optionally, the pollen tube length of the MsWRKY49 overexpressing strain is reduced by more than 30% compared with wild-type alfalfa under normal boron conditions.
[0015] Alternatively, MsWRKY49 regulates pollen tube elongation through the following mechanisms:
[0016] Under low boron stress, the ROS level in the pollen tubes of the MsWRKY49 overexpressing strain increased significantly, inhibiting the excessive elongation of the pollen tubes.
[0017] In the mswrky49 gene knockout mutant, the ROS level in pollen tubes was significantly reduced and the pollen tube elongation rate was accelerated.
[0018] Optionally, the MsWRKY49 gene is specifically expressed during the pollen development stage of alfalfa, and its expression level is upregulated by at least 2 times under low boron conditions.
[0019] A method for improving low boron tolerance of plant reproductive organs comprises the following steps:
[0020] Overexpression of the MsWRKY49 gene in recipient plants or editing of its endogenous gene to regulate expression levels was achieved through genetic transformation.
[0021] Transgenic plants were cultured under low-boron conditions and their pollen tube growth indices were measured;
[0022] Screen out transgenic lines with enhanced pollen tube elongation ability.
[0023] Optionally, the target plant is alfalfa or boron-sensitive economic crops, including but not limited to soybean, rapeseed, and cotton.
[0024] Beneficial effects:
[0025] Reduce the use of boron fertilizers and reduce the risk of agricultural pollution
[0026] Gene regulation technology replaces the traditional application of boron fertilizers, which not only reduces dependence on chemical fertilizers, but also avoids soil / water pollution caused by excessive boron, taking into account both economic benefits and ecological protection.
[0027] Elucidating the molecular regulatory mechanisms mediated by ROS
[0028] For the first time, the mechanism by which the MsWRKY49 gene balances elongation and stress resistance by regulating the ROS level in pollen tubes was revealed, providing a key theoretical basis and genetic manipulation target for plant breeding for boron stress tolerance.
[0029] Genetic engineering enables precise improvement of crop traits
[0030] By using genetic engineering technologies such as Agrobacterium transformation, new low-boron-tolerant crop varieties can be efficiently created. This strategy is universal and provides a replicable technical path for the genetic improvement of crops such as soybeans and wheat. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is a schematic diagram showing the effect of low boron on alfalfa pollen germination and pollen tube elongation according to an embodiment of the present invention;
[0032] Figure 2 This is a schematic diagram of an embodiment of the present invention in which low boron inhibits pollen tube elongation by increasing the ROS level in alfalfa;
[0033] Figure 3 Schematic diagram of the phylogeny and gene expression profile analysis of the alfalfa pollen-specific MsWRKY gene family in the present invention;
[0034] Figure 4 Schematic diagram of expression profile analysis of nine MsWRKY genes under different boron concentrations according to an embodiment of the present invention;
[0035] Figure 5 This is a schematic diagram of an embodiment of the present invention showing that the mswrky49 mutant promotes pollen tube elongation by reducing ROS accumulation under low boron conditions;
[0036] Figure 6 This is a schematic diagram showing that overexpression of MsWRKY49 in alfalfa shortens pollen tube length by increasing ROS accumulation in an embodiment of the present invention;
[0037] Figure 7 Schematic diagram showing that MsWRKY49 has transcription factor autoactivation activity according to an embodiment of the present invention. DETAILED DESCRIPTION
[0038] The following describes preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.
[0039] This study uses genetic engineering techniques to analyze and utilize the MsWRKY49 gene to regulate pollen tube elongation under low-boron stress. Focusing on Xinjiang alfalfa, the study systematically constructed a gene function verification system to address the issues of pollen tube elongation inhibition and seed yield reduction caused by boron deficiency. The system revealed the molecular mechanism by which MsWRKY49 regulates pollen tube elongation through the reactive oxygen species (ROS) signaling pathway and proposed a crop improvement strategy based on this gene.
[0040] During the experimental material preparation phase, the inventive team selected Xinjiang large-leaf alfalfa seeds and pre-germinated them under strictly controlled greenhouse conditions. After germination for nine days at 24°C, 16 hours of light / 8 hours of darkness, the seeds were transplanted into a mixture of vermiculite and nutrient soil, maintaining a growth temperature of 22-24°C and 60% humidity. After a three-month growth cycle, root, stem, leaf, and flower organ samples were collected, quickly frozen with liquid nitrogen, and stored in a -80°C ultra-low temperature freezer, providing high-quality material for subsequent genetic manipulation.
[0041] The core components of the gene function invention include vector construction and genetic transformation. The invention uses homologous recombination technology to insert the full-length coding sequence of the MsWRKY49 gene into the pCAMBIA1300 vector, constructing an expression vector driven by the Lat52 promoter. Simultaneously, the CRISPR / Cas9 system is used to design specific target sites and construct a gene-editing vector. Genetic transformation is carried out using the Agrobacterium EHA105-mediated method. After hygromycin resistance screening and PCR genotyping, homozygous transgenic lines are obtained. qRT-PCR analysis confirmed that the expression level of MsWRKY49 in the overexpression line is significantly upregulated, while the expression level of the gene in the mutant plant is significantly reduced.
[0042] The pollen tube elongation phenotype was analyzed using an in vitro culture system. Fresh open flowers were collected and pollen germination experiments were carried out in liquid culture media containing different boron concentrations (0mM, 0.03235mM, and 1.62mM). The pollen germination rate, pollen tube length, and rupture rate were accurately measured using a Nikon microscope imaging system combined with ImageJ software. The results showed that under low boron conditions (0.03235mM), the average length of wild-type pollen tubes was 60% shorter than that under normal boron concentrations (1.62mM), and 10% of the pollen tubes showed tip rupture. The pollen tube length of the overexpression strain was further shortened to 60% of that of the wild type under normal boron conditions, while the mutant recovered to 128% of the length under normal boron conditions.
[0043] To understand the regulatory mechanism of MsWRKY49, the authors conducted in-depth testing of ROS levels in pollen tubes. Using two fluorescent probes, CM-H2DCFDA and PO1, for staining, confocal microscopy revealed that the ROS fluorescence intensity of wild-type pollen tubes under low-boron conditions was 2.3 times higher than that under normal boron conditions. The ROS level of the overexpression strain further increased to 2 times under normal boron conditions, while the ROS accumulation of the mutant was significantly reduced to 0.6 times under low-boron conditions. Exogenous application of H2O2 can simulate the inhibitory effect of pollen tube elongation caused by low boron, while the ROS scavenger ascorbic acid (AsA) can restore the phenotype of pollen tubes under low-boron conditions.
[0044] Gene expression analysis revealed that MsWRKY49 expression was upregulated fourfold in pollen tubes treated with low boron compared to normal boron conditions. Subcellular localization experiments confirmed that the protein was localized to the nucleus, and yeast two-hybrid analysis demonstrated its transcriptional activation activity.
[0045] This study innovatively established a regulatory model for MsWRKY49, ROS, and pollen tube elongation: Under low boron stress, MsWRKY49 gene expression is upregulated, and its encoded protein promotes ROS production in pollen tubes through a transcriptional regulatory network. Excessive ROS causes oxidative damage to cells, inhibiting pollen tube elongation and increasing the risk of rupture. This discovery not only clarifies the molecular mechanism by which boron affects plant reproductive development but also opens up a new strategy for regulating pollen tube elongation through gene editing.
[0046] Example 1: Construction of alfalfa plants overexpressing the MsWRKY49 gene and their performance under low boron conditions
[0047] Purpose of the experiment:
[0048] This example aims to construct alfalfa plants overexpressing the MsWRKY49 gene through genetic transformation methods and verify the changes in their pollen tube elongation ability under low boron conditions to clarify the function of this gene in the response to low boron stress.
[0049] Materials and methods
[0050] Vector construction:
[0051] The full-length coding sequence (CDS) of MsWRKY49 gene was amplified using Xinjiang Medicago truncatula cDNA as template and inserted into pCAMBIA1300 vector by homologous recombination to construct the Lat52 promoter-driven overexpression vector (pCAMBIA1300-MsWRKY49).
[0052] Genetic transformation:
[0053] The recombinant vector was introduced into Agrobacterium EHA105 competent cells, and transgenic alfalfa plants were obtained through genetic transformation.
[0054] Plant screening and identification:
[0055] Genomic DNA was extracted from the leaves of transgenic plants, and the integration of the MsWRKY49 gene was verified by PCR amplification.
[0056] qRT-PCR was used to detect the relative expression level of MsWRKY49 in the overexpression lines, and high-expression lines (OE-1 and OE-4) were selected for subsequent experiments.
[0057] Phenotypic analysis:
[0058] Pollen germination experiment: Flowers of transgenic plants and wild type (WT) at full flowering stage were collected and cultured in vitro in pollen germination medium containing different boron concentrations (B0: 0 mM, B1: 0.03235 mM, B2: 1.62 mM) for 2 hours.
[0059] Index determination: The pollen tube morphology was observed using a Nikon microscope, and the pollen tube length was measured using ImageJ software. The germination rate (pollen tube length ≥ pollen grain diameter was considered germination) and the rupture rate (pollen tube tip swelling or breakage) were calculated.
[0060] result:
[0061] Pollen tube phenotype: Under normal boron conditions (B2), the average pollen tube lengths of OE-1 and OE-4 strains were 229.6 μm and 218 μm, respectively, which were significantly shorter than WT (328.6 μm).
[0062] in conclusion:
[0063] Overexpression of MsWRKY49 significantly inhibited pollen tube elongation in alfalfa under low-boron conditions. This result confirmed that MsWRKY49 plays a negative regulatory role in pollen tube development under low-boron stress.
[0064] Example 2: Construction of the mswrky49 mutant and its performance under low-boron conditions
[0065] Purpose of the experiment:
[0066] In this example, the CRISPR / Cas9 technology was used to construct a MsWRKY49 gene mutant, analyze its phenotypic changes under low-boron conditions, and verify its functional complementation with overexpression plants to further analyze the biological function of this gene.
[0067] Materials and methods
[0068] Vector construction:
[0069] The CRISPOR online tool was used to design the MsWRKY49 gene-specific sgRNA target (5'-GTTCGAGATCGACGACTCGG-3') and cloned into the p6401 vector to construct the gene editing vector (p6401-mswrky49).
[0070] Genetic transformation and mutant screening:
[0071] The vector was transferred into Agrobacterium EHA105, and transgenic plants were obtained by infecting alfalfa through Agrobacterium-mediated genetic transformation.
[0072] The target region of the resistant plants was amplified by PCR and sequenced, and homozygous mutants (mswrky49-L4 and mswrky49-L6) were obtained by screening.
[0073] Phenotypic analysis:
[0074] Pollen germination experiment: The method was the same as in Example 1, and the pollen tube phenotypes of the mutant and WT were compared under low boron conditions.
[0075] ROS level detection: The CM-H2DCFDA staining method was used to detect the accumulation level of reactive oxygen species (ROS) in pollen tubes. The specific steps refer to the technical plan.
[0076] result:
[0077] Pollen tube phenotype: Under low boron conditions (B1), the average pollen tube lengths of mswrky49-L4 and mswrky49-L6 lines were 230 μm and 243.5 μm, respectively, which were significantly longer than WT (197.5 μm).
[0078] ROS levels: Under low boron conditions, the ROS fluorescence intensity of the mutant pollen tubes was 42.6% lower than that of the WT, indicating that the loss of MsWRKY49 led to reduced ROS accumulation.
[0079] in conclusion:
[0080] The mswrky49 mutant exhibited enhanced pollen tube elongation and reduced ROS levels under low-boron conditions. This result fully complemented the phenotype of the overexpressing plant, confirming that MsWRKY49 inhibits pollen tube elongation under low-boron stress by regulating the ROS signaling pathway. This example provides direct evidence for improving crop tolerance to boron stress through gene editing.
[0081] The implementation of the technical solution provides multiple benefits for agricultural applications: the cultivation of MsWRKY49-deficient crops through molecular breeding; the invention of the ROS regulatory mechanism provides a theoretical basis for the development of new plant growth regulators; and the cross-species conservation of this gene gives it the potential for application in improving a variety of crops. The inventive team predicts that planting mswrky49 mutant alfalfa in the field will show significant ecological and economic benefits. The above shows and describes the basic principles, main features, and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from all perspectives, the embodiments should be regarded as illustrative and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims be included in the present invention. Any reference signs in the claims should not be construed as limiting the claims to which they relate.
[0082] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.
Claims
1. An application of the MsWRKY49 gene in regulating pollen tube elongation under low boron stress in alfalfa, characterized in that: The following steps are involved: The MsWRKY49 gene was overexpressed in alfalfa through genetic transformation to create a MsWRKY49 overexpression strain. The MsWRKY49 overexpressing strain and wild-type alfalfa were cultured under low-boron conditions, and their pollen tube lengths were measured and compared; It was verified that the MsWRKY49 gene enhances the elongation ability of pollen tubes under low boron stress by regulating the level of reactive oxygen species (ROS) in pollen tubes.
2. The use according to claim 1, characterized in that The genetic transformation method comprises the following steps: inserting the full-length coding sequence of the MsWRKY49 gene into a plant expression vector by homologous recombination technology, and introducing the vector into the alfalfa genome by Agrobacterium-mediated method to obtain a stably inherited MsWRKY49 overexpression strain.
3. The use according to claim 1, characterized in that The low-boron condition is: the boric acid concentration in the pollen culture medium is 0 mM to 0.03235 mM.
4. The use according to any one of claims 1 to 3, characterized in that The pollen tube length of the MsWRKY49 overexpressing strain is increased by more than 30% compared with that of wild-type alfalfa under low-boron conditions.
5. The use according to any one of claims 1 to 4, characterized in that MsWRKY49 regulates pollen tube elongation through the following mechanisms: Under low boron stress, the ROS level in the pollen tubes of the MsWRKY49 overexpressing strain increased significantly, inhibiting the excessive elongation of the pollen tubes. In the mswrky49 gene knockout mutant, the ROS level in pollen tubes was significantly reduced and the pollen tube elongation rate was accelerated.
6. The use according to any one of claims 1 to 5, characterized in that The MsWRKY49 gene is specifically expressed during the pollen development stage of alfalfa, and its expression level is upregulated by at least 2 times under low boron conditions.
7. A method for improving low boron tolerance of plant reproductive organs, characterized in that: The following steps are involved: Overexpression of the MsWRKY49 gene in recipient plants or editing of its endogenous gene to regulate expression levels was achieved through genetic transformation. Transgenic plants were cultured under low-boron conditions and their pollen tube growth indices were measured; Screen out transgenic lines with enhanced pollen tube elongation ability.
8. The method according to claim 7, characterized in that The target plant is alfalfa or boron-sensitive economic crops, including but not limited to soybean, rapeseed, and cotton.