Application of MsUBP13.3 gene in improvement of plant salt stress resistance

Abstract

The invention discloses application of an MsUBP13.3 gene in improving salt stress resistance of plants, and belongs to the technical field of biology. Experiments prove that the MsUBP13.3 gene is related to the salt tolerance of the plant, and the salt stress resistance of the plant can be regulated by regulating the expression of the MsUBP13.3 gene. The method provided by the invention has important reference value for cultivating and obtaining salt-tolerant plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of the MsUBP13.3 gene in improving plant salt stress resistance. Background Art

[0002] Alfalfa (Medicago sativa L.) is known as the "king of forage grasses" and is an important perennial leguminous forage grass. It is highly regarded for its rich nutrition and good taste. Alfalfa is grown all over the world, with a total global production area of ​​32 million hm2. 2 , China's alfalfa planting area reached 5.5 million hm2 2 .

[0003] Studies have reported that planting alfalfa on saline-alkali land can not only increase forage production 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 global soil salinization area is expanding year by year, and it was a difficult problem in China at that time to cultivate new alfalfa varieties on saline-alkali land with harsh environment. Therefore, analyzing the tolerance mechanism of plants to high salinity is an important issue in agricultural production.

[0004] UBP (Ubiquitin-specific protease) is the largest family of deubiquitinases in plants. It can reverse ubiquitination modification by hydrolyzing peptide bonds between ubiquitin molecules or between ubiquitin and substrate proteins, and jointly regulate ubiquitination modification homeostasis with E3 ubiquitin ligase, affecting the stability and activity of target proteins. According to their conserved domains and sequence similarities, the UBPs family of Arabidopsis can be divided into 14 subfamilies, and the gene functions of different subfamilies are slightly different. It has been reported that subfamilies S1, S4, S5, S7 and S11 are related to stress tolerance in Arabidopsis. At present, only AtUBP16 of the S7 subfamily and AtUBP24 of the S11 subfamily play a regulatory role in the process of salt stress tolerance. UBP12 and UBP13 belong to the S5 subfamily. Although studies in Arabidopsis have shown that their functions are diverse and studies have shown that they are involved in biological stress responses, there are no reports that they regulate Arabidopsis salt stress tolerance, and there are no reports that their homologous genes are involved 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 the present invention is to provide application of MsUBP13.3 gene in improving plant salt stress resistance.

[0006] The present invention is achieved in that:

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

[0008] (a) a protein having an amino acid sequence of SEQ ID No. 2;

[0009] (b) A protein derived from (a) with one or more amino acid residues substituted and / or deleted and / or added to the amino acid sequence shown in SEQ ID NO. 2 and related to plant resistance to salt stress.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned alfalfa MsUBP13.3 protein.

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

[0012] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0013] In a fourth aspect, the present invention provides a recombinant bacterium comprising the above-mentioned recombinant vector.

[0014] In a fifth aspect, the present invention provides the use of alfalfa MsUBP13.3 protein and its encoding gene in improving the salt stress tolerance of plants.

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

[0016] In some embodiments, the above-mentioned use includes regulating lateral root development of plants under salt stress.

[0017] In a sixth aspect, the present invention provides a method for improving the salt stress tolerance of plants, which comprises introducing the above-mentioned nucleic acid or the 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 above-mentioned alfalfa MsUBP13.3 protein in a plant, thereby enhancing the plant's resistance to salt stress; the above-mentioned plants include Arabidopsis thaliana and alfalfa.

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

[0020] The invention constructs an overexpression fusion vector, and transiently transforms tobacco epidermal cells for subcellular analysis, and the results show that MsUBP13.3 is located in the cell nucleus and cytoplasm; then, the floral immersion transformation technology is used to transform MsUBP13.3 into Arabidopsis thaliana, and the root length and the number of lateral roots of MsUBP13.3 transgenic Arabidopsis thaliana under salt stress are significantly better than those of the wild type, which verifies that overexpression of MsUBP13.3 can improve the salt tolerance of Arabidopsis thaliana; and then, the instantaneous transformation technology of alfalfa hairy roots is used to verify that the MsUBP13.3 gene plays a positive regulatory role in the process of responding to salt stress; under salt stress, the root phenotype and the relative expression level of the MsUBP13.3 gene of the overexpression strain overexpressing MsUBP13.3 are significantly better than those of the wild type; and the correlation of overexpression of MsUBP13.3 inducing an antioxidant system to protect cells from oxidative damage is also confirmed. Therefore, it can be shown that the MsUBP13.3 gene can improve the tolerance of plants to salt stress, which has important reference and reference value for breeding and obtaining salt-tolerant plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

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

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

[0024] Figure 3 It is the identification of the expression level of MsUBP13.3 transgenic Arabidopsis in Example 5;

[0025] Figure 4Figure 5 shows the root length phenotype of MsUBP13.3 transgenic Arabidopsis thaliana under salt stress on the plate. A: Growth phenotype of MsUBP13.3 transgenic Arabidopsis thaliana and Col-0 wild-type Arabidopsis thaliana on the control and 125 mM NaCl plates; B is the relative lateral root length statistics of the growth phenotype of MsUBP13.3 transgenic lines in A under normal conditions and 125 mM NaCl conditions; C is the lateral root number statistics of the growth phenotype of MsUBP13.3 transgenic lines in A under normal conditions and 125 mM NaCl conditions; the error is the standard error of three biological replicates, P<0.05;

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

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

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

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

[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0031] In the present invention, the inventors cloned the MsUBP13.3 gene from Medicago sativa L. of the Leguminosae family for the first time. 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] Among them, the amino acid sequence of MsUBP13.3 protein is as shown in SEQ ID NO.2, but is not limited to the amino acid sequence as shown in SEQ ID NO.2, and can also be a protein derived from SEQ ID NO.2 that is related to plant salt stress resistance and has one or more amino acid residues substituted and / or deleted and / or added as shown in SEQ ID NO.2.

[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, in order to facilitate the purification or detection of the above protein, a tag protein may be connected to the amino terminus 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 the present invention is shown in SEQ ID NO.1.

[0056]

[0057] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein MsUBP13.3 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 90% or more identity with the nucleotide sequence of the protein MsUBP13.3 isolated from the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein MsUBP13.3 and have the function of the protein MsUBP13.3.

[0058] The present invention also provides a recombinant vector containing the nucleic acid molecule.

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

[0060] The present invention also provides a recombinant bacterium, which contains the recombinant vector.

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

[0062] The invention also provides the application of the above-mentioned protein with salt stress resistance and its coding gene in improving the salt stress resistance of plants.

[0063] Specifically, the method for improving the salt stress resistance of plants includes: introducing the above nucleic acid or the CDS region of the nucleic acid having the base sequence shown in SEQ ID NO.1 into the target plant genome to obtain a transgenic plant; and artificially cultivating the transgenic plant to allow it to grow naturally.

[0064] Furthermore, the above nucleic acid or CDS region is introduced into the plant via a recombinant expression vector.

[0065] Such plants include Arabidopsis thaliana and alfalfa.

[0066] The protein and its encoding gene provided by the present invention can be used to optimize plant varieties from a genetic perspective, which is beneficial to the growth and development of plants under salt stress conditions, especially the normal growth of lateral roots. Therefore, plants overexpressing the MsUBP13.3 gene can be constructed through transgenic technology and can be used in the breeding of salt-tolerant plants.

[0067] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0068] In the experiment of the present invention, Nicotiana benthamiana, alfalfa 'Zhongmu 1' seeds and Columbia wild-type Arabidopsis thaliana (Col-0) were used, all of which were stored in the National Key Laboratory of Grass Seed Innovation and Grassland Agricultural Ecosystem of Lanzhou University.

[0069] The Agrobacterium GV3101 and Escherichia coli DH5α used were purchased from Beijing TransGen Biotech Co., Ltd. The rhizogenes Agrobacterium 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 Agricultural Ecosystems of Lanzhou University. The Saccharomyces cerevisiae INVSc1 strain was purchased from Yikeshu Company.

[0070] Example 1

[0071] This example is about gene cloning and expression vector construction, which is as follows:

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

[0073] The CDS sequence data of candidate genes were extracted from the genome database of alfalfa, and the gene primers were designed using Snapgene software. When connecting to the pYES2 vector, the primers designed selected the BamHI and KpnI restriction sites, and when connecting to the pBI121::EGFP vector, the primers designed selected the BamHI and SacI restriction sites. The forward and reverse primers were designed using the double enzyme digestion method. The primer sequences are shown in Table 1. The primers used were synthesized by Xi'an Qingke Biotechnology Co., Ltd. Using MCLAB1-5 TM 2× High-Fidelity Master Mix (Qingke, Xi'an) was used for amplification, and 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 program

[0079]

[0080]

[0081] The PCR reaction procedure is shown in Table 3. Prepare an agarose gel with a concentration of 1.5%, an electrophoresis detection current of 400 mA, a voltage of 135 V, and an electrophoresis time of 20 min. Use the WD-9413B gel imaging analysis system (Six One, Beijing) to scan and cut the target DNA fragment, and use a DNA gel recovery kit (Quanshijin, Beijing) to recover it. Connect with the pEASY-BluntSimpleCloningKit vector at 37°C for 20 min, and transform the ligation product into Escherichia coli DH5α. Use M13-F / M13-R universal primers to perform PCR detection on the bacterial solution, and then confirm by sequencing.

[0082] (2) Construction of yeast expression vector

[0083] The pYES2 yeast expression vector and the target fragment were digested according to the enzyme digestion systems in Table 4 and Table 5, respectively. After the reaction system was shaken and mixed, the enzyme digestion was carried out in a water bath for 3 hours, and then the gel was run for detection, and the target band was cut and recovered. Using T4 DNA ligase (M0202, New England Biolabs Beijing Co., Ltd.), the target fragment after gel recovery was connected to the pYES2 yeast expression vector fragment according to the system in Table 6. Then Escherichia coli was transformed, and a single clone was grown overnight for PCR detection, and then the single clone with correct sequencing was stored and the plasmid was extracted for standby use.

[0084] Table 4 Expression vector double restriction enzyme digestion reaction system

[0085]

[0086] Table 5 Target fragment double restriction enzyme digestion reaction system

[0087]

[0088]

[0089] Table 6 Ligation reaction system

[0090]

[0091] According to the empty vector map and gene sequence, two restriction sites, BamHI and KpnI, were selected to amplify the CDS sequence of the MsUBP13.3 gene, and the pYES2 vector and the target gene fragment were connected by double restriction digestion to construct an expression vector. After transformation of E. coli, colony PCR identification was performed, and the primers for colony PCR identification were T7-F / MsUBP13.3-R1 (chimeric primers, non-CDS sequence full-length primers). The correct positive single clones were shaken and sent for sequencing, and the single clones that were successfully sequenced were subjected to plasmid extraction and preservation.

[0092] Example 2

[0093] This example is a yeast heterologous expression and salt tolerance function verification, as follows:

[0094] (1) The constructed vector plasmid and pYES2 empty plasmid were transformed into the competent cells of INVSc1 yeast.

[0095] (2) Identify the correct pYES2 empty vector and positive yeast strains containing the target gene. Shake the strains in 10 mL SC-Ura glucose liquid medium and incubate on a shaker at 28°C and 200 rpm for 24 h.

[0096] (3) Determination of bacterial solution OD 600 Then, the bacterial solution was diluted to 0.4 with 10 mL SC-Ura galactose induction expression medium and cultured at 28°C and 200 rpm in a shaking incubator for 36 h to induce exogenous gene expression.

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

[0098] (5) The centrifuged yeast cells were added to an equal volume of 4 M NaCl, 4 M sorbitol, 15 mM H2O2 and sterile water (control) and resuspended. The cells were cultured at 28°C and 200 rpm in a shaking incubator for 72 h, 120 h, 120 h and 96 h, respectively. The control culture time was consistent with the treatment culture time.

[0099] (6) The treated bacterial solution was diluted to 1, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 These six gradients were diluted with sterile water in 2 mL tubes, 5 μL of bacterial solution was drawn, and plated on SC-Ura glucose solid medium. The experiment was set up with three technical replicates and three biological replicates, which were sealed and placed in a 28°C incubator for inverted culture for 3-4 days. During the culture process, real-time photography was taken to record the growth status of the colonies.

[0100] The results showed that in the control group, there was no significant difference in the growth of transgenic yeast cells and empty yeast cells. After stress with 4M NaCl, transgenic yeast cells grew significantly better than empty yeast cells ( Figure 1 ). This indicates that MsUBP13.3 enhances the tolerance of yeast strains to salt stress through its transgenic expression, and preliminarily proves that the MsUBP13.3 salt-drought tolerance candidate gene plays a positive regulatory role in yeast cells responding to salt stress.

[0101] Example 3

[0102] This example is the cloning and overexpression vector construction of MsUBP13.3, as follows:

[0103] The overexpression vector pBI121::EGFP stored in our laboratory was selected. According to the vector map and gene sequence, two restriction sites, Sal I and Spe I, were finally selected to construct the vector using the double restriction method. Primers were designed using Snapgene software (Table 1) and synthesized by Xi'an Qingke Biotechnology Co., Ltd. Using MCLAB 1-5 TM 2× High-Fidelity Master Mix amplification enzyme (Qingke, Xi'an) was used to amplify the target fragment. The amplification system was referred to Example 1. The DNA product in the gel was recovered using a gel recovery kit (Quanshijin, Beijing). The pEASY-Blunt Simple Cloning Kit vector was connected, and the ligation product was transformed into Escherichia coli DH5α. A single clone was picked and tested using M13-F and MsUBP13.3-R1 (non-full-length primer sequence) and then sent to the company for sequencing confirmation. After the sequencing was correct, the plasmid was extracted and subjected to double restriction digestion reaction with the pBI121::EGFP expression vector plasmid. The reaction system was referred to the content of Example 1. After the restriction digestion reaction was completed, the fragments were confirmed by electrophoresis. The target fragment after restriction digestion was ligated with the fragment after restriction digestion of the pBI121::EGFP expression vector using T4 DNA ligase (M0202, New England Biolabs Beijing Co., Ltd.), and then transformed into Escherichia coli competent cells. After overnight culture at 37°C, colony PCR detection was performed. After the bacterial test was correct, the sequencing was sent and the single clone with correct sequencing was stored and the plasmid was extracted for standby 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 generate the MCLAB1-5 TM PCR amplification was performed using 2× High-Fidelity Master Mix high-fidelity enzyme, and the PCR product was recovered using a gel recovery kit and set aside for later use.

[0106] Table 7 Primer information

[0107]

[0108] (2) Target fragment ligated to entry vector

[0109] The ligation reaction system is as follows:

[0110]

[0111] Incubate at 25°C overnight.

[0112] The ligation product was transformed into Escherichia coli DH5α. After plating, the plate was inverted and incubated at 37°C for 16 hours. The positive single clone was detected using universal primers M13F and M13R and sent for sequencing. The single clone with correct sequencing was shaken, and the plasmid was 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 chimeric primers 35S-F / MsUBP13.3-R1 (full-length primer without CDS sequence).

[0114] (3) LR reaction to construct RNAi recombinant vector

[0115] First, transfer the target vector pANDA35HK into E. coli DH5α for propagation, and detect the correct plasmid. Determine the concentration of the target vector and the entry vector Entryclone to ensure that the concentration reaches 100ng / μL. Then perform the LR reaction, the system is as follows:

[0116] 2×GatewayLRCloneEnzymemix 5μL

[0117] EntryClone (100ng / μL) 3μL

[0118] Target vector (0.5μg / μL) 2μL

[0119] After fully mixing the above reaction system, centrifuge quickly and incubate at 25℃ overnight, add 1μL of 10×ProteinaseKSolution, shake and mix, and incubate at 37℃ for 20min. Then transform it into E. coli DH5α. After the transformation is completed, take 200μL of culture and spread it on LB solid medium containing kanamycin Kan antibiotic with a final concentration of 50mg / L, invert it in a 37℃ incubator and culture for 16 hours. The single clone that succeeded in bacterial inspection was shaken to extract plasmid and stored at -20℃ for use.

[0120] The RNAi vector was constructed using the Gateway method. The attB-PCR product of MsUBP13.3 was obtained by PCR and connected to the entry vector Entryclone. After the positive clone was identified, the correctly sequenced entry clone was subjected to LR reaction with the target expression vector pANDA35HK. After single clone detection, the positive bacterial liquid was used to extract the plasmid, which is the RNAi recombinant vector of the MsUBP13.3 gene (RNAi-MsUBP13.3).

[0121] (4) Transformation and identification of Agrobacterium

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

[0123] 2) Transformation by electroporation

[0124] 3) Heat shock transformation

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

[0126] Example 4

[0127] This example is tobacco subcellular localization, and the specific steps are as follows:

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

[0129] (2) Use a UV spectrophotometer to measure the OD in the bacterial solution 600 The value should be between 0.5 and 1. After centrifugation at 2400g for 15 minutes, the cells were collected and the supernatant was removed.

[0130] (3) To make the OD of the new bacterial solution 600 The value reached the range of 0.4-0.6. We used a resuspension solution to resuspend the bacteria and prepared it using MS liquid medium. In every 100mL concentration, we added 1M MgCl2·6H2O 1mL (the final concentration was 10mM) 1M 2-(N-morpholino)ethanesulfonic acid (MES) 1mL (the final concentration was 10mM) 1M acetosyringone (AS) 10μL (the final concentration was 100μM) and adjusted the pH value to 5.8. After storing it at room temperature for 2-3 hours, it can start to infect.

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

[0132] (7) Use a syringe without a needle to draw up 1 mL of the bacterial suspension and slowly inject it into the infected area on the back of the leaf.

[0133] (8) Spray the infected tobacco leaves with water and incubate them in the dark for 12 hours, then transfer them to normal light conditions for incubation.

[0134] (9) 2-3 days after injection, we used a hole punch to collect the infected area of ​​the leaf and then prepared slides. We then took photos using a confocal microscope after adjusting the optimal field of view.

[0135] The results are as follows Figure 2 As shown, it was observed that the pBI121::MsUBP13.3::EGFP fusion protein was localized in both the cell membrane and the nucleus, indicating that the MsUBP13.3 protein may function both in the cell membrane and in the nucleus.

[0136] Example 5

[0137] This embodiment is

[0138] (1) Arabidopsis genetic transformation: Arabidopsis transgenic seedlings were obtained by floral dipping method. The main steps refer to (Identification of ERF transcription factor in Melilotus officinalis and analysis of its function in response to drought stress, Wei Na, 2024).

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

[0140] The Arabidopsis DNA of 10 transgenic lines of the harvested T1 generation was extracted using a plant genomic DNA extraction kit (Tiangen, Beijing). The chimeric primer 35S-F / MsUBP13.3-R1 was used for PCR detection to verify whether it was a positive plant. After the positive plant was identified, its RNA was extracted using the RNAEasyFast Plant Tissue RNA Rapid Extraction Kit (Tiangen, Beijing). Next, we used the FastKing cDNA First Chain Synthesis Kit (Tiangen, Beijing) for reverse transcription to obtain cDNA, and used qRT-PCR technology to determine the relative expression level of MsUBP13.3 in transgenic Arabidopsis. The specific method is shown in the following table. The qRT-PCR reaction system and procedure are shown in Tables 8 and 9. The reaction was performed for a total of 38 cycles, with 3 technical replicates, using 2 -ΔΔCT The calculation method processes the data to obtain the relative expression of the gene.

[0141] Table 8 qRT-PCR reaction system

[0142]

[0143] Table 9 qRT-PCR reaction procedure

[0144]

[0145]

[0146] The transgenic MsUBP13.3 gene Arabidopsis was extracted and PCR was performed to detect positive plants. The kanamycin resistance gene detection primers NPT-F / NPT-R and the promoter and target gene chimeric primers 35S-F / MsUBP13.3-R1 were used to detect positive plants. A total of 9 overexpression positive Arabidopsis lines were obtained ( Figure 3 ). RNA from the nine transgenic Arabidopsis and wild-type Arabidopsis was further extracted and reverse transcribed into cDNA, and the expression level of MsUBP13.3 was detected using qRT-PCR technology. The results showed that the expression levels of MsUBP13.3 in the nine transgenic lines were significantly higher than those in the Col-0 line, among which the highest expression levels were in the three overexpression lines OE1, OE4 and OE8 ( Figure 3 ).

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

[0148] The seeds of the three representative strains of Arabidopsis thaliana with the highest expression of the MsUBP13.3 gene, OE1, OE4, OE8, and wild-type Col-0, were sterilized and evenly spread on 1 / 2MS medium and 1 / 2MS medium containing 125mMNaCl using a 10μL pipette tip. After sealing, they were treated at a low temperature of 4℃ for 3d, and then moved to a light incubator at 22℃ and 60% relative humidity for 7d. Three transgenic strains with consistent root lengths and the wild-type Col-0 strain were selected and transferred to 1 / 2MS medium and 1 / 2MS medium containing 125mmol / LNaCl, respectively. The plates with Arabidopsis were placed vertically in a light incubator, and the growth environment was set to 22℃, 16 hours of light and 8 hours of darkness. The root growth of Arabidopsis seedlings was observed at any time, and the growth phenotype was photographed and the root length was measured after about a week. The results were averaged and the standard deviation was calculated.

[0149] The results are as follows Figure 4 As shown, on 1 / 2MS and 125mMNaCl plates, the root length and lateral root number of the wild type and transgenic lines showed significant differences, and the root length and lateral root number of the transgenic lines were significantly higher than those of the wild type.

[0150] Example 6

[0151] This example is a test of the salt resistance of alfalfa hairy roots, as follows:

[0152] 1. Transient transformation of alfalfa hairy roots

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

[0154] (1) Seed cleaning: Select full alfalfa seeds of 'Zhongmu No. 1', add concentrated sulfuric acid and shake for 3 minutes, then aspirate the concentrated sulfuric acid, wash with ddH2O in a clean bench for 5 times, then shake with 6% sodium hypochlorite solution for 5 minutes, then wash with ddH2O water until there is no color, spread it on a FA plate, and move it to a 25°C light incubator for two weeks.

[0155] (2) Infection: Two weeks later, when the seedlings grow the third cotyledon, use sterilized tweezers to gently clamp the alfalfa seedlings and use a scalpel to cut the root-stem connection. Use tweezers to gently clamp the rooted seedlings and dip a small amount of Agrobacterium rhizogenes transformed with EV, MsUBP13.3 and RNAi, then spread them on water agar plates, with about 12 seedlings on each plate. After sealing the plates, place them vertically in a 22°C culture room until roots grow.

[0156] (3) Salt stress: The infected plants were divided into two groups. The treatment group was cultured on water agar medium supplemented with 100 mM NaCl, and the control group was cultured on water agar medium. After two weeks, the lateral root phenotype was observed and photographed, and then samples were taken from the treatment group and the control group. The expression level of the transgenic hairy root MsUBP13.3 was measured. 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 alfalfa hairy roots using alfalfa rooting transformation technology, with the empty vector pBI121::EGFP control as the control. After alfalfa was infected with Agrobacterium rhizogenes, it was placed vertically in a water agar medium and cultured in a light culture room. After two weeks, when roots were grown, a small amount of hairy roots infected with Agrobacterium rhizogenes transformed with pBI121::MsUBP13.3:EGFP expression vector and RNAi vector were cut for positive root identification.

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

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

[0160] In order to detect the ROS level of transgenic alfalfa hairy roots, use dyestuff OxyBURST green H2DCFDA, the alfalfa seedling hairy roots that will not be processed and that are processed 5 hours with 100mM NaCl are used for analysis.First, collect the lateral root of the seedling of salt treatment and control treatment respectively with 1.5mL centrifuge tube, first H2DCFDA is dissolved in dimethyl sulfoxide (DMSO) to 10mM, then use sample buffer (10mM Tris-HCl, 50mM KCl, pH 7.2) to be diluted to the final concentration of 50mM for use.Sample is soaked in 0.01% Tween 20 and vacuumized 30 minutes, rinsed twice with distilled water, then washed with washing buffer (10mM Tris-HCl pH 7.2 and 50mM KCl).Subsequently sample and 50mM H2DCFDA staining solution are at room temperature hatched 10min in the dark, and remove excessive dye with distilled water twice. 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 simultaneously and analyzed under a confocal microscope by using the same settings.

[0161] This experiment detected the level of reactive oxygen species in the hairy roots of MsUBP13.3 transgenic alfalfa, EV, and RNAi transgenic alfalfa by staining with H2DCFDA (cytosolic redox state) after salt treatment. The results showed that under normal conditions, the content of ROS in the hairy roots of alfalfa was very low and the difference was small; but after salt stress treatment, compared with the wild-type control, less ROS was detected in the hairy roots of overexpressing MsUBP13.3, while the ROS in the hairy roots of RNAi transgenic alfalfa increased significantly ( Figure 8 A), These results suggest that overexpression of MsUBP13.3 may have the function of regulating intracellular ROS levels and protect plant roots from salt stress.

[0162] In this experiment, we found that overexpression of MsUBP13.3 reduced the accumulation of ROS in the root system through reactive oxygen species staining, and found that MsUBP13.3 has the potential to regulate ROS homeostasis. The phenotype was observed on a medium treated with 125 mM salt and exogenously applied reduced glutathione (GSH). It was found that the lateral roots with MsUBP13.3 functional deficiency resumed growth, and the phenotype was not significantly different from the control ( Figure 8 B). This indicates that under salt stress conditions, ROS are generated and accumulated, 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 that maintaining intracellular ROS homeostasis under salt stress is important for lateral root growth and development.

Claims

1. Alfalfa MsUBP13.3 protein, characterized in that is (a) or (b) as follows: (a) a protein having an amino acid sequence of SEQ ID No. 2; (b) A protein derived from (a) with one or more amino acid residues substituted and / or deleted and / or added to the amino acid sequence shown in SEQ ID NO. 2 and related to plant resistance to salt stress.

2. A nucleic acid molecule, characterized in that It encodes the alfalfa MsUBP13.3 protein 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 as SEQ ID NO.

1.

4. A recombinant vector, characterized in that: Containing the nucleic acid molecule according to claim 2 or 3.

5. A recombinant bacterium, characterized in that: Contains the recombinant vector according to claim 4.

6. Use of the alfalfa MsUBP13.3 protein and its encoding gene as claimed in claim 1 in improving the salt stress tolerance of plants.

7. The use according to claim 6, characterized in that: The plants include Arabidopsis thaliana and alfalfa.

8. The use according to claim 7, characterized in that: The application includes regulating the lateral root development of plants under salt stress.

9. A method for improving the salt stress resistance of plants, characterized in that: The method comprises introducing the nucleic acid according to claim 2 or 3 or the 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 plants include Arabidopsis thaliana and alfalfa.

10. A plant breeding method, characterized in that: include: Increasing the content and / or activity of the alfalfa MsUBP13.3 protein according to claim 1 in a plant, thereby enhancing the plant's resistance to salt stress; The plants include Arabidopsis thaliana and alfalfa.

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