Application of mnERF23 gene in enhancing drought resistance of plants

Abstract

The application discloses application of MnERF23 gene in enhancing drought resistance of plants and belongs to the technical field of genetic engineering. The application first confirms that the MnERF23 gene has the function of positively regulating the drought resistance of plants. The application also establishes a double-function verification method of "Arabidopsis heterologous overexpression + mulberry homologous transient overexpression", which effectively avoids the technical bottleneck of the stable genetic transformation system of the mulberry. Experiments prove that overexpression of the MnERF23 gene can significantly enhance the root growth ability of Arabidopsis under mannitol simulated drought stress, reduce the wilting degree of leaves of the mulberry under natural drought treatment, reduce the accumulation of malondialdehyde and improve the antioxidant enzyme activity. The application discloses a molecular mechanism of the MnERF23 gene in enhancing the drought resistance of plants by activating the expression of osmotic stress related genes and the antioxidant enzyme system. The application has important significance for promoting the stress resistance genetic breeding of the mulberry and other crops.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to the application of the MnERF23 gene in enhancing plant drought resistance. Background Technology

[0002] mulberry( Morus notabilis Mulberry is an important economic tree species. Its leaves are not only the sole feed source for silkworms but are also increasingly being widely used as a healthy food ingredient, possessing significant economic value and application prospects. However, drought stress is one of the main factors affecting the quality and yield of mulberry leaves, severely restricting the stable development of the mulberry industry. Currently, the molecular mechanisms by which mulberry responds to drought stress are not fully understood, which greatly limits the progress of using molecular breeding techniques to improve stress-resistant mulberry varieties.

[0003] Plant responses to drought stress constitute a complex molecular regulatory network involving multiple stages, including signal sensing, transduction, transcriptional regulation, and physiological responses. Transcription factors play a crucial role in regulating the expression of drought-related genes. The AP2 / ARE transcription factor family is an important class of transcription factors in plants, with members widely involved in regulating plant growth and development as well as responses to abiotic stresses. Previous studies have shown that members of the ARE subfamily can enhance plant drought resistance through mechanisms such as regulating reactive oxygen species scavenging, proline synthesis, and the ABA signaling pathway. Although the whole-genome identification of the mulberry AP2 / ARE gene family has been completed, there is still a significant gap in research on the specific functions of ARE transcription factors in mulberry in response to drought stress, and a lack of key gene resources that can effectively enhance the drought resistance of mulberry.

[0004] In existing technologies, the closest research to this invention on drought-resistant genes in mulberry includes functional analysis of transcription factors such as MnARE2 and MnDREB6E. These studies typically employ heterologous overexpression of the target gene in Arabidopsis thaliana, combined with phenotypic observation and physiological and biochemical index determination to verify gene function. However, these studies do not involve the MnERF23 gene and generally suffer from a core technical bottleneck: a stable and efficient mulberry genetic transformation system is not yet mature. Problems encountered during mulberry tissue culture, such as weak explant differentiation ability, easy browning of callus tissue, long transformation cycle, and low positive plant yield, restrict the gene function verification process and thus limit the direct guiding value of the research conclusions for mulberry breeding.

[0005] Therefore, developing a gene function verification method that can circumvent the above-mentioned bottlenecks and is efficient and accurate, and discovering new genes with clear drought resistance functions, is of great practical significance for promoting molecular breeding of mulberry trees for stress resistance, and is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of the MnERF23 gene in enhancing plant drought resistance, fills the gap in the research on the drought resistance function of the MnERF23 gene in the ARE family of mulberry, and provides an efficient and accurate gene function verification method that can effectively avoid the technical bottleneck of stable genetic transformation system, thereby providing new gene resources and reliable technical path for molecular breeding of drought resistance in mulberry.

[0007] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0008] The primary objective of this application is to provide the application of the MnERF23 gene in enhancing plant drought resistance, wherein the CDS sequence of the MnERF23 gene is shown in SEQ ID NO:1; and the plant is mulberry or Arabidopsis thaliana.

[0009] As a preferred technical solution, the amino acid sequence encoded by the MnERF23 gene is shown in SEQ ID NO.2.

[0010] Another object of this application is to provide: a biomaterial, said biomaterial being any of the following: 1) An expression cassette containing the MnERF23 gene; 2) A recombinant vector containing the MnERF23 gene, or a recombinant vector containing the expression cassette described in 1); 3) Recombinant bacteria containing the MnERF23 gene, or recombinant bacteria containing the expression cassette described in 1), or recombinant bacteria containing the recombinant vector described in 2); 4) A transgenic plant containing the MnERF23 gene, or a transgenic plant containing the expression cassette described in 1), or a transgenic plant containing the recombinant vector described in 2), or a transgenic plant containing the recombinant bacteria described in 3).

[0011] Another object of this application is to provide: the application of the biomaterial, wherein the application is any one of the following: (a) Enhance the drought resistance of plants; (b) Enhance the activity of antioxidant enzymes in plants under drought stress; (c) Reduce malondialdehyde content in plants under drought stress and mitigate membrane lipid peroxidation damage; (d) Activate the expression of osmotic stress-related genes in plants; (e) Improve the root growth capacity of plants under drought stress; (f) Developing transgenic plants with enhanced drought resistance; (g) Screening of materials for use as molecular markers in plant drought resistance breeding.

[0012] Another object of this application is to provide: a PCR primer pair for amplifying the CDS sequence of the MnERF23 gene, said primer pair being designed according to the sequence shown in SEQ ID NO:1, and used for homologous recombination cloning, the specific sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0013] Another objective of this application is to provide: a method for verifying the function of genes used to enhance the drought resistance of mulberry trees, comprising the following steps: (a) Construct a recombinant expression vector containing drought-resistant genes; (b) Transform the recombinant expression vector from step (a) into Agrobacterium to obtain engineered bacteria; (c) Using the engineered bacteria from step (b), transform the model plant Arabidopsis thaliana by Agrobacterium infection to obtain transgenic Arabidopsis thaliana with heterologous overexpression of drought resistance gene, and subject it to drought stress treatment to verify the drought resistance function of drought resistance gene. (d) Using the engineered bacteria from step (b), the drought resistance gene was transiently overexpressed in mulberry leaves via Agrobacterium injection. The mulberry plants with transient overexpression were then subjected to drought stress treatment to further verify the drought resistance function of the drought resistance gene in mulberry trees.

[0014] As a preferred technical solution, the drought-resistant gene is the MnERF23 gene.

[0015] Another object of this application is to provide a method for screening or breeding transgenic plants with enhanced drought resistance, comprising the steps of transferring the MnERF23 gene into a target plant and overexpressing the gene.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention fills a technological gap by clarifying for the first time the specific function of the MnERF23 gene in drought stress response in mulberry, demonstrating that it can significantly enhance the drought resistance of plants (Arabidopsis thaliana and mulberry), providing a new functional gene with independent intellectual property rights for molecular breeding of drought resistance in mulberry, and enriching the theoretical system of molecular mechanisms of stress resistance in mulberry.

[0017] Overcoming technical bottlenecks: This invention innovatively employs a dual validation strategy of "Arabidopsis heterologous overexpression + mulberry homologous transient overexpression." This strategy first utilizes a mature and efficient Arabidopsis system to rapidly complete preliminary validation of gene function, and then uses a simple and short-cycle homologous transient expression system to perform precise functional validation on mulberry. This design effectively avoids the technical shortcomings of low efficiency and long cycles in stable mulberry genetic transformation systems, significantly improving the reliability and relevance of research conclusions.

[0018] The mechanism of action was elucidated: This invention, through multi-dimensional experimental data corroborating each other, clearly revealed the pathway by which the MnERF23 gene regulates plant drought resistance: by activating the expression of osmotic stress-related genes (such as AtDREB1A, AtABI5, and AtNCED3), enhancing the activity of antioxidant enzymes (CAT, POD, and SOD), and reducing the accumulation of malondialdehyde (MDA), a membrane lipid peroxidation product, it comprehensively improves the plant's drought tolerance. This provides clear molecular mechanistic support for the application of this gene.

[0019] This invention provides a technical paradigm: The functional verification method established by this invention is simple to operate, has a short cycle, and is low in cost. It not only quickly realizes the functional verification of the MnERF23 gene, but also provides a technical paradigm that can be directly referenced for the study of gene function of other mulberry stress resistance genes or other plants with difficult genetic transformation. It has important practical significance for breeding new crop varieties with strong drought resistance and promoting the sustainable development of related industries. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 Here is a diagram of the amino acid sequence encoded by the CDS sequence of the MnERF23 gene.

[0022] Figure 2 Image: Agarose gel electrophoresis of the PCR amplification products of the MnERF23 gene.

[0023] Figure 3 The vector spectrum of the pCAMBIA1305 vector is shown.

[0024] Figure 4 Image showing enzyme digestion verification of the pCAMBIA1305-MnERF23 recombinant vector.

[0025] Figure 5 Here is an electrophoresis image of the PCR product of the transgenic Arabidopsis MnERF23 gene.

[0026] Figure 6 Here is an electrophoresis diagram of the PCR product of the transgenic Arabidopsis thaliana GFP gene.

[0027] Figure 7 Here is an electrophoresis image of the semi-quantitative product of the transgenic Arabidopsis MnERF23 gene.

[0028] Figure 8Here is an electrophoresis diagram of the semi-quantitative product of the transgenic Arabidopsis thaliana GFP gene.

[0029] Figure 9 Here are the comparison images of root length in Arabidopsis thaliana under drought treatment; where a and b are comparison images of root length in different groups under different drought treatment conditions.

[0030] Figure 10 To: The expression of osmotic stress-related genes in different groups before and after drought treatment.

[0031] Figure 11 The image shows a comparison of mulberry trees under drought treatment. In the image, a and c show the wilting and yellowing of leaves in the TOE-1305 control line before and after drought treatment, respectively. In the image, b and d show the wilting and yellowing of leaves in the TOE-MnERF23 line before and after drought treatment, respectively.

[0032] Figure 12 The effect of different groups of natural drought treatment on physiological indicators of MDA, CAT, POD and SOD. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] In the examples, all raw materials not mentioned are commercially available, and all experimental methods not mentioned are conventional experimental methods, which will not be described in detail here.

[0035] Example 1 Cloning of the MnERF23 gene in mulberry and construction of its expression vector (1) Extraction of total RNA from mulberry tree Using the Novozymes Biotechnology Co., Ltd. Nucleic Acid Extraction Kit (RC122-01), strictly follow the instructions to obtain total RNA from mulberry trees. Measure the purity and concentration of the extracted RNA using a nucleic acid detector, and check the integrity of the RNA using agarose gel electrophoresis. Store the remaining RNA at -80℃.

[0036] (2) RNA reverse transcription to obtain cDNA The reverse transcription reaction was performed using the Novozymes Reverse Transcription Kit (323) from Novizan Biotechnology Co., Ltd. The procedure was strictly followed according to the actual instructions. The product after the reverse transcription reaction was completed was cDNA, which was stored at -20℃ for later use.

[0037] (3) Cloning of the MnERF23 gene The published mulberry MnERF23 CDS sequence (gene number MnotChr1G00045910, sequence shown in SEQ ID NO.1, encoding amino acid sequences shown in SEQ ID NO.2 and...) was used in the database. Figure 1 Primers were designed using homologous recombination, and cDNA obtained from reverse transcription was used as a template for PCR amplification. The PCR reaction system is shown in Table 1, the PCR reaction procedure is shown in Table 2, and the PCR products were detected by 1% agarose gel electrophoresis (results are shown in Table 2). Figure 2 (As shown), the target fragment was recovered and purified.

[0038] Table 1. PCR reaction system for target gene amplification

[0039] Table 2. PCR reaction process for target gene amplification

[0040] >MnotChr1G00045910 TAA, SEQ ID NO.1.

[0041] The amino acid sequence encoded by the MnERF23 gene: MCGGAIISDFISTPRSSRLTADYLWPDLKKSGSGKRFSKPVRSVIVDIDDDFEADFQGFKDDSDVDDDDEVIDVKPFAFSARKPTSSRGSTTVKYTESDGQAEKSAKRKRKNQYRGIRQRPWGKWAAEIRDPRKGVRVWLGTFNTAEEAARAYDAEARRIRGKKAKVNFPDETPRALPKHPVKEG PKRSLPKENSNSSESNLNNQSFNFVNNSDQDYYNAMGFLEEKPLTNQYEHVETLPAKAGAGLKSNAPAATTPMYFSSDQGSNSFECSDFGLGEHGSKTPEISSVFSATSENDDSLSLEDTNPTKKLKSDSENVVLPEENHAKTLSEELSAFESQMKFLQMPYLEGSALLWDDTFLAGDSTQDGGNSINLWSFDDFSTMSGEAF , SEQ ID NO.2.

[0042] Upstream primer F: 5'-ATGTGTGGAGGTGCTATAATCTCCG-3', SEQ ID NO.3; Upstream primer R: 5'-TTAGAAGGCTTCGCCAGACATGGTG-3', SEQ ID NO.4.

[0043] (4) Construction of plant expression vectors The purified MnERF23 gene CDS fragment was inserted into the pCAMBIA1305 vector, which had been digested with SpeI and BamHI, via homologous recombination (vector map shown below). Figure 3 The recombinant plasmid pCAMBIA1305-MnERF23 was constructed as shown in the figure. The recombinant plasmid was transformed into competent *E. coli* cells, positive clones were screened, cultured, propagated, and the plasmid was extracted. Double enzyme digestion was performed for verification, and the digestion products were detected by 1% agarose gel electrophoresis (results are shown in the figure). Figure 4 As shown in the image, the plasmid recombination was initially determined to be complete, and subsequent sequencing verification was performed. The correctly sequenced recombinant plasmid was extracted using a plasmid extraction kit (Novizan DC201) and stored at -20℃ for later use.

[0044] The pCAMBIA1305 vector is an existing technology vector that has been publicly used in the literature Bo C, Cai R, Fang X, Wu H, MaZ, Yuan H, Cheng B, Fan J, Ma Q. Transcription factor ZmWRKY20 interacts with ZmWRKY115 to repress expression of ZmbZIP111 for salt tolerance in maize. Plant J. 2022 Sep;111(6):1660-1675. doi: 10.1111 / tpj.15914. Epub 2022 Aug 1.PMID: 35861696.

[0045] Example 2 Heterologous overexpression of MnERF23 gene in Arabidopsis thaliana and verification of drought resistance function (1) Transformation of recombinant plasmid into Agrobacterium The following steps were followed to transform competent cells: 1) Take the competent Agrobacterium cells stored at -80℃ and let them partially melt at room temperature or in your palm for a moment. When they are in an ice-water mixture, insert them into ice.

[0046] 2) Add 0.01-1 μg of recombinant plasmid pCAMBIA1305-MnERF23 to every 100 μl of competent cells, mix by hand by tapping the bottom of the tube, and incubate on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0047] 3) Add 700 μl of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2-3 hours.

[0048] 4) Centrifuge at 6000 rpm for one minute to collect the bacteria. Take about 100 μl of supernatant, gently pipette and resuspend the bacterial block, spread it on LB containing Kan Rif antibiotic, invert and incubate at 28℃ for 2-3 days. After the growth of bacterial plaques, verify the colony by PCR, shake the bacteria, and amplify to obtain Agrobacterium tumefaciens containing recombinant plasmids. Store for later use.

[0049] (2) Cultivation of wild-type Arabidopsis: After vernalization, the seeds were sown on 1 / 2 MS solid medium and cultured at 25℃ under 16h light / 8h darkness. When the seedlings grew for 6-8 days and the roots were about 6cm long, they were transplanted into a mixture of vermiculite and sterilized black soil in a 3:1 volume ratio. The seedlings were covered with a film to keep them moist. After one week, the film was removed and the seedlings were cared for normally until they flowered.

[0050] (3) Agrobacterium infection of Arabidopsis: Agrobacterium bacterial suspension containing recombinant plasmid was activated and cultured in liquid LB medium containing kanamycin and rifampin; the bacterial suspension was collected by centrifugation and suspended and mixed with Arabidopsis transformation buffer (Ms medium 0.22g, MES 0.05g, sucrose 5g, Silwet L-77 30μl); the bacterial suspension was dripped onto the stigma of Arabidopsis before flowering using a Pasteur dropper, and the stigma was covered with a black plastic bag for 24 hours after infection; the infection was repeated once a week later to improve the transformation efficiency; watering and fertilization were carried out in a timely manner after infection, and pests and diseases were controlled. Watering was stopped after most of the siliques matured and turned yellow, and T0 generation transgenic seeds were harvested.

[0051] (4) Screening of transgenic positive lines of Arabidopsis thaliana: T0 generation transgenic Arabidopsis thaliana seeds were disinfected, vernalized, and then evenly sown on 1 / 2 MS solid medium containing 25 mg / L hygromycin. The seeds were placed vertically in an artificial climate chamber and cultured under 25℃, 16h light / 8h darkness conditions. After 7-10 days of greenhouse culture, Arabidopsis thaliana plants that grew normally on the medium were selected for preliminary screening of transgenic positive seedlings, which were then transplanted into nutrient soil for further cultivation. When the transgenic positive seedlings reached near flowering stage, 1-2 leaves were selected from each plant, and leaf DNA was extracted and molecularly verified using PCR technology. The results are as follows: Figure 5 As shown, the MnERF23 gene was amplified in all five lines, further confirming the authenticity of the positive lines. Subsequently, following the aforementioned initial screening and molecular verification methods, the empty vector control transgenic Arabidopsis lines were tested, and the results are as follows. Figure 6 As shown, all transgenic lines amplified the GFP gene. Following the aforementioned initial screening and molecular verification methods, positive lines were subjected to continuous single-plant seed collection, continuous screening, and subculture until genetically stable homozygous T3 generation seeds were obtained.

[0052] (5) Screening of target gene overexpression lines and control lines: After obtaining the corresponding lines by sowing and culturing T3 generation seeds, the expression level of the MnERF23 gene in each line was detected by semi-quantitative technology. The results are as follows: Figure 7 As shown, transgenic Arabidopsis lines overexpressing the MnERF23 gene, oeERF23-1 to oeERF23-5, were obtained through screening. Lines with high MnERF23 gene expression levels, namely oeERF23-1, oeERF23-2, and oeERF23-3, were selected for subsequent experiments. The same screening method was used to screen Arabidopsis lines transformed with the empty vector pCAMBIA1305. The expression level of the GFP gene (nucleotide sequence shown in SEQ ID NO. 5) was detected using semi-quantitative techniques. The results are shown below. Figure 8 As shown, empty vector control transgenic Arabidopsis thaliana lines were obtained and named oe1305-1 to oe1305-5 respectively. oe1305-1 was selected for subsequent experiments and named oe1305.

[0053] >The GFP sequence: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTAC, SEQ ID NO.5.

[0054] (6) Drought stress treatment and phenotypic analysis The MnERF23 overexpressing lines oeERF23-1, oeERF23-2, and oeERF23-3 were used as experimental materials, and the Arabidopsis line oe1305 transformed with the pCAMBIA1305 vector was used as the control material. Both experimental and control materials were subjected to drought stress. The drought stress treatment was as follows: Arabidopsis seedlings that had germinated for 3 days and exhibited uniform growth were transplanted into MS solid medium containing different concentrations of mannitol. Three mannitol concentration gradients were set: 0 mM (simulating normal growth conditions), 100 mM (simulating mild drought stress), and 200 mM (simulating moderate drought stress). During the culture process, the growth status of each line was continuously observed and recorded, with a focus on root growth, to provide experimental data support for subsequent analysis of the drought resistance function of the MnERF23 gene. The results are as follows: Figure 9 As shown.

[0055] When the mannitol concentration in the culture medium was 0 mM (normal growth conditions), there was no significant difference in root growth between the oe1305 line and the oeERF23-1, oeERF23-2, and oeERF23-3 lines. When the mannitol concentration was 100 mM (mild drought stress), the root length of all lines was shortened, but the root length of the oeERF23-1, oeERF23-2, and oeERF23-3 lines was significantly longer than that of the oe1305 line. When the mannitol concentration was increased to 200 mM (moderate drought stress), the root growth of all Arabidopsis plants was further inhibited, and the root growth of the oeERF23-1, oeERF23-2, and oeERF23-3 lines was affected compared with the oe1305 line.

[0056] (7) Molecular mechanism analysis RNA was extracted from Arabidopsis seedlings subjected to the above stress treatment, and the expression levels of osmotic stress-related marker genes were detected by RT-qPCR. RT-qPCR results showed that the expression levels of osmotic stress-related genes such as AtDREB1A, AtABI5, and AtNCED3 were significantly activated in the oeERF23-1, oeERF23-2, and oeERF23-3 lines. Figure 10 This indicates that the MnERF23 gene can enhance the drought resistance of Arabidopsis thaliana by activating the expression of genes related to osmotic stress.

[0057] Example 3 Homologous transient overexpression of MnERF23 gene in mulberry and verification of its drought resistance function (1) Preparation of Agrobacterium suspension The Agrobacterium tumefaciens containing pCAMBIA1305-MnERF23 and the empty vector pCAMBIA1305 preserved in Example 2 was cultured in LB medium containing the corresponding resistance until the OD600 reached 1.0-1.5. The cells were collected by centrifugation at 5000 rpm for 10 min, and the cells were resuspended in an equal volume of the prepared resuspension to obtain the infection permeate (containing 10 mM MgCl2, 10 mM MMEs, 150 μM acetylsyl syringone, pH = 5.6). The solution was allowed to stand in the dark at room temperature for 1.5-2 h (no more than 3 h).

[0058] (2) Preparation of mulberry seedlings Plant mulberry seeds and let them grow until the four cotyledons are fully expanded. Take them out of the light room and place them under a fluorescent lamp for about 1 hour to allow the stomata of the leaves to open. Select healthy mulberry seedlings.

[0059] (3) Instantaneous transformation of mulberry leaves Agrobacterium resuspension containing the plant expression vector of the target gene was drawn into a needle-free syringe. The prepared resuspension was injected into the back of a mulberry leaf using a 1 mL syringe (needle removed). Successful injection was indicated by the injection site becoming moist. The mulberry plant was then covered with plastic wrap to maintain moisture and incubated in the dark for 2 days to allow Agrobacterium to successfully infect the leaf cells. MnERF23 was transiently overexpressed in the leaves of mulberry seedlings and named TOE-MnERF23. The control group used the empty vector pCAMBIA1305 and named it TOE-1305.

[0060] (4) Treatment of natural drought stress and determination of physiological indicators TOE-MnERF23 and TOE-1305 plants were placed under the same environmental conditions and then subjected to natural drought treatment for 10 consecutive days without watering. During the treatment, leaf morphological changes (such as wilting and yellowing) were continuously observed and recorded. After the treatment, the contents of MDA (malondialdehyde), CAT (catalase), POD (peroxidase), and SOD (superoxide dismutase) in the leaves were detected using the micro-method kit from Solarbio Science & Technology Co., Ltd., strictly following the instructions, to clarify the regulatory role of the MnERF23 gene on drought-related physiological indicators of mulberry trees.

[0061] After 10 days of natural drought treatment, the leaves of the TOE-1305 control line showed obvious wilting and yellowing, while the wilting and yellowing of the leaves of the TOE-MnERF23 line were significantly reduced. Figure 11 Physiological index tests showed that, compared with TOE-1305, the MDA content in the leaves of the TOE-MnERF23 strain was significantly reduced, while the activities of three antioxidant enzymes, CAT, POD, and SOD, were significantly increased. Figure 12This indicates that the MnERF23 gene can improve the drought tolerance of mulberry trees by reducing membrane lipid peroxidation and enhancing antioxidant enzyme activity.

[0062] In summary, this invention, through a dual verification strategy of "Arabidopsis heterologous overexpression + mulberry homologous transient overexpression," has for the first time confirmed the positive regulatory function of the mulberry TOE-MnERF23 gene in plant drought resistance, and revealed its molecular mechanism of enhancing drought resistance by activating osmotic stress genes and the antioxidant system. The gene resources and methods provided by this invention offer an important theoretical and practical foundation for molecular breeding of stress-resistant mulberry and other crops.

[0063] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the MnERF23 gene in enhancing plant drought resistance, characterized by, The CDS sequence of the MnERF23 gene is shown in SEQ ID NO:1; the plant is mulberry or Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The amino acid sequence encoded by the MnERF23 gene is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, The biomaterial is any one of the following: 1) An expression cassette containing the MnERF23 gene as described in claim 1; 2) A recombinant vector containing the MnERF23 gene as described in claim 1, or a recombinant vector containing the expression cassette as described in 1); 3) Recombinant bacteria containing the MnERF23 gene as described in claim 1, or recombinant bacteria containing the expression cassette as described in 1), or recombinant bacteria containing the recombinant vector as described in 2); 4) A transgenic plant containing the MnERF23 gene as described in claim 1, or a transgenic plant containing the expression cassette as described in 1), or a transgenic plant containing the recombinant vector as described in 2), or a transgenic plant containing the recombinant bacteria as described in 3).

4. The application of the biomaterial according to claim 3, characterized in that, The application is any one of the following: (a) Enhance the drought resistance of plants; (b) Enhance the activity of antioxidant enzymes in plants under drought stress; (c) Reduce malondialdehyde content in plants under drought stress and mitigate membrane lipid peroxidation damage; (d) Activate the expression of osmotic stress-related genes in plants; (e) Improve the root growth capacity of plants under drought stress; (f) Developing transgenic plants with enhanced drought resistance; (g) Screening of materials for use as molecular markers in plant drought resistance breeding.

5. A PCR primer pair for amplifying the CDS sequence of the MnERF23 gene as described in claim 1, characterized in that, The primer pair is designed based on the sequence shown in SEQ ID NO:1 and is used for homologous recombination cloning. The specific sequences are shown in SEQ ID NO:3 and SEQ ID NO:

4.

6. A method for verifying gene function to enhance drought resistance in mulberry trees, characterized in that, Includes the following steps: (a) Construct a recombinant expression vector containing the MnERF23 drought resistance gene; (b) Transform the recombinant expression vector from step (a) into Agrobacterium to obtain engineered bacteria; (c) Using the engineered bacteria from step (b), transform the model plant Arabidopsis thaliana by Agrobacterium infection to obtain transgenic Arabidopsis thaliana with heterologous overexpression of drought resistance gene, and subject it to drought stress treatment to verify the drought resistance function of drought resistance gene. (d) Using the engineered bacteria from step (b), the drought resistance gene was transiently overexpressed in mulberry leaves via Agrobacterium injection. The mulberry plants with transient overexpression were then subjected to drought stress treatment to further verify the drought resistance function of the drought resistance gene in mulberry trees.

7. The method for verifying gene function to enhance drought resistance in mulberry trees according to claim 6, characterized in that, The drought-resistant gene is the MnERF23 gene.

8. A method for screening or cultivating transgenic plants with enhanced drought resistance, characterized in that, The method includes the steps of transferring the MnERF23 gene as described in claim 1 into a target plant and overexpressing the gene.

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