Application of MAPKKK20 gene in improving seed germination of plants under ABA stress, plant breeding method

Abstract

The application belongs to the field of plant genetic engineering, and particularly relates to application of a MAPKKK20 gene in improving seed germination of plants under ABA stress and a plant breeding method. The application specifically improves the seed germination rate of plants under ABA stress through overexpression of the MAPKKK20 gene; the gene sequence number of the MAPKKK20 gene in NCBI is NM_114891.2. The application first verifies and discloses the function of the MAPKKK20 gene in improving the seed germination rate, and discloses the action mechanism, and finds that overexpression of the MAPKKK20 gene can improve the tolerance of the seed germination stage of plants to ABA stress, and improve the seed germination rate of plants under ABA stress. Based on this, the application can provide a gene resource for crop ABA tolerance molecular breeding, and can provide a novel approach for plant ABA tolerance breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the application of the MAPKKK20 gene in improving seed germination in plants under ABA stress and plant breeding methods. Background Technology

[0002] Seed germination is the process by which mature, viable seeds, under suitable conditions, restart biosynthesis and metabolic activities, and the radicle breaks through the seed coat. It is the first step in plant growth and development and is crucial for the reproduction of seed plants. Many species' seeds can remain dormant until conditions meet the requirements for germination. This is an adaptive strategy that serves as a buffer against negative environmental impacts, and this characteristic is essential for protecting species continuity and maintaining biodiversity.

[0003] Seed germination is influenced by a variety of factors, including the seed's own state and the external environment. Studies have shown that the plant hormone abscisic acid (ABA) plays a crucial role in regulating seed germination; it can induce seed dormancy and inhibit germination. For example, in the ABA synthesis triple mutants nced5, nced6, and nced9, seed dormancy is reduced and germination occurs earlier. In the ABA metabolism mutants cyp707a1, cyp707a2, and cyp707a3, ABA content is increased, dormancy is enhanced, and germination is inhibited.

[0004] In Arabidopsis thaliana, the mitogen-activated protein kinase (MAPK) family amplifies and transmits cellularly sensed external signals to downstream targets via a three-tiered cascade pathway, responding to external stressors or growth and development signals. The MAPK family has been reported to include approximately 20 MAPKs, 10 MKKs, and 80 MAPKKKs. Among them, mitogen-activated protein kinase kinase 20 (MAPKKK20) is a member of the Arabidopsis thaliana mitogen-activated protein kinase family. However, current research on the function and mechanism of action of the MAPKKK20 gene is limited, and there are no reports on the association between the MAPKKK20 gene and ABA regulation of seed germination.

[0005] Therefore, given the important role of seed germination in the reproduction of seed plants, exploring new ways to effectively weaken ABA inhibition in seed germination is of great significance for improving the seed germination rate of plants tolerating various stresses. Summary of the Invention

[0006] One of the objectives of this invention is to provide an application of the MAPKKK20 gene in improving seed germination under ABA stress. By overexpressing the MAPKKK20 gene, the tolerance of plant seeds to ABA stress during the germination stage can be improved, thereby increasing the germination rate of plant seeds under ABA stress.

[0007] The second objective of this invention is to provide an application of a recombinant expression vector in improving seed germination under ABA stress. This vector carries the MAPKKK20 gene, which can achieve MAPKKK20 gene overexpression, thereby improving the tolerance of plant seeds to ABA stress during the germination stage and increasing the germination rate of plant seeds under ABA stress.

[0008] The third objective of this invention is to provide a plant breeding method that can obtain plant varieties with improved seed germination rates under ABA stress.

[0009] One of the objectives of this invention is achieved through the following technical solution:

[0010] Application of MAPKKK20 gene in improving seed germination under ABA stress: Overexpression of MAPKKK20 gene improves seed germination rate under ABA stress; the gene sequence number of MAPKKK20 gene in NCBI is NM_114891.2.

[0011] The MAPKKK20 gene used in this invention has the sequence number NM_114891.2 (downloadable from https: / / www.ncbi.nlm.nih.gov / nuccore / NM_114891.2?report=genbank). Its messenger RNA (mRNA) sequence is 1447 bp long, and its gene coding sequence is 1029 bp long, including 342 amino acids.

[0012] This invention innovatively clones mitogen-activated protein kinase kinase 20 (MAPKKK20) from Arabidopsis thaliana, and constructs a MAPKKK20 gene overexpression vector and a MAPKKK20 gene lacking kinase activity. K36M Overexpression vectors were used to transform wild-type Arabidopsis thaliana (Col-0, WT) using the Agrobacterium inflorescence infection method to obtain overexpressing plants. The results showed that, under exogenous abscisic acid (ABA) treatment, the seed germination rate of MAPKKK20 overexpressing plants was significantly higher than that of the wild-type line (WT group), while overexpressing MAPKKK20 lacked kinase activity. K36MThe seed germination rate of the plants was not significantly different from that of the WT group. Further analysis revealed that the gibberellin (GA) content in the MAPKKK20 overexpression material was higher under ABA treatment than in the WT group. This indicates that the MAPKKK20 gene can negatively regulate ABA-repressed seed germination, and this function depends on its kinase activity.

[0013] Furthermore, the specific application is as follows: by constructing a MAPKKK20 gene overexpression vector through genetic engineering, transgenic plants with improved seed germination rate under ABA stress are obtained.

[0014] In this invention, there are no particular limitations on the plants suitable for gene transformation, as long as they are suitable for gene transformation operations, such as various crops, flowering plants, or forestry plants. The plants may be (but are not limited to): dicotyledons, monocotyledons, or gymnosperms. As a preferred embodiment, the plant is Arabidopsis thaliana, and any plant possessing this gene or a homologous gene is applicable.

[0015] The biological function of the MAPKKK20 gene in the tolerance of plant seeds to ABA stress during the germination stage is disclosed in this invention. Specifically, it improves the germination rate of plant seeds under ABA stress. Specifically, under ABA stress, the germination rate of MAPKKK20 gene overexpression lines is significantly higher than that of wild-type lines, while the germination rate of overexpression lines that lose MAPKKK20 kinase activity is not significantly different from that of wild-type lines.

[0016] Furthermore, the concentration of ABA is 0.5–2.0 μM.

[0017] The functions and related applications of the genes protected by this invention not only include the aforementioned MAPKKK20 gene, but can also be extended in the future to the functions of homologous genes with high homology in ABA stress tolerance during seed germination.

[0018] The second objective of this invention is achieved by the following technical solution:

[0019] Application of recombinant expression vector in improving seed germination in plants under ABA stress: The recombinant expression vector contains the MAPKKK20 gene; the gene sequence number of the MAPKKK20 gene in NCBI is NM_114891.2; the recombinant expression vector is constructed using the MAPKKK20 gene, and the germination rate of plant seeds under ABA stress is improved by overexpressing the MAPKKK20 gene.

[0020] Preferably, the method for preparing the recombinant expression vector includes: designing primers based on the nucleotide sequence of the MAPKKK20 gene, cloning the MAPKKK20 gene, and then ligating the MAPKKK20 gene into a plant expression vector to obtain the recombinant expression vector.

[0021] This invention constructs a recombinant expression vector, which can be used to transform plants using conventional biological methods such as Agrobacterium-mediated transformation, followed by screening and cultivation of the transformed plants. Because this recombinant expression vector contains the aforementioned MAPKKK20 gene, it also possesses the function of improving the tolerance of plant seeds to ABA stress during the seed germination stage.

[0022] The third objective of this invention is achieved by the following technical solution:

[0023] The plant breeding method of the present invention specifically obtains plants with a higher seed germination rate under ABA stress by increasing the activity of the MAPKKK20 gene expression protein in the target plant; or, the plant breeding method obtains plants with a higher seed germination rate under ABA stress by promoting the expression of the MAPKKK20 gene in the target plant; the gene sequence number of the MAPKKK20 gene in NCBI is NM_114891.2.

[0024] In this invention, there are no particular limitations on the plants suitable for gene transformation, as long as they are suitable for gene transformation operations, such as various crops, flowering plants, or forestry plants. The plants may be (but are not limited to): dicotyledons, monocotyledons, or gymnosperms. As a preferred embodiment, the target plant is Arabidopsis thaliana, and any plant possessing this gene or a homologous gene is applicable.

[0025] Furthermore, promoting the expression of the MAPKKK20 gene in the target plant can be achieved by overexpressing the MAPKKK20 gene or by targeted mutation of the MAPKKK20 gene. Specifically, this can be achieved by, for example: (1) introducing the MAPKKK20 gene into the target plant; (2) introducing a strong promoter and / or enhancer; or (3) other common methods in the art.

[0026] Furthermore, the operation process of the plant breeding method of the present invention includes: regulating the expression of MAPKKK20 using DNA homologous recombination technology and Agrobacterium-mediated transformation system to obtain transgenic plant lines.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention utilizes plant genetic engineering technology, employing Agrobacterium inflorescence infection to transform wild-type Arabidopsis thaliana (Col-0, WT), obtaining Arabidopsis plants overexpressing the MAPKKK20 gene. The function of this gene is verified and disclosed for the first time, along with its related mechanism of action. Experiments revealed that MAPKKK20 gene overexpression enhances the tolerance of plant seeds to ABA stress during seed germination. Furthermore, the MAPKKK20 gene enhances Arabidopsis seed tolerance to exogenous ABA during germination by influencing GA content; this function depends on MAPKKK20 kinase activity. Therefore, this invention provides genetic resources for molecular breeding of crops to tolerate ABA.

[0029] Furthermore, this invention introduces the MAPKKK20 gene into the target plant to obtain a transgenic plant that exhibits higher ABA tolerance than WT during the seed germination stage, thus providing a new approach for breeding plants to tolerate ABA. Attached Figure Description

[0030] Figure 1 This is a diagram showing the sequence analysis results of the MAPKKK20 protein in Experiment Example 1 of this invention;

[0031] Figure 2 This is a diagram showing the construction results of the MAPKKK20 overexpression material in Experiment Example 1 of this invention;

[0032] Figure 3 This is a graph showing the germination rate of the MAPKKK20 overexpression material in Experiment Example 2 of this invention;

[0033] Figure 4 This is a diagram showing the early growth of seedlings of the MAPKKK20 overexpression material in Experiment Example 3 of this invention.

[0034] Figure 5 The material MAPKKK20, which loses its kinase activity, was overexpressed in Experimental Example 4 of this invention. K36M Seed germination rate results;

[0035] Figure 6 The material MAPKKK20, which loses its kinase activity, was overexpressed in Experimental Example 5 of this invention. K36M Analysis diagram of early seedling growth;

[0036] Figure 7 This is a seed coat permeability analysis diagram of the MAPKKK20 gene overexpression material in Experiment Example 6 of this invention;

[0037] Figure 8 This is a graph showing the changes in ABA and GA hormone content in the MAPKKK20 gene overexpression material in Experiment Example 6 of this invention;

[0038] Figure 9This is a graph showing the expression levels of GA synthesis-related genes in the MAPKKK20 gene overexpression material of Experiment Example 6 of this invention. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely further illustrative of the invention and not intended to limit its scope. Unless otherwise specified, some of the technical means mentioned in the embodiments are conventional means well known to those skilled in the art. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods. Unless otherwise specified, the reagents and materials used are commercially available. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0040] In this invention, there are no particular limitations on the plants suitable for gene transformation, such as various crops, flowering plants, or forestry plants. The plants may be (but are not limited to): dicotyledons, monocotyledons, or gymnosperms. As a preferred embodiment, the target plant is Arabidopsis thaliana, and any gene possessing this gene or a homologous gene is applicable. The term "plant" in this invention includes the whole plant, its parent and offspring plants, and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues, and organs, all of which contain our target gene or nucleic acid. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores; similarly, each of the aforementioned objects contains the target gene / nucleic acid.

[0041] The following examples include some of the biomaterials, reagents, and equipment used in the experiments:

[0042] Biological materials: Arabidopsis thaliana Col-0 seeds were preserved in the laboratory; overexpression vector p35S-1300-GFP was preserved in the laboratory; Escherichia coli DH5α and Agrobacterium GV3101 were preserved in the laboratory; primer synthesis and sequencing were completed by Zhengzhou Qingke Biotechnology Co., Ltd.

[0043] The MAPKKK20 gene involved in the following examples has the NCBI sequence number NM_114891.2 (downloadable from https: / / www.ncbi.nlm.nih.gov / nuccore / NM_114891.2?report=genbank). The MAPKKK20 gene's messenger RNA (mRNA) sequence is 1447 bp long, and its coding sequence is 1029 bp long, containing 342 amino acids.

[0044] Experimental reagents: Plasmid extraction kit, RNA extraction kit, reverse transcription kit, real-time fluorescence kit, and one-step cloning enzyme were purchased from Novizan Biotechnology Co., Ltd.; sucrose, NaCl, TTC powder, and ABA powder were purchased from Solarbio; hygromycin was purchased from Solarbio; MS medium was purchased from Beijing Coolerbott Technology Co., Ltd.; and endonucleases were purchased from Mona Biotechnology Co., Ltd.

[0045] Experimental equipment: Heraeus Pico21 high-speed centrifuge (Thermo Scientific); 22331 PCR instrument (Eppendorf); Nanodrop 2000C nucleic acid detector (Thermo Scientific); HH-1 water bath (Kewei Yongxing); C1000 quantitative PCR instrument (Thermo Scientific); MLS-3750 autoclave (Sanyo, Japan); SpectraMax iD5 microplate reader (Thermo Scientific).

[0046] Example 1: Application of the MAPKKK20 gene in improving seed germination under ABA stress

[0047] In this embodiment, overexpression of the MAPKKK20 gene can improve the tolerance of plant seeds to ABA stress during the germination stage and increase the germination rate of plant seeds under ABA stress. The MAPKKK20 gene has the NCBI sequence number NM_114891.2.

[0048] Specifically, in this embodiment, the application of the MAPKKK20 gene involves constructing an overexpression vector for the MAPKKK20 gene using genetic engineering techniques to obtain transgenic plants with improved seed germination rates under ABA stress. The plant used is Arabidopsis thaliana; the ABA concentrations are 0.5, 1.0, and 2.0 μM. The improved seed germination under ABA stress is specifically manifested in the following: under ABA stress, the germination rate of the MAPKKK20 gene overexpression line is significantly higher than that of the wild-type line, and the overexpression of the MAPKKK20 line, which loses kinase activity, is significantly higher. K36MThe germination rate was not significantly different from that of the wild-type strain.

[0049] Example 2: Application of recombinant expression vector in improving plant tolerance to ABA stress

[0050] The recombinant expression vector in this embodiment contains the MAPKKK20 gene; the MAPKKK20 gene sequence number in NCBI is NM_114891.2. This embodiment uses the MAPKKK20 gene to construct a recombinant expression vector, and by overexpressing the MAPKKK20 gene, the germination rate of plant seeds under ABA stress is improved.

[0051] The recombinant expression vector was prepared by designing primers based on the nucleotide sequence of the MAPKKK20 gene, cloning the MAPKKK20 gene, and then ligating the MAPKKK20 gene into the plant expression vector p35S-1300-GFP to obtain the recombinant expression vector. The plant used was Arabidopsis thaliana; the ABA concentrations were 0.5, 1.0, and 2.0 μM. The tolerance to ABA stress was as follows: under ABA stress, the germination rate of the MAPKKK20 gene overexpression line was significantly higher than that of the wild-type line, and the overexpression of the MAPKKK20 line, which lost kinase activity, was significantly higher. K36M The germination rate was not significantly different from that of the wild-type strain.

[0052] Example 3 Plant breeding methods

[0053] The plant breeding method in this embodiment specifically involves increasing the activity of the MAPKKK20 gene expression protein in the target plant to obtain plants with a higher seed germination rate under ABA stress than the target plant. The MAPKKK20 gene has the NCBI sequence number NM_114891.2. The target plant is Arabidopsis thaliana.

[0054] In other embodiments, the plant breeding method involves promoting the expression of the MAPKKK20 gene in the target plant to obtain plants with a higher seed germination rate under ABA stress than the target plant. The MAPKKK20 gene has the NCBI sequence number NM_114891.2. The target plant is Arabidopsis thaliana. Promoting the expression of the MAPKKK20 gene in the target plant is specifically achieved by overexpressing the MAPKKK20 gene.

[0055] Experiment 1: MAPKKK20 protein sequence analysis and construction of recombinant expression vector

[0056] RNA was extracted from Arabidopsis thaliana plants that had grown for 4 weeks, and cDNA was obtained by reverse transcription. Using the cDNA as a template, the MAPKKK20 gene sequence (gene sequence number NM_114891.2) obtained from the NCBI database was used to design specific primers in Primer Premier 5.0. The MAPKKK20 gene was cloned via PCR, and the MAPKKK20 protein sequence was analyzed using an online tool (http: / / www.ebi.ac.uk / interpro / ). The analysis results are as follows: Figure 1 As shown.

[0057] Figure 1 The results showed that the MAPKKK20 protein is 342 amino acids in length, with the kinase domain located at amino acids 3-268 from the N-terminus, containing one ATP-binding site and one active site. Experimental verification and literature review revealed that mutating lysine 36 of the MAPKKK20 gene to methionine resulted in a different MAPKKK20 protein. K36M The gene then loses its kinase activity.

[0058] To investigate the function of the MAPKKK20 gene, this invention ligated the MAPKKK20 gene into the plant expression vector p35S1300GFP, constructing the overexpression vector p35S1300GFP-MAPKKK20. Then, using the Agrobacterium-mediated inflorescence infection method, p35S1300GFP-MAPKKK20 was successfully transformed into the wild-type Arabidopsis line Col-0 (WT group). After successive generations of hygromycin resistance screening, homozygous Arabidopsis materials overexpressing MAPKKK20 (denoted as MAPKKK20OE-2 and MAPKKK20OE-3, respectively) were successfully obtained. Subsequently, RNA was extracted from Arabidopsis seedlings to quantitatively analyze the expression level of the MAPKKK20 gene. The results are as follows: Figure 2 As shown.

[0059] Depend on Figure 2 It can be seen that the expression level of the MAPKKK20 gene in the overexpressed Arabidopsis materials MAPKKK20OE-2 and MAPKKK20OE-3 is significantly higher than that in the WT group, indicating that the MAPKKK20 overexpression material of the present invention has been successfully constructed.

[0060] Experiment 2 verifies the ABA sensitivity of MAPKKK20 overexpression materials during seed germination.

[0061] To investigate the function of MAPKKK20, MAPKKK20OE-2, MAPKKK20OE-3, and WT seeds harvested at 60 days old were sown on MS medium containing different concentrations of abscisic acid (ABA concentrations of 0 μM, 0.5 μM, 1 μM, and 2 μM). Germination rate was measured every 12 hours (germination was defined as when the radicle broke through more than half of the seed coat). The results are as follows: Figure 3 As shown.

[0062] Depend on Figure 3 It can be seen that under normal growth conditions without ABA treatment (CK group), the germination rates of MAPKKK20OE-2, MAPKKK20OE-3, and WT all reached 100% at 48h, showing no significant difference. Figure 3 A). However, regardless of whether exogenous ABA was treated with 0.5 μM, 1 μM, or 2 μM, the germination rates of MAPKKK20OE-2 and MAPKKK20OE-3 were higher than those of the WT wild group ( Figure 3 (B, 3C, 3D). This indicates that MAPKKK20 overexpression materials are insensitive to ABA during germination, meaning that MAPKKK20 negatively regulates ABA-inhibited seed germination, and overexpression of MAPKKK20 can improve seed tolerance to ABA stress during the germination stage.

[0063] Experiment 3 further verified the ABA sensitivity of MAPKKK20 overexpression materials during the early growth stage of seedlings.

[0064] Furthermore, this invention harvested 60-day-old MAPKKK20OE-2, MAPKKK20OE-3, and WT seeds and sowed them on MS medium containing different concentrations of ABA (0 μM, 0.5 μM, 1 μM, and 2 μM). The early growth of the seedlings was observed and analyzed on days 14 and 21, respectively. The results are as follows: Figure 4 As shown.

[0065] Figure 4 The results showed that, under normal conditions, the green cotyledon ratio of the MAPKKK20 overexpression material was not significantly different from that of the WT material, regardless of whether it was day 14 or day 21. However, under ABA treatment, the green cotyledon ratio of the MAPKKK20 overexpression material was significantly higher than that of the WT material. Figure 4 (A, 4B, 4C) This indicates that during the early growth stage of seedlings, the cotyledon greening of MAPKKK20 overexpression material seedlings is not sensitive to ABA, that is, MAPKKK20 gene overexpression can improve the tolerance of plant seedlings to ABA stress in the early growth stage.

[0066] Experiment 4 validates the overexpression of the loss-of-kinase-activity material MAPKKK20 K36M ABA sensitivity during seed germination

[0067] Based on the protein sequence characteristics of MAPKKK20, this invention creates an Arabidopsis material, MAPKKK20, that loses its kinase activity through overexpression. K36M OE-3 and MAPKKK20 K36M OE-5, and will harvest 60 days of MAPKKK20. K36M OE-3, MAPKKK20 K36M OE-5 and WT seeds were sown on MS medium containing different concentrations of ABA (0 μM, 0.5 μM, 1 μM, 2 μM), and the germination rate was measured every 12 hours. Results are as follows: Figure 5 As shown.

[0068] Depend on Figure 5 It can be seen that regardless of whether ABA processing is performed, MAPKKK20 K36M OE-3, MAPKKK20 K36M No significant difference was observed in the germination rates of OE-5 and WT seeds. Figure 5 (A, 5B, 5C, 5D) Overexpression of MAPKKK20, which loses its kinase activity, showed that the material was sensitive to ABA during germination, just like WT, indicating that MAPKKK20's role in regulating ABA-inhibited seed germination depends on its kinase activity.

[0069] Experiment 5 further validated the overexpression of the kinase-inactive material MAPKKK20. K36M ABA sensitivity during early seedling growth

[0070] This invention will yield 60 days of MAPKKK20 K36M OE-3, MAPKKK20 K36M OE-5 and WT seeds were sown on MS medium containing different concentrations of ABA (0 μM, 0.5 μM, 1 μM, 2 μM), and the early growth of seedlings was analyzed on days 14 and 21. Results are as follows: Figure 6 As shown.

[0071] Depend on Figure 6 It can be seen that regardless of whether ABA processing is performed, MAPKKK20 K36M OE-3, MAPKKK20 K36M There was no significant difference in the green cotyledon rate between OE-5 and WT. Figure 6 A, 6B, 6C), overexpression of the kinase-free material MAPKKK20 K36M Like WT, it is sensitive to ABA during germination, indicating that the negative regulation of ABA-inhibited seedling growth by MAPKKK20 also depends on its kinase activity.

[0072] Analysis of the mechanism by which MAPKKK20 participates in ABA inhibition of seed germination and early seedling growth in Experiment Example 6

[0073] Seed germination is the first step in plant growth and development and is regulated by many factors. To elucidate the mechanism by which MAPKKK20 negatively regulates ABA's inhibition of seed germination, this invention analyzed seed coat permeability using TTC staining solution. The results are as follows: Figure 7 As shown.

[0074] Depend on Figure 7 It can be seen that MAPKKK20OE-2, MAPKKK20OE-3, and MAPKKK20 K36M OE-3, MAPKKK20 K36M There was no significant difference in seed staining between OE-5 and WT. Figure 7 A and 7B) indicate that MAPKKK20 does not participate in the regulation of seed coat permeability, and the germination of MAPKKK20 overexpression materials earlier than WT is due to other mechanisms.

[0075] Abscisic acid (ABA) and gibberellin (GA) are two important hormones affecting seed germination. To further understand the mechanism of action of MAPKKK20, this invention analyzed the ABA and GA contents in MAPKKK20 overexpression materials and WT. The results are as follows: Figure 8 As shown.

[0076] Depend on Figure 8 It can be seen that, without ABA treatment, there was no significant difference in ABA and GA content between the MAPKKK20 overexpression material and WT. Figure 8 (A and 8B). Under ABA treatment, the ABA content in the MAPKKK20 overexpression material was not significantly different from that in the WT material, but the GA content in the MAPKKK20 overexpression material was higher than that in the WT material. Figure 8 A and 8B) indicate that MAPKKK20 may regulate the synthesis of GA in the presence of ABA.

[0077] To verify this conclusion, this invention further detected the expression levels of GA synthesis-related genes (GA20ox1, GA20ox3), and the results are as follows: Figure 9 As shown.

[0078] Figure 9 The results showed that in the absence of ABA, there was no significant difference in the expression levels of GA20ox1 and GA20ox3 between the MAPKKK20 overexpression material and the WT material. However, under ABA treatment, the expression levels of GA20ox1 and GA20ox3 in the MAPKKK20 overexpression material were significantly higher than those in the WT material. Figure 9A, 9B). This indicates that MAPKKK20 participates in regulating seed germination by affecting the expression of GA synthesis genes GA20ox1 and GA20ox3, thereby influencing GA synthesis.

[0079] In summary, overexpression of the MAPKKK20 gene significantly increased the seed germination rate of Arabidopsis thaliana under ABA treatment compared to wild-type Arabidopsis thaliana (WT), indicating that the MAPKKK20 gene negatively regulates ABA-repressed seed germination. Furthermore, overexpression of the MAPKKK20 gene in Arabidopsis thaliana material that loses kinase activity... K36M The seed germination rate under ABA treatment was not significantly different from that under WT, indicating that the negative regulation of ABA-repressed seed germination by the MAPKKK20 gene depends on its kinase activity. Furthermore, under ABA treatment, the GA content in MAPKKK20-overexpressing seeds was significantly higher than that in WT seeds. Analysis of GA synthesis-related genes revealed that the expression levels of GA20ox1 and GA20ox3 in MAPKKK20-overexpressing materials were higher than those in WT seeds under ABA treatment. Therefore, MAPKKK20 negatively regulates ABA-repressed seed germination by affecting the expression of GA synthesis genes, thereby influencing GA content. This research is crucial for the propagation of seed plants.

[0080] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The application of the MAPKKK20 gene in improving seed germination in plants under ABA stress, characterized in that, Overexpression of the MAPKKK20 gene upregulated the expression of GA20ox1 and / or GA20ox3, which are related to GA synthesis, thereby increasing the endogenous gibberellin content and improving the germination rate of Arabidopsis seeds under ABA stress. The MAPKKK20 gene has the NCBI sequence number NM_114891.

2.

2. The application of the MAPKKK20 gene according to claim 1 in improving seed germination in plants under ABA stress, characterized in that, The specific application is as follows: by constructing a MAPKKK20 gene overexpression vector through genetic engineering, transgenic plants with improved seed germination rate under ABA stress are obtained.

3. The application of the MAPKKK20 gene according to claim 1 in improving seed germination in plants under ABA stress, characterized in that, Improving the germination rate of plant seeds under ABA stress is specifically manifested in the following ways: Under ABA stress, the germination rate of MAPKKK20 gene overexpression lines was significantly higher than that of wild-type lines, while the germination rate of lines that overexpressed and lost MAPKKK20 kinase activity was not significantly different from that of wild-type lines.

4. The application of the MAPKKK20 gene according to claim 1 in improving seed germination in plants under ABA stress, characterized in that, The concentration of ABA is 0.5~2.0 μM.

5. The application of recombinant expression vectors in improving seed germination in plants under ABA stress, characterized in that, The recombinant expression vector contains the MAPKKK20 gene; the gene sequence number of the MAPKKK20 gene in NCBI is NM_114891.2; the recombinant expression vector is constructed using the MAPKKK20 gene, and by overexpressing the MAPKKK20 gene, the expression of GA synthesis-related genes GA20ox1 and / or GA20ox3 is upregulated, thereby increasing the endogenous gibberellin content and improving the germination rate of plant seeds under ABA stress; the plant is Arabidopsis thaliana.

6. The application of the recombinant expression vector according to claim 5 in improving seed germination under ABA stress, characterized in that, The method for preparing the recombinant expression vector includes: designing primers based on the nucleotide sequence of the MAPKKK20 gene, cloning the MAPKKK20 gene, and then ligating the MAPKKK20 gene into a plant expression vector to obtain the recombinant expression vector; the plant is Arabidopsis thaliana.

7. A plant breeding method, characterized in that, The plant breeding method described herein is to obtain plants with a higher seed germination rate under ABA stress than wild-type plants by increasing the activity of the MAPKKK20 gene expression protein in Arabidopsis thaliana; or, the plant breeding method described herein is to obtain plants with a higher seed germination rate under ABA stress than wild-type plants by promoting the expression of the MAPKKK20 gene in Arabidopsis thaliana, upregulating the expression of GA synthesis-related genes GA20ox1 and / or GA20ox3, and increasing the endogenous gibberellin content; the gene sequence number of the MAPKKK20 gene in NCBI is NM_114891.2; the plant described herein is Arabidopsis thaliana.

8. The plant breeding method according to claim 7, characterized in that, The expression of the MAPKKK20 gene in Arabidopsis thaliana was promoted by overexpressing the MAPKKK20 gene.

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