Application of MsUBP13.3 gene in improving salt stress resistance of plants

Abstract

The application discloses application of MsUBP13.3 genes in improving plant salt stress resistance, and belongs to the technical field of biology. The application verifies that the MsUBP13.3 genes are related to plant salt stress resistance through experiments, and the ability of plant salt stress resistance can be regulated by regulating expression of the MsUBP13.3 genes. The above method provided by the application has important reference and reference value for cultivating and obtaining salt-tolerant plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of the MsUBP13.3 gene in improving plant salt stress resistance. Background Technology

[0002] Alfalfa (Medicago sativa L.), known as the "King of Forage," is an important perennial leguminous forage crop, highly valued for its rich nutrition and excellent taste. Alfalfa is cultivated worldwide, with a total global production area of ​​32 million hectares. 2 China's alfalfa planting area has reached 5.5 million hectares. 2 .

[0003] Existing studies have reported that planting alfalfa on saline-alkali land can not only increase forage yield and alleviate the contradiction of insufficient protein feed, but also improve the utilization rate of saline-alkali land and reduce the cost of saline-alkali land transformation. However, the area of ​​global soil salinization is expanding year by year, and cultivating new alfalfa varieties on harsh saline-alkali land was a challenge in China at that time. Therefore, understanding the plant's tolerance mechanism to high salinity is an important issue in agricultural production.

[0004] UBPs (Ubiquitin-specific proteases) are the largest family of deubiquitinating enzymes in plants. They reverse ubiquitination by hydrolyzing peptide bonds between ubiquitin molecules or between ubiquitin and substrate proteins. Together with E3 ubiquitination ligases, they regulate ubiquitination homeostasis, affecting the stability and activity of target proteins. Based on their conserved domains and sequence similarity, the Arabidopsis UBP family can be divided into 14 subfamilies, with slightly different gene functions in each subfamily. Subfamilies S1, S4, S5, S7, and S11 have been reported to be associated with stress tolerance in Arabidopsis. Currently, only AtUBP16 of the S7 subfamily and AtUBP24 of the S11 subfamily play a regulatory role in salt stress tolerance. UBP12 and UBP13 belong to the S5 subfamily. Although studies in Arabidopsis have shown functional diversity and they have been shown to participate in biotic stress responses, there are no reports of them regulating salt stress tolerance in Arabidopsis, nor are there any reports of their homologs participating in salt tolerance in other plants. Given the functional importance of the ABA signaling pathway in abiotic stresses such as salt and drought, UBP12 / 13 may also be involved in the formation of plant salt tolerance. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the MsUBP13.3 gene in improving the salt stress resistance of plants.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides the MsUBP13.3 protein from alfalfa, as shown in (a) or (b) below:

[0008] (a) A protein with the amino acid sequence SEQ ID No. 2;

[0009] (b) Proteins derived from (a) whose amino acid sequence shown in SEQ ID NO.2 has been modified by substitution and / or deletion and / or addition of one or more amino acid residues and are associated with plant salt stress resistance.

[0010] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned alfalfa MsUBP13.3 protein.

[0011] In some embodiments, the nucleotide sequence of the above-mentioned nucleic acid molecule is shown in SEQ ID NO.1.

[0012] Thirdly, the present invention provides a recombinant vector containing the aforementioned nucleic acid molecules.

[0013] Fourthly, the present invention provides a recombinant bacterium containing the above-mentioned recombinant vector.

[0014] Fifthly, this invention provides the application of alfalfa MsUBP13.3 protein and its encoding gene in improving the salt stress tolerance of plants.

[0015] In some embodiments, the above-mentioned plants include Arabidopsis thaliana and alfalfa.

[0016] In some embodiments, the above applications include regulating lateral root development in plants under salt stress.

[0017] In a sixth aspect, the present invention provides a method for improving the salt stress tolerance of plants, comprising introducing the above-mentioned nucleic acid or a CDS region of the nucleic acid having the base sequence shown in SEQ ID NO.1 into the genome of a target plant to obtain a transgenic plant; the above-mentioned plants include Arabidopsis thaliana and alfalfa.

[0018] In a seventh aspect, the present invention provides a plant breeding method comprising: increasing the content and / or activity of the aforementioned alfalfa MsUBP13.3 protein in plants, thereby enhancing the plants' resistance to salt stress; wherein the aforementioned plants include Arabidopsis thaliana and alfalfa.

[0019] The present invention has the following beneficial effects:

[0020] This invention constructs an overexpression fusion vector and transiently transforms tobacco epidermal cells for subcellular analysis. The results show that MsUBP13.3 is located in the nucleus and cytoplasm. Then, using the flower immersion transformation technique, MsUBP13.3 is transformed into Arabidopsis thaliana. Under salt stress, the root length and number of lateral roots of MsUBP13.3 transgenic Arabidopsis thaliana are significantly better than those of wild type, verifying that overexpression of MsUBP13.3 can improve the salt tolerance of Arabidopsis thaliana. Furthermore, the transient transformation technique of alfalfa hairy roots verifies that the MsUBP13.3 gene plays a positive regulatory role in the response to salt stress. Under salt stress, the root phenotype and relative expression level of MsUBP13.3 in the overexpressing lines are significantly better than those of wild type. This also confirms the correlation between overexpression of MsUBP13.3 and the induction of an antioxidant system to protect cells from oxidative damage. Therefore, it can be shown that the MsUBP13.3 gene can improve the plant's tolerance to salt stress, which has important reference and reference value for cultivating and obtaining salt-tolerant plants. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This refers to the growth phenotype of MsUBP13.3 transformed yeast cells under salt stress in Example 2;

[0023] Figure 2 Subcellular localization of MsUBP13.3 protein in Example 4: EGFP: protein with green fluorescence; Marker: plasma membrane and nuclear localization marker protein; Bright field: bright field; Merge: overlay of GFP, Marker and Bright field.

[0024] Figure 3 To identify the expression level of MsUBP13.3 transgenic Arabidopsis thaliana in Example 5;

[0025] Figure 4The table data shows the root length phenotype of MsUBP13.3 transgenic Arabidopsis thaliana under salt stress in Example 5. A: Growth phenotypes of MsUBP13.3 transgenic Arabidopsis thaliana and Col-0 wild-type Arabidopsis thaliana on control and 125 mM NaCl plates; B: Relative lateral root length statistics of the MsUBP13.3 transgenic lines in A under normal and 125 mM NaCl conditions; C: Lateral root number statistics of the MsUBP13.3 transgenic lines in A under normal and 125 mM NaCl conditions; the error is the standard error of three biologically repeatable occurrences, P < 0.05.

[0026] Figure 5 The protein abundance of MsUBP13.3 transgenic alfalfa hairy roots in Example 6 under normal and salt stress conditions is shown in Figure 6. **P<0.05.

[0027] Figure 6 The table shows the root length phenotype of MsUBP13.3 transgenic alfalfa hairy roots under salt stress in Example 6; A: growth phenotypes of EV, MsUBP13.3, and RNAi transgenic alfalfa under control and 125 mM NaCl salt stress conditions; B: lateral root length statistics of corresponding genotype plants in A under control and 125 mM NaCl salt stress conditions; C: lateral root number statistics of corresponding genotype plants in A under control and 125 mM NaCl salt stress conditions; the error is the standard error of three biologically repeatable occurrences; **P < 0.01;

[0028] Figure 7 To identify the relative expression level of the MsUBP13.3 gene transformed into hairy roots under salt stress in Example 6;

[0029] Figure 8 Figure 6 shows the detection of reactive oxygen species (ROS) in the control and salt-treated EV and transgenic hairy roots. Figure A shows the ROS fluorescence intensity; Figure B shows the root length phenotype of MsUBP13.3 transgenic alfalfa hairy roots under salt treatment and exogenous GSH stress. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] In this invention, the inventors cloned the MsUBP13.3 gene for the first time from alfalfa (Medicago sativa L.) of the genus Alfalfa in the family Leguminosae. Experiments have shown that the MsUBP13.3 protein is related to the salt stress tolerance of plants, providing new ideas and methods for studying salt tolerance-related regulatory genes.

[0032] The amino acid sequence of MsUBP13.3 protein is shown in SEQ ID NO.2, but is not limited to the amino acid sequence shown in SEQ ID NO.2. It can also be a protein derived from SEQ ID NO.2 with one or more amino acid residues substituted and / or deleted and / or added, and associated with plant salt stress resistance.

[0033] Alfalfa MsUBP13.3 protein sequence SEQ ID NO.2:

[0034] MTIMTPAPIDVTSLFLRHSRIVFLVDRKQCDGDFVYVLVISFLFSFWFCSNNS

[0035] RRMKRCLCRTRFRMRIWLRTIIINQWMCLHLQQPLLILFFHSEQACTYFPKG

[0036] NNVDYLSMYLDVADSTSLPYGWSRYAQFSLAVVNQIHNKYTVRKDTQHQF

[0037] NARESDWGFTSFMPLGELYDPSRGYLMNDTLIIEAEVLVRRIVDYWTYDSK

[0038] KETGYVGLKNQGATCYMNSLLQTLYHIPYFRKLQYSDTSVATKELTKSFGW

[0039] DTYDSFLQHDVQELNRVLCEKLEDKMKATVVEGTIQKLFEGHHMNYIECIN

[0040] VDYKSTRKESFYDLQLDVKGCPDVYASFDKYVEVERLEGDNKYHAEQYGL

[0041] QDAKKGVLFIDFPPVLQLQLKRFEYDFMRDTMVKINDRYEFPLQLDLDRDN

[0042] GNIYHLMLIGMSAIFTHFIADKEKVICNVDEKDIAEHLRERLKKEQEEKEHK

[0043] KKEAEAHLYTIIKVARDEDLGEQIGKDIYFDLVDHDKVRSFRVQKQTPFNVF

[0044] KEEVAKEFGIPVQFQRFWLWAKRQNHTYRPNRPLTQIEEAQSVGQLREISNK

[0045] VHNAELKLFLEVERGPDLCPIAPPEKTKDDILLFFKLYDPEKEELRYVGRLFV

[0046] NNTGKPSEILARLNKMAGYDPEEEIGLYEEIKFEPNVMCEPIDKKLTFRASQL

[0047] EDGDIICFQKAPATDNEEHIRYPDVPSYLEYVHNRQVVHFRSLDKPKEDDFC

[0048] LEMSRLFTYDDVVERVAEQLGLDDPSKIRLTPHNCYSQQPKPQPIKYRGVEH

[0049] LSDMLVHYNQTSDILYYEVLDIPLPELQGLKTLKVAFHHATKDEAVIHTIRLP

[0050] KQSTVGDVLEDLKKKVELSRPDTELRLLEVFYHKIYKVFPPNEKIENINDQY

[0051] WTLRAEEIPEEEKNLGPHDRLIHVYHFTKDTTQNQMQIQNFGEPFFLVIHECE

[0052] TLAEIRLRIQKKLQVPDDEFVKWKFAFFSLGRPEYLEDSEVVSNRFQRRDVY

[0053] GAWEQYLGLEHTDNAPKRSYAANQNRHTFEKPVKIYN

[0054] In addition, to facilitate the purification or detection of the above proteins, a tag protein can be attached to the amino or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.2.

[0055] The nucleotide sequence of the MsUBP13.3 gene in this invention is shown in SEQ ID NO.1.

[0056]

[0057] Those skilled in the art can readily mutate the nucleotide sequence of the protein MsUBP13.3 encoding the present invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 90% or more of the nucleotide sequence identity with the protein MsUBP13.3 isolated in the present invention, provided they encode and function as protein MsUBP13.3, are derived from and equivalent to the nucleotide sequence of the present invention.

[0058] The present invention also provides a recombinant vector containing the above-mentioned nucleic acid molecules.

[0059] Specifically, the recombinant vectors mentioned above include the following vectors: target gene cloning vectors (for preserving and cloning target genes, such as E. coli plasmids), intermediate cloning vectors (constructed by inserting T-DNA fragments into E. coli plasmids along with target genes, marker genes, etc., and serving as the basic plasmids for constructing intermediate expression vectors), intermediate expression vectors (intermediate vectors containing plant-specific promoters, used as plasmids for constructing transformation vectors), and / or plant gene transformation vectors (vectors used for introducing target genes into plant cells).

[0060] The present invention also provides a recombinant bacterium containing the above-mentioned recombinant vector.

[0061] The present invention also provides a method for preparing the above-mentioned MsUBP13.3 protein, comprising: encoding the above-mentioned nucleic acid and preparing it through biological expression and / or artificial synthesis.

[0062] The present invention also provides the application of the above-mentioned salt-tolerant protein and its encoding gene in improving the salt-tolerant ability of plants.

[0063] Specifically, methods to improve the salt stress resistance of plants include: introducing the above-mentioned nucleic acid or the CDS region of the nucleic acid containing the base sequence shown in SEQ ID NO.1 into the genome of the target plant to obtain transgenic plants; and artificially cultivating the transgenic plants to allow them to grow naturally.

[0064] Furthermore, the aforementioned nucleic acid or CDS regions are introduced into plants using a recombinant expression vector.

[0065] The aforementioned plants include Arabidopsis thaliana and alfalfa.

[0066] The protein and its encoding gene provided by this invention can be used to genetically optimize plant varieties, which is beneficial to the growth and development of plants under salt stress, especially the normal growth of lateral roots. Therefore, plants overexpressing the MsUBP13.3 gene can be constructed using transgenic technology and applied to the breeding of salt-tolerant plants.

[0067] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0068] The experiments of this invention used seeds of Nicotiana benthamiana, alfalfa 'Zhongmu No. 1', and Arabidopsis thaliana (Col-0), all of which were stored in the National Key Laboratory of Grass Seed Innovation and Grassland Agro-ecosystems at Lanzhou University.

[0069] The Agrobacterium GV3101 and Escherichia coli DH5α competent cells used were purchased from Beijing TransGenBiotech Co., Ltd. The Agrobacterium rhizogenes Ar.Qual strain, expression vector plasmid pBI121::EGFP vector and RNAi vector pANDA35HK were all preserved by the National Key Laboratory of Grass Seed Innovation and Grassland Agro-ecosystem at Lanzhou University. The Saccharomyces cerevisiae INVSC1 strain was purchased from Yikeshu Company.

[0070] Example 1

[0071] This example demonstrates gene cloning and expression vector construction, as detailed below:

[0072] (1) Primer design and candidate gene cloning

[0073] CDS sequence data of candidate genes were extracted from the alfalfa genome database, and gene primers were designed using Snapgene software. Primers were designed with BamHI and KpnI restriction sites for ligation into the pYES2 vector, and with BamHI and SacI restriction sites for ligation into the pBI121::EGFP vector. A double digestion method was used to design forward and reverse primers. Primer sequences are shown in Table 1. All primers were synthesized by Xi'an Qingke Biotechnology Co., Ltd. MCLAB1-5 was used... TM The reaction system was amplified using 2×High-FidelityMasterMix (Qingke, Xi'an). Table 2 shows the reaction system.

[0074] Table 1 Gene Primer Information

[0075]

[0076] Table 2 Gene Cloning PCR Reaction System

[0077]

[0078] Table 3 PCR reaction procedure

[0079]

[0080]

[0081] The PCR reaction procedure is shown in Table 3. A 1.5% agarose gel was prepared, and the electrophoresis detection current was 400 mA, the voltage was 135 V, and the electrophoresis time was 20 min. After scanning with a WD-9413B gel imaging analysis system (Liuyi, Beijing), the target DNA fragment was excised from the gel and recovered using a DNA gel recovery kit (TransGen, Beijing). The fragment was ligated with the pEASY-BluntSimpleCloningKit vector at 37℃ for 20 min, and the ligation product was transformed into *E. coli* DH5α. PCR detection of the bacterial culture was performed using the M13-F / M13-R universal primers, followed by sequencing confirmation.

[0082] (2) Construction of yeast expression vector

[0083] The pYES2 yeast expression vector and the target fragment were digested with enzymes according to the enzyme digestion systems in Tables 4 and 5, respectively. After vortexing and mixing, the reaction mixtures were incubated in a water bath for 3 hours, followed by gel electrophoresis for detection. The target band was then excised and recovered from the gel. Using T4 DNA ligase (M0202, New England Biolabs Beijing Co., Ltd.), the recovered target fragment was ligated to the pYES2 yeast expression vector fragment according to the system in Table 6. The ligation was then performed on *E. coli*, and after overnight culture to produce single colonies, PCR detection was performed. The correctly sequenced single colonies were then stored and plasmids were extracted for later use.

[0084] Table 4. Double enzyme digestion reaction system for expression vector

[0085]

[0086] Table 5. Double enzyme digestion reaction system for the target fragment

[0087]

[0088]

[0089] Table 6 Connection Reaction System

[0090]

[0091] Based on the empty vector map and gene sequence, two restriction enzyme sites, BamHI and KpnI, were selected to amplify the CDS sequence of the MsUBP13.3 gene. The pYES2 vector and the target gene fragment were then ligated using double enzyme digestion to construct the expression vector. After transformation into *E. coli*, colony PCR was performed for identification. The primers for colony PCR identification were T7-F / MsUBP13.3-R1 (chimeric primers, not the full-length CDS sequence primers). Correctly identified positive single clones were sent for sequencing after shaking, and plasmids were extracted and preserved from the successfully sequenced single clones.

[0092] Example 2

[0093] This example demonstrates the heterologous expression and salt tolerance function of yeast, as detailed below:

[0094] (1) Transform the constructed vector plasmid and the pYES2 empty vector plasmid into competent cells of INVSC1 yeast.

[0095] (2) To identify the correct pYES2 empty vector and positive yeast strain containing the target gene, single clones were shaken in 10 mL of SC-Ura glucose liquid medium and incubated on a shaker at 28°C and 200 rpm for 24 h.

[0096] (3) Determination of bacterial culture OD 600 The bacterial culture was then diluted to 0.4 with 10 mL of SC-Ura galactose-induced expression medium and cultured at 28°C and 200 rpm for 36 h to induce exogenous gene expression.

[0097] (4) Adjust the bacterial culture to OD 600 =1.0, take 500 μL, centrifuge briefly at 12000 rpm, and then discard the supernatant.

[0098] (5) After centrifugation, the yeast cells were resuspended in equal volumes of 4M NaCl, 4M sorbitol, 15mM H2O2 and sterile water (control), and cultured in a shaker at 28℃ and 200rpm for 72h, 120h, 120h and 96h respectively. The culture time of the control was the same as that of the treatment.

[0099] (6) Dilute the above-treated bacterial solutions according to dilution gradients of 1, 10, and 10. -1 10 -2 10 -3 10 -4 10 -5 These six gradients were diluted with sterile water in 2 mL tubes, and 5 μL of bacterial suspension was taken and spotted onto SC-Ura glucose solid medium. The experiment was conducted with three technical replicates and three biological replicates. The tubes were sealed and incubated upside down at 28°C for 3-4 days. During the incubation process, real-time photographic records were taken of the colony growth.

[0100] The results showed that in the control group, there was no significant difference in growth between transgenic yeast cells and empty yeast cells. However, after stress with 4M NaCl, the transgenic yeast cells grew significantly better than the empty yeast cells. Figure 1 This indicates that MsUBP13.3 enhances the yeast strain's tolerance to salt stress through its transgenic expression, preliminarily demonstrating that the MsUBP13.3 salt-drought tolerant candidate gene plays a positive regulatory role in yeast cells' response to salt stress.

[0101] Example 3

[0102] This example demonstrates the cloning and overexpression vector construction of MsUBP13.3, as detailed below:

[0103] The overexpression vector pBI121::EGFP, preserved in our laboratory, was selected. Based on the vector map and gene sequence, Sal I and Spe I restriction enzyme sites were chosen, and the vector was constructed using a double digestion method. Primers were designed using Snapgene software (Table 1) and synthesized by Xi'an Qingke Biotechnology Co., Ltd. MCLAB 1-5 was used... TM The target fragment was amplified using 2× High-FidelityMasterMix amplification enzyme (Qingke, Xi'an), following the amplification system described in Example 1. DNA products from the gel were recovered using a gel extraction kit (TransGen, Beijing). The pEASY-Blunt Simple Cloning Kit vector was ligated, and the ligation product was transformed into *E. coli* DH5α. Single clones were picked and tested using M13-F and MsUBP13.3-R1 (non-full-length primer sequences) before being sent to the company for sequencing confirmation. After successful sequencing, the plasmid was extracted and subjected to double enzyme digestion with the pBI121::EGFP expression vector plasmid. The reaction system was as described in Example 1. After the enzyme digestion was completed, the fragment was confirmed by electrophoresis. The digested target fragment was ligated with the digested fragment of the pBI121::EGFP expression vector using T4 DNA ligase (M0202, New England Biolabs Beijing Co., Ltd.). Subsequently, the fragment was transformed into competent E. coli cells and cultured overnight at 37°C. After colony PCR detection, the colony was sent for sequencing after successful detection. The single clone with correct sequencing was stored and the plasmid was extracted for later use.

[0104] (1) Obtaining the target fragment

[0105] Gateway cloning technology was used to design primers, and the constructed overexpression vector plasmid was used as a template to utilize MCLAB1-5. TM PCR amplification was performed using 2×High-FidelityMasterMix high-fidelity enzyme, and the PCR products were recovered using a gel extraction kit for later use.

[0106] Table 7 Primer Information

[0107]

[0108] (2) Target fragment connection entry carrier

[0109] The connection reaction system is as follows:

[0110]

[0111] React at 25°C overnight.

[0112] The ligation product was transformed into Escherichia coli DH5α, plated, and incubated upside down in a 37°C incubator for 16 h. Positive single clones were detected using universal primers M13F and M13R and sent for sequencing. Single clones with correct sequencing were cultured, and plasmids were extracted and stored at -20°C for later use.

[0113] The fusion expression vector pBI121-MsUBP13.3:EGFP was successfully constructed. The gene cloning primers were MsUBP13.3-F / MsUBP13.3-R, and the colony PCR detection primers were the chimeric primers 35S-F / MsUBP13.3-R1 (non-CDS sequence full-length primers).

[0114] (3) Constructing RNAi recombinant vectors via LR reaction

[0115] First, the target vector pANDA35HK was transformed into E. coli DH5α for propagation. After verification, plasmid extraction was performed. The concentrations of the target vector and the entry vector Entryclone were determined to ensure a concentration of 100 ng / μL. Then, an LR reaction was performed, with the following system:

[0116] 2×GatewayLRCloneEnzymemix 5μL

[0117] EntryClone (100 ng / μL) 3 μL

[0118] Target vector (0.5 μg / μL) 2 μL

[0119] After thoroughly mixing the above reaction system, centrifuge rapidly and incubate overnight at 25°C. Add 1 μL of 10× Proteinase K Solution, vortex to mix, and incubate at 37°C for 20 min. Then transform it into Escherichia coli DH5α. After transformation, take 200 μL of culture and spread it on LB solid medium containing kanamycin (Kan) antibiotic at a final concentration of 50 mg / L. Invert the medium and incubate at 37°C for 16 hours. Extract plasmids from successfully colonized single clones and store them at -20°C for later use.

[0120] The RNAi vector was constructed using the Gateway method. The attB-PCR product of MsUBP13.3 was obtained by PCR and ligated into the entry vector Entryclone. After positive clone identification, the sequenced entry clone was subjected to LR reaction with the target expression vector pANDA35HK. After single-clone detection, plasmid was extracted from the positive bacterial culture to obtain the RNAi recombinant vector of the MsUBP13.3 gene (RNAi-MsUBP13.3).

[0121] (4) Transformation and identification of Agrobacterium

[0122] 1) Preparation of Agrobacterium tumefaciens Ar.Qual competent cells

[0123] 2) Electrocution method conversion

[0124] 3) Thermal shock conversion

[0125] The specific steps of the above method are referenced from the literature (Identification of ERF transcription factor in Osmanthus fragrans and its functional analysis in response to drought stress, Wei Na, 2024).

[0126] Example 4

[0127] This embodiment describes subcellular localization in tobacco, and the specific steps are as follows:

[0128] (1) The Agrobacterium strain containing plasmids that was successfully detected in Example 3 was shaken in 10 mL of LB liquid medium (containing 50 mg / L Kan and 50 mg / L rifampin) and cultured overnight on a shaker at 28°C and 200 rpm.

[0129] (2) OD in bacterial culture was measured using an ultraviolet spectrophotometer. 600 The value should be between 0.5 and 1. After centrifuging at 2400g for 15 minutes, collect the bacterial cells and remove the supernatant.

[0130] (3) To increase the OD of the new bacterial culture 600 When the pH reached the range of 0.4-0.6, we resuspended the bacterial cells in a resuspension solution and prepared the culture using MS liquid medium. For each 100 mL volume, we added 1 mL of 1M MgCl₂·6H₂O (final concentration 10 mM), 1 mL of 1M 2-(N-morpholino)ethanesulfonic acid (MES) (final concentration 10 mM), and 10 μL of 1M acetylsyringone (AS) (final concentration 100 μM), and adjusted the pH to 5.8. After storing at room temperature for 2-3 hours, infection could begin.

[0131] (5) Before the tobacco seedlings enter the soaking stage, they should be sprayed with enough water and cultivated under appropriate light conditions to ensure that the stomata of the tobacco leaves can be fully opened.

[0132] (7) Using a syringe with the needle removed, draw 1 mL of the suspension and slowly inject it into the infected area on the underside of the leaf.

[0133] (8) After spraying water on the soaked tobacco leaves, they were dark-treated for 12 hours and cultured overnight before being moved to normal light conditions.

[0134] (9) Two to three days after injection, we collected leaf samples from the stained area using a perforator and prepared slides. We then took photographs using a confocal microscope after adjusting to the optimal field of view.

[0135] The results are as follows Figure 2 As shown, the pBI121::MsUBP13.3::EGFP fusion protein was observed to be localized on both the cell membrane and the nucleus. This indicates that the MsUBP13.3 protein may perform its function in both the cell membrane and the nucleus.

[0136] Example 5

[0137] This embodiment is

[0138] (1) Arabidopsis genetic transformation: transgenic Arabidopsis seedlings were obtained by the flower dipping method. The main steps are referred to (Identification of ERF transcription factor in Osmanthus fragrans and its function in response to drought stress, Wei Na, 2024).

[0139] (2) Identification and expression level analysis of transgenic Arabidopsis thaliana seedlings

[0140] DNA was extracted from 10 transgenic Arabidopsis thaliana lines of generation T1 using a plant genomic DNA extraction kit (Tiangen, Beijing). PCR was performed using chimeric primers 35S-F / MsUBP13.3-R1 to verify positive plants. After identification as positive plants, RNA was extracted using the RNAEasyFast Plant Tissue RNA Rapid Extraction Kit (Tiangen, Beijing). Next, reverse transcription was performed using the FastKing cDNA First-Strand Synthesis Kit (Tiangen, Beijing) to obtain cDNA. The relative expression level of MsUBP13.3 in transgenic Arabidopsis thaliana was then determined using qRT-PCR technology. The specific methods are shown in the tables below. The qRT-PCR reaction system and procedure are shown in Tables 8 and 9. The reaction was performed for 38 cycles with 3 technical replicates. -ΔΔCT The computational method processes the data to obtain the relative expression levels of genes.

[0141] Table 8 qRT-PCR reaction system

[0142]

[0143] Table 9 qRT-PCR reaction procedure

[0144]

[0145]

[0146] Positive Arabidopsis thaliana transgenic with the MsUBP13.3 gene were identified by PCR using genomic DNA extraction. The kanamycin resistance gene detection primers NPT-F / NPT-R and the promoter / target gene chimeric primer 35S-F / MsUBP13.3-R1 were used to detect positive plants, resulting in nine overexpressing positive Arabidopsis thaliana lines. Figure 3 RNA was further extracted from these nine transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana, then reverse transcribed into cDNA, and the expression level of MsUBP13.3 was detected using qRT-PCR. The results showed that the expression level of MsUBP13.3 in the nine transgenic lines was significantly higher than that in the Col-0 line, with the highest expression levels in the three overexpression lines OE1, OE4, and OE8. Figure 3 ).

[0147] (3) Evaluation of salt tolerance in transgenic Arabidopsis thaliana

[0148] Seeds from three representative Arabidopsis lines (OE1, OE4, and OE8) with the highest expression levels of the MsUBP13.3 gene, as well as the wild-type Col-0, were sterilized and then evenly spread on 1 / 2 MS medium and 1 / 2 MS medium containing 125 mM NaCl using a 10 μL pipette tip. After sealing, the seeds were treated at 4°C for 3 days, then transferred to a light incubator at 22°C and 60% relative humidity for 7 days. Three transgenic lines with consistent root length and the wild-type Col-0 line were selected and transferred to 1 / 2 MS medium and 1 / 2 MS medium containing 125 mmol / L NaCl, respectively. The Arabidopsis-containing plates were placed vertically in the light incubator, with the growth environment set at 22°C, 16 hours of light, and 8 hours of darkness. Root growth of the Arabidopsis seedlings was observed continuously. After approximately one week, growth phenotypic photographs were taken, and root length was measured. The results were averaged, and the standard deviation was calculated.

[0149] The results are as follows Figure 4 As shown, on 1 / 2 MS and 125 mM NaCl plates, there were significant differences in root length and lateral root number between wild-type and transgenic lines, with transgenic lines showing significantly higher root length and lateral root number than wild-type lines.

[0150] Example 6

[0151] This example verifies the salt tolerance of alfalfa hairy roots, as detailed below:

[0152] 1. Transient transformation of alfalfa hairy roots

[0153] The function of the MsUBP13.3 gene in alfalfa was validated using the established hairy root transformation system in the laboratory. The specific steps are as follows:

[0154] (1) Seed cleaning: Select plump alfalfa 'Zhongmu No. 1' seeds, add concentrated sulfuric acid and shake for 3 minutes, then remove the concentrated sulfuric acid. Wash the seeds 5 times with ddH2O in a clean bench, then treat with 6% sodium hypochlorite solution and shake for 5 minutes, then wash with ddH2O water until colorless. Spread the seeds on FA plates and incubate in a 25℃ light incubator for two weeks.

[0155] (2) Infection: Two weeks later, when the seedlings have grown their third cotyledon, gently grasp the alfalfa seedlings with sterilized tweezers and cut them at the root-stem junction with a scalpel. Gently grasp the seedlings with cut roots with tweezers and dip them in a small amount of Agrobacterium rhizogenes transgenic EV, MsUBP13.3 and RNAi. Then spread them on water agar plates, placing about 12 seedlings on each plate. Seal the plates and place them vertically in a 22°C incubator until roots develop.

[0156] (3) Salt stress: Infected plants were divided into two groups. The treatment group was cultured on water agar medium supplemented with 100 mM NaCl, while the control group was cultured on water agar medium. After two weeks, the lateral root phenotype was observed and photographed. Then, samples were taken from both the treatment and control groups. The expression level of transgenic hairy roots MsUBP13.3 was determined. The reaction system and procedure were the same as in Example 5.

[0157] The constructed pBI121::MsUBP13.3::EGFP expression vector and RNAi vector were first transformed into *Agrobacterium rhizogenes*, and then transiently expressed in the hairy roots of alfalfa using alfalfa hairy root transformation technology. The control was an empty vector pBI121::EGFP control. After infecting *Agrobacterium rhizogenes* with alfalfa, the roots were placed vertically in a light-controlled culture room on water agar medium. After two weeks of root emergence, a small number of hairy roots infected with the transformed *Agrobacterium rhizogenes* vectors pBI121::MsUBP13.3::EGFP and RNAi vectors were harvested for positive root identification.

[0158] To further investigate the function of MsUBP13.3 in lateral root development, transgenic lines that had developed roots were transferred to control and salt-stressed media. After one week, their phenotypes were observed and photographed. The results showed that, compared to the control treatment, the number of lateral roots in the MsUBP13.3-overexpressing transgenic hairy roots under salt stress was significantly greater than that in EV and RNAi (…). Figure 6This indicates that overexpression of MsUBP13.3 positively regulates lateral root development. We measured the relative expression levels of MsUBP13.3 in the hairy roots of MsUBP13.3 transgenic roots under control and salt stress treatments. We found that the relative expression levels of MsUBP13.3 in transgenic hairy roots under both normal and salt stress conditions were significantly higher than those under wild type and RNAi, and the relative expression level of MsUBP13.3 under salt stress was higher than that under normal conditions. Figure 7 ).

[0159] 2. Reactive oxygen species staining of transgenic alfalfa hairy roots

[0160] To detect ROS levels in the hairy roots of transgenic alfalfa, the dye OxyBURST green H2DCFDA was used. Hairy roots of untreated and control alfalfa seedlings treated with 100 mM NaCl for 5 hours were analyzed. First, lateral roots from salt-treated and control seedlings were collected separately in 1.5 mL centrifuge tubes. H2DCFDA was first dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM, then diluted to a final concentration of 50 mM with sample buffer (10 mM Tris-HCl, 50 mM KCl, pH 7.2). The samples were then immersed in 0.01% Tween 20 under vacuum for 30 minutes, rinsed twice with distilled water, and then washed with washing buffer (10 mM Tris-HCl, pH 7.2 and 50 mM KCl). Subsequently, the samples were incubated with 50 mM H2DCFDA staining solution in the dark at room temperature for 10 minutes, and excess dye was removed twice with distilled water. All samples were examined using a confocal microscope (Olympus FV3000, Japan) with excitation at 488 nm and emission at 530 nm. To compare fluorescence intensity, all parameter axes from different experimental conditions were fixed and analyzed under a confocal microscope using the same settings.

[0161] This experiment examined the reactive oxygen species (ROS) levels in the hairy roots of MsUBP13.3 transgenic alfalfa, EVs, and RNAi transgenic alfalfa, using H2DCFDA (cytosol redox state) staining after salt treatment. The results showed that under normal conditions, the ROS content in alfalfa hairy roots was low and showed little difference; however, after salt stress treatment, compared with the wild-type control, fewer ROS were detected in the hairy roots overexpressing MsUBP13.3, while the ROS content in the hairy roots of RNAi transgenic alfalfa was significantly increased. Figure 8 A) These results indicate that overexpression of MsUBP13.3 may have the function of regulating intracellular ROS levels and can protect plant roots from salt stress damage.

[0162] This experiment, using reactive oxygen species (ROS) staining, revealed that overexpression of MsUBP13.3 reduced ROS accumulation in roots, indicating a potential role for MsUBP13.3 in regulating ROS homeostasis. Phenotypic observation on a medium treated with 125 mM salt and exogenously applied reduced glutathione (GSH) showed that lateral roots lacking MsUBP13.3 function recovered growth, with no significant difference in phenotype compared to the control. Figure 8 (B) This indicates that under salt stress, ROS are produced and accumulate, leading to oxidative stress in plants and reducing their salt tolerance, while applying GSH levels helps improve their salt tolerance. The results suggest that overexpression of MsUBP13.3 may protect cells from oxidative damage by regulating intracellular ROS homeostasis, and highlight the importance of maintaining intracellular ROS homeostasis for lateral root growth and development under salt stress.

Claims

1. Alfalfa MsUBP13.3 protein, characterized in that, It is a protein with the amino acid sequence SEQ ID No.

2.

2. A nucleic acid molecule, characterized in that, It encodes the MsUBP13.3 protein of alfalfa as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

1.

4. A recombinant vector, characterized in that, It contains the nucleic acid molecule as described in claim 2 or 3.

5. A recombinant bacterium, characterized in that, It contains the recombinant vector as described in claim 4.

6. The application of the alfalfa MsUBP13.3 protein as described in claim 1, or the nucleic acid molecule as described in claim 2 or 3, in improving the salt stress tolerance of plants; The plants mentioned are Arabidopsis thaliana and alfalfa.

7. The application according to claim 6, characterized in that, The applications include regulating lateral root development in plants under salt stress.

8. A method for improving the salt stress resistance of plants, characterized in that, This includes introducing the nucleic acid molecule described in claim 2 or 3 into the genome of a target plant to obtain a transgenic plant; The plants mentioned are Arabidopsis thaliana and alfalfa.

9. A plant breeding method, characterized in that, include: Introducing the nucleic acid molecule described in claim 2 or 3 into the genome of the target plant increases the content and / or activity of the alfalfa MsUBP13.3 protein described in claim 1 in the plant, thereby enhancing the plant's resistance to salt stress. The plants mentioned are Arabidopsis thaliana and alfalfa.

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