Application of GmRPN11d gene in improving plant salt resistance and sensitivity to ABA

Abstract

The present invention discloses the application of GmRPN11d gene in improving the salt resistance and ABA sensitivity of plants. The present invention belongs to the field of plant biotechnology. The present invention provides a method for improving the salt resistance and ABA sensitivity of plants. The present invention provides the application of GmRPN11d protein or GmRPN11d gene in improving the salt stress resistance of plants or improving the ABA sensitivity of plants. The amino acid sequence of GmRPN11d protein is shown in SEQ ID NO.2; the nucleotide sequence of GmRPN11d gene is shown in SEQ ID NO.1. The present invention provides a foundation for the research on soybean stress resistance.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to application of a GmRPN11d gene in improving plant salt resistance and sensitivity to ABA. Background Art

[0002] Soybean (Glycine max) is an important oil crop and food crop in my country, but it often faces various abiotic stresses during its growth. Under high salt stress, Na inside and outside plant cells + / K + Imbalance will result in limited nutrient absorption and excessive accumulation of reactive oxygen species (ROS) causing osmotic stress and ionic stress. High salt will affect soybean germination, root establishment, and seedling growth, causing leaf damage, hindering nutrient transport and photosynthesis, and thus affecting soybean yield and product quality.

[0003] In the long-term natural selection, plants have evolved a variety of physiological and biochemical defense mechanisms to cope with stress. Among them, non-functional and abnormal proteins will accumulate in large quantities in cells under salt stress. Plants can identify and remove them in time through the ubiquitin-proteasome pathway, which can effectively change the proteome of plants and thus improve their adaptability to salt stress.

[0004] Abscisic acid (ABA), as an important plant hormone, is one of the important signals in the salt stress response process. Both deficiency and excess will affect plant development. Summary of the invention

[0005] The purpose of the present invention is to provide a method for improving the salt resistance of plants and improving the sensitivity of plants to ABA.

[0006] The present invention provides an application of a GmRPN11d protein or a GmRPN11d gene in improving plant resistance to salt stress or improving plant sensitivity to ABA.

[0007] It is further defined that the amino acid sequence of the GmRPN11d protein is shown as SEQ ID NO.2; and the nucleotide sequence of the GmRPN11d gene is shown as SEQ ID NO.1.

[0008] Further defined, the plant is soybean or Arabidopsis thaliana.

[0009] It is further defined that the concentration of salt applied in the salt stress resistance is 100 mM-200 mM; the concentration of ABA in the improvement of plant sensitivity to ABA is 1 μM-3 μM.

[0010] The present invention provides a method for cultivating plants resistant to salt stress, and the method comprises the following steps:

[0011] (1) Amplify the GmRPN11d gene and insert the gene sequence into a plant overexpression vector;

[0012] (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transfer into Arabidopsis or soybean to obtain transgenic plants;

[0013] (3) Identify the transgenic soybeans obtained in step (2) to obtain positive transgenic plants.

[0014] The present invention provides a method for cultivating plants with improved ABA sensitivity, and the method comprises the following steps:

[0015] (1) Amplify the GmRPN11d gene and insert the gene sequence into a plant overexpression vector;

[0016] (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transfer into Arabidopsis or soybean to obtain transgenic plants;

[0017] (3) Identify the transgenic soybeans obtained in step (2) to obtain positive transgenic plants.

[0018] The present invention provides a method for improving the antioxidant enzyme activity of plants under salt stress environment. The method comprises placing transgenic plants overexpressing the GmRPN11d gene under a 100 mM NaCl environment for stress treatment, and then detecting the antioxidant enzyme activity in the transgenic plants.

[0019] It is further defined that the antioxidant enzyme is SOD, POD or CAT.

[0020] The invention provides a method for increasing the number of lateral roots of seedlings, fresh weight of aboveground parts and chlorophyll content of plants under salt stress. The method comprises placing transgenic plants over-expressing the GmRPN11d gene in a 100 mM NaCl environment for stress treatment.

[0021] The present invention provides an application of a plant over-expressing a GmRPN11d gene in improving the plant's resistance to salt stress or improving the plant's sensitivity to ABA.

[0022] Beneficial effects: Heterologous expression of GmRPN11d can enhance the resistance of Arabidopsis to salt stress, and heterologous expression of GmRPN11d enhances the sensitivity of Arabidopsis to ABA. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The results of the analysis of the expression pattern of the GmRPN11d gene; A: the expression of GmRPN11d in different tissues of soybean; B: the expression of GmRPN11d in different developmental stages of soybean; CG: the expression of GmRPN11d in leaves under different stress treatments (C: 200mM NaCl, D: 50mM NaHCO3, E: 100μM ABA, F: 1mM SA, G: 100μM MeJA); H: the subcellular localization of GmRPN11d protein; the expression of the GmRPN11d gene in the 0h treatment group was used as the benchmark 1, and the values ​​measured in the figure are the average values ​​of three independent biological replicates; "*" indicates a significant difference (P>0.005), and "**" indicates an extremely significant difference (P>0.0001);

[0024] Figure 2 The results of positive PCR detection of transgenic Arabidopsis; Note: M: 2000Marker; -: ddH2O; +: positive plasmid; A, B, C represent the positive PCR detection results of T1, T2 and T3 generations, respectively. 1-18: 18 GmRPN11d transgenic Arabidopsis T1 lines obtained; The red box in Figure A represents the lines used for T2 generation propagation; The primers used in Figures A and B are GmPRN11d specific primers, and the primers used in Figure C are hygromycin primers.

[0025] Figure 3 Figure 1 shows the results of heterologous expression of GmRPN11d to enhance the resistance of Arabidopsis to salt stress; Note: A: RT-PCR detection of the expression of GmRPN11d in the overexpression line; B: Western blot detection of the expression of GmRPN11d protein level in the overexpression line; C: Germination of GmRPN11d overexpressing Arabidopsis grown in 1 / 2MS, 100mM NaCl and 125mM NaCl medium for 11 days; D: Root length statistics of GmRPN11d overexpressing lines on salt stress vertical culture medium; E: Statistics of lateral roots of GmRPN11d overexpressing lines; F: Statistics of germination rate; G: Statistics of green leaf rate; H: Statistics of root length; I: Phenotype of 23-day-old Arabidopsis treated with 200mM NaCl for 5 days; J: Statistics of chlorophyll content; K: Statistics of fresh weight of aboveground and underground parts; L: Statistics of Na on the 3rd day after salt stress treatment. + and K + The values ​​in the figure are the average values ​​of three independent biological replicates; "*" indicates significant difference (P>0.005), "**" indicates extremely significant difference (P>0.0001);

[0026] Figure 4The results of the physiological index measurement of overexpressed Arabidopsis under salt stress treatment; Note: A: DAB staining; B: NBT staining; C: MDA, H2O2 content and SOD, POD, CAT activity measurement. The measured values ​​in the figure are the average of three independent biological replicates; "*" indicates a significant difference (P>0.005), "**" indicates an extremely significant difference (P>0.0001);

[0027] Figure 5 Detection of related gene expression under salt stress; Note: The values ​​measured in the figure are the average values ​​of three independent biological replicates; "*" indicates significant difference (P>0.005), "**" indicates extremely significant difference (P>0.0001);

[0028] Figure 6 The results of heterologous expression of GmRPN11d to enhance the sensitivity of Arabidopsis to ABA; Note: A: germination of GmRPN11d-overexpressing Arabidopsis grown in 1 / 2MS, 1μM ABA and 3μM ABA medium for 4 days; B: germination rate statistics; C: green leaf rate statistics; D: statistics of 15 days of growth on ABA-treated vertical medium; E: root length statistics; F: chlorophyll content statistics; J: fresh weight statistics of aboveground and underground parts. The values ​​measured in the figure are the average of three independent biological replicates; "*" indicates significant difference (P>0.005), "**" indicates extremely significant difference (P>0.0001);

[0029] Figure 7 The results of the sensitivity analysis of Arabidopsis thaliana heterologously expressing GmRPN11d to ABA are shown in the figure; Note: The values ​​measured in the figure are the average values ​​of three independent biological replicates; "*" indicates a significant difference (P>0.005), and "**" indicates an extremely significant difference (P>0.0001);

[0030] Figure 8 The results of GmRPN11d's involvement in regulating ABA-induced stomatal closure; Note: A: Guard cells of WT, OE#15 and OE#18 leaves grown under normal light conditions for 23 days, Bar = 50 μm; B: Statistics of stomatal apertures of Arabidopsis before and after ABA treatment, 110 stomata were counted for each sample;

[0031] Fig. 9 The relative expression results of chlorophyll degradation and senescence-related genes in Arabidopsis before and after ABA treatment; Note: The values ​​measured in the figure are the average values ​​of three independent biological replicates; "*" indicates significant difference (P>0.005), "**" indicates extremely significant difference (P>0.0001);

[0032] Fig.10The figure shows the effect of ABA treatment on the chloroplast structure of Arabidopsis thaliana overexpressing GmRPN11d; Note: C: chloroplast; GL: grana lamella; OG: osmophilic granules; SG: starch granules.

[0033] Fig.11 The results of overexpressing the GmRPN11d gene to enhance the resistance of soybean hairy roots to salt stress; Note A: Positive detection of K599-GmRPN11d transgenic soybean hairy root chimera; B: Phenotype of 2-week-old soybean composite plants treated with 200mM NaCl for 12h; C: Partial photograph of a single plant; D: Detection results of MDA content, SOD, POD and CAT activity of soybean hairy roots under 12h salt stress. DETAILED DESCRIPTION

[0034] The Arabidopsis thaliana variety “Colombia Wild Type” and the soybean (Glycine max) variety “Dongnong 50” are both preserved in our laboratory, and the tobacco (Nicotiana tabacum) variety “Benshi” was kindly donated by Researcher Guo Yushuang.

[0035] (1) Cloning vector: pFAST-Blunt Simple Vector (Cat: DT 124-101) purchased from Beijing Dinning Biotechnology Co., Ltd.;

[0036] (2) Expression vectors: plant expression vector: pCAM35S-GFP, prokaryotic expression vector: PGEX-4T-1, pET32a are all preserved in this laboratory;

[0037] (3) E. coli strains: DH5α, BL21 Star (DE3);

[0038] (4) Agrobacterium strains: GV3101 (Cat: AC1001), EHA105 (Cat: AC1010), K599 (Cat: AC1080);

[0039] Escherichia coli strains and Agrobacterium tumefaciens strains were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0040] 2xTaq PCR Master Mix (Cat: DN1056), 10xEnhancer (Cat: DT 124-103), Loading buffer, agarose gel recovery & DNA purification kit (Cat: DP 105-01) and were purchased from Beijing Dinning Biotechnology Co., Ltd.; DNA ligase was purchased from Nanjing Vinozan Biotechnology Co., Ltd.; 2xSuper Pfx Master Mix (Cat: CW2965M) and Ultrapure RNAKit ultrapure RNA extraction kit (Cat: CW0581M) were purchased from Kangwei Biotechnology Co., Ltd.; cDNA reverse transcription kit and fluorescent quantitative enzyme were purchased from Spark Jade; EcoR I, BamH I and other restriction endonucleases were purchased from New England Biolabs; ampicillin (Amp), kanamycin (Km), rifampicin (Rif), streptomycin (Sm) and hygromycin (Hygromycin B, Hyg) were purchased from Beijing Biotoda Technology Co., Ltd.

[0041] Preparation methods of major drugs

[0042]

[0043]

[0044] LB medium (1L): weigh 10g tryptone, 5g yeast extract powder, 10g NaCl, add a little distilled water and place on a magnetic stirrer to fully dissolve, add distilled water to make up to 1L, adjust the pH to 7.4. If it is solid LB medium, add 15g agar powder to each liter of liquid LB, sterilize at 120℃ for 20min, and use after cooling.

[0045] 1 / 2MS solid medium (1L): weigh 2.185g MS salts, 30g sucrose, 8.5g agar powder, pH 5.8.

[0046] All culture media involving hormones and antibiotics are sterilized at 120℃ for 20 minutes and allowed to cool (not hot when held in the hand for 20 seconds) before adding. It is best to prepare them before use. Otherwise, store them in a 4℃ refrigerator.

[0047] Example 1. GmRPN11d gene expression pattern

[0048] 1. RT-qPCR analysis of GmRPN11d under different stresses

[0049] 1. Extraction of Total RNA from Soybean Leaves

[0050] For details on the extraction steps, please refer to the Ultrapure RNA Kit (Cat: CW0581M) manual. Store the extracted RNA in a -80°C refrigerator to prevent RNA degradation.

[0051] 2. First-strand cDNA Synthesis

[0052] The concentration of each sample was detected by In-finite 200pro E-PLEX using the RNA extracted in step 1, and the concentration of each sample was adjusted to 1 ng / μl. The first-strand cDNA was synthesized according to the instructions of Spark Jade's SPARKscriptⅡRT Plus Kit (With gDNAEraser) (AG0304).

[0053] The cDNA synthesized in step 2 was used with Spark Jade's 2xSYBR Green qPCR Mix (With ROX) (AH0104) kit and a fluorescent quantitative PCR instrument QuantStudioTM 3Real-Time PCR Instrument (96-Well 0.2ml Block) to measure the relative expression level of the GmRPN11d gene. The relative expression level of the GmRPN11d gene was calculated using the 2-ΔΔCt method. The experiment was set up with three biological replicates and three technical replicates. The primers used are shown in Table 1 below. For details of the amplification system, see the Spark Jade 2xSYBR Green qPCR Mix (With ROX) (AH0104) manual. GmTUA5 is a soybean internal reference gene.

[0054] Table 1 GmRPN11d RT-qPCR primer sequences

[0055]

[0056] Table 2 RT-qPCR reaction solution system

[0057]

[0058]

[0059] Amplification Procedure

[0060]

[0061] The expression of GmRPN11d gene in different tissues (leaves, roots, cotyledons, young roots, flowers, pods, beans) of soybean variety "Dongnong 50" and at different developmental stages and under different stress treatments was analyzed by RT-qPCR. The expression of GmRPN11d gene in different tissues of soybean was detected, and it was found that GmRPN11d gene was expressed in young roots, young leaves, cotyledons, roots, flowers, pods and beans of soybean, and the expression level was the highest in young roots, while the expression level in leaves and beans was lower (such as Figure 1 The expression level of GmRPN11d gene was analyzed by counting the first trifoliate leaf from top to bottom at the second node stage (V2), initial flowering stage (R1), initial pod stage (R2), full pod stage (R4), initial grain stage (R5), grain filling stage (R6), early maturity stage (R7) and full maturity stage (R8) of soybean under normal growth state. It was found that the expression level of GmRPN11d gene increased with the completion of soybean development process (as shown in Figure 1). Figure 1 ); soybean seedlings at the second node stage (V2) were treated with 200mM NaCl, 50mM NaHCO3, 100μM ABA, 1mM SA, and 100μM MeJA respectively. It was found that under 200mM NaCl stress, the expression level of GmRPN11d gene reached the highest level at 1h, and the expression level decreased significantly with the extension of stress treatment time; under NaHCO3 stress, the expression level increased slightly only in the 1h treatment period, while there was no significant difference between the other time periods and the treatment time of 0h; under ABA stress, the expression level decreased continuously before 12h, and then increased sharply; under SA treatment, the expression level continued to increase with the extension of treatment time; under MeJA treatment, the expression level of GmRPN11d decreased first and then increased, reaching the highest level at 12h, and then decreased (as shown in Figure 2A). Figure 1 (C, D, E, F, and G in Figure 5). Based on the expression pattern of the GmRPN11d gene, it was preliminarily concluded that the GmRPN11d gene may be related to the plant growth cycle process and may respond to salt, ABA, SA, and MeJA at the transcriptional level.

[0062] Example 2. Construction of pCAM35S-GmRPN11d-eGFP plant expression vector

[0063] 1. Cloning of GmRPN11d gene

[0064] Cloning of 1GmRPN11d gene

[0065] The cDNA obtained in Example 1 was used as a template, and the full-length primer of the GmRPN11d gene sequence was used as a template. The primer sequences are shown in Table 3 below.

[0066] GmRPN11d gene sequence: (SEQ ID NO.1) atgtcgggcatggagaggcttcagaggatgttcgctggtgcaggtggagcgctgggtcatcccccaccggattctcccaccctcgattcctccgagcaggtctacatctcctccctcgcccttctcaaaatgctcaaacacggaagagctggagtaccgatggaagtgatgggtctgatgttgggggagttcgtggatgagtacaccgttcgcgttgtggatgtgtttgcgatgcctcagagtgggaccggtgttagcgttgaagctgttgatcatgtttttcagactaatatgcttgatatgttgaagcagactggaaggccggagatggttgtggggtggtatcactcgcatcctggatttgggtgttggctttctggagtggacattaatacacaacagagttttgaggctttaaatcaacgggcagtggctgtggtggtggaccctattcagagtgttaaaggcaaagtggtaattgatgctttccggctgattaatccacaaactatgatgctgggtcaggaaccacgacagacaacttccaacctgggccacctaaacaagccatctatccaagctctgatccatggcttgaatagacattattactcgatagcgattaattacagaaagaatgaacttgaagagaagatgttacttaaccttcacaaaaagaagtggactgatggtttgacactcaggcattttgattcacattctaaaactaatgaacagactgttcaggaaatgctaagtttagcagccaagtacaacaaggcagtgcaggaggaagatgagctgccgccagaaaaacttgcaattgctaatgtggggaggcaagatgctaagaagcaccttgaagagcatgtttccaatttgatgtcatcaaacattgttcagactttgggaatgatgcttgacacggttgttttctag;

[0067] GmRPN11d amino acid sequence: (SEQ ID NO.2)

[0068] MSGMERLQRMFAGAGGALGHPPPDSPTLDSSEQVYISSLALLKMLKHGRAGVPMEVMGLMLGEFVDE

[0069] YTVRVVDVFAMPQSGTGVSVEAVDHVFQTNMLDMLKQTGRPEMVVGWYHSHPGFGCWLSGVDINT

[0070] QQSFEALNQRAVAVVVDPIQSVKGKVVIDAFRLINPQTMMLGQEPRQTTSNLGHLNKPSIQALIHGLNR

[0071] HYYSIAINYRKNELEEKMLLNLHKKKWTDGLTLRHFDSHSKTNEQTVQEMLSLAAKYNKAVQEEDELPPEKLAIANVGRQDAKKHLEEHVSNLMSSNIVQTLGMMLDTVVF;

[0072] Table 3 Primers for cloning sequence of GmRPN11d gene

[0073]

[0074]

[0075] Table 4 PCR amplification system of GmRPN11d gene

[0076] Reagents 50μl reaction system cDNA 2.5μl 2xSuper Pfx Master Mix 25μl PFBS-GmRPN11d-S 2.5μl PFBS-GmRPN11d-AS 2.5μl <![CDATA[ddH2O]]> 17.5μl

[0077] The amplification procedure is as follows:

[0078]

[0079] 2GmRPN11d agarose gel fragment recovery

[0080] The target fragment of GmRPN11d gene was recovered using the agarose gel recovery & DNA purification kit (Cat: DP 105-01) of Beijing Dinning Biotechnology Co., Ltd. The operation steps are detailed in the instruction manual. 3 The ligation cloning vector system is shown in Table 5 below:

[0081] Table 5 pFAST-Blunt Simple Vector System

[0082] Reagent name 2.5 μl reaction system GmRPN11d gene target fragment 2μl pFAST-Blunt Simple Vector 0.25μl 10x Enhancer 0.25μl

[0083] After all components were added, they were gently pipetted with a 2.5 μl pipette to mix them evenly, and then centrifuged in a low-speed instant centrifuge and reacted at 37°C for 30 min.

[0084] 3. Transformation of E. coli

[0085] The transformation process of E. coli was carried out using the routine freeze-thaw method in the laboratory.

[0086] 4. Positive detection of transformants

[0087] After transformation, the transformant grows a single colony on the plate of LB solid medium containing antibiotics. A sterilized white pipette tip is used to prick a single colony of normal size and shape in the clean bench and shake overnight. The bacterial solution is used as a template and PCR amplification is performed with gene-specific primers. After the bacterial solution PCR test is positive, the positive bacterial solution is sent to Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0088] 5. Plasmid Extraction

[0089] For details on the plasmid extraction steps, please refer to the plasmid mini-prep kit EasyPure plasmid MiniPrep Kit (Code: EM101-02).

[0090] 2. Connection of target gene fragment and plant expression vector

[0091] (1) Linearization of vector

[0092] The plant expression vector pCAM35S-eGFP (PCG3301, hygromycin resistance) was digested with BamH Ⅰ restriction endonuclease and reacted at 37.0°C overnight. The next day, the digestion reaction solution was subjected to agarose gel electrophoresis, and the fragments were purified and recovered. The digestion system is shown in Table 6 below:

[0093] Table 6 BamH Ⅰ restriction enzyme digestion system

[0094]

[0095]

[0096] (2) Ligation of the target fragment and expression vector

[0097] The GmRPN11d target gene was amplified by PCR using pCAM35S-GmRPN11d-eGFP specific primers. The primers are shown in Table 7 to obtain the GmRPN11d gene target fragment with the homology arms of the pCAM35S-eGFP vector. Then, the target fragment GmRPN11d gene was connected to the pCAM35S-eGFP (PCG3301, hygromycin resistance) vector. The connection reaction system is shown in Table 8.

[0098] Table 7 GmRPN11d specific primers

[0099]

[0100] Table 8 pCAM35S-GmRPN11d-eGFP homologous recombination system

[0101] Reagents 2.5 μl reaction system Exnase ΙΙ 0.25μl 5×CEΙΙ Buffer 0.25μl Target gene fragment 1μl Linearized vector 1μl

[0102] After all components were added, they were gently pipetted with a 2.5 μl pipette to mix them evenly, and then centrifuged in a low-speed instant centrifuge and reacted at 37°C for 30 min.

[0103] Example 3. Subcellular localization analysis of GmRPN11d

[0104] 1. The recombinant vector pCAM35S-GmRPN11d-eGFP obtained in Example 1 and the empty vector control plasmid pCAM35S-eGFP were transformed into Agrobacterium tumefaciens GV3101 and activated once and twice until the OD of the secondary activated bacterial solution reached 600nm =0.6, the activated bacterial solution was centrifuged at 4°C, 2000 rpm for 20 min, and the precipitate was resuspended in a resuspension solution (10 mM MgCl2, 10 mM MES-KOH (ph 5.7), 150 μM acetosyringone) (OD 600nm =0.6) suspension was cultured in the dark at 28°C for 2h, and the prepared suspension was injected into Nicotiana benthamiana leaves with a syringe. The tobacco infected with pCAM35S-eGFP was used as the control group. The infected tobacco was cultured in the dark for 1 day and cultured in the light for 2 days. The fluorescence was observed using a laser confocal microscope at an excitation wavelength of 488nm. The results showed that the fluorescence in the tobacco leaves of the control group was distributed in the cytoplasm, nucleus, and cell membrane, while the green fluorescence produced by the fusion of GmRPN11d and GFP appeared in the cytoplasm and nucleus (such as Figure 1 H), indicating that GmRPN11d protein is localized in the cytoplasm and nucleus.

[0105] Example 4. Identification and acquisition of GmRPN11d transgenic Arabidopsis

[0106] 1. The recombinant vector pCAM35S-GmRPN11d-eGFP plasmid obtained in Example 1 was transformed into Agrobacterium tumefaciens GV3101, and then the Arabidopsis thaliana was infected by the Agrobacterium floral dip method.

[0107] 2. Screening of GmRPN11d transgenic Arabidopsis

[0108] The Arabidopsis seeds obtained after infection were sterilized in a clean bench and vernalized. The vernalized seeds were inoculated on a 1 / 2MS solid medium containing hygromycin B (Hyg) resistance for screening. The leaves of transgenic seedlings containing the target gene on the resistance medium were green. The selected transgenic seedlings were transplanted into nutrient soil for growth. The transgenic Arabidopsis seedlings that had grown for about a week were tested for PCR positive. The seeds of the positive seedlings were collected and screened repeatedly until the T3 generation was homozygous.

[0109] Results: Detection and acquisition of transgenic Arabidopsis thaliana with GmRPN11d gene

[0110] The frozen bacterial solution of pCAM35S-GmRPN11d-eGFP (GV3101) was taken out from the -80℃ refrigerator, added into 10ml of liquid LB containing kanamycin and rifampicin antibiotics, and cultured overnight at 28℃ 2000rpm (i.e., primary activation). 1ml of the primary activated bacterial solution was aspirated and added into 20ml of liquid LB containing kanamycin and rifampicin antibiotics, and cultured at 28℃ 2000rpm. The OD value of the bacterial solution was detected by UV spectrophotometer at a wavelength of 600nm. When it was 0.6, the culture was stopped and the OD 600 = 0.6, centrifuge at 4℃ and 4000rpm for 10min, discard the supernatant, resuspend the bacteria with sucrose solution containing Silweet-77 as the infection solution, immerse the flower buds of wild-type Arabidopsis thaliana in the infection solution for 20S, put the infected Arabidopsis thaliana in a fresh-keeping cover, culture it in the dark for 24h, remove the fresh-keeping cover, and transfer it to a normal incubator for growth. One week later, infect it again in the same way, wait for it to be inoculated and harvest the seeds. The harvested seeds are sterilized and vernalized and inoculated on 1 / 2MS medium containing 20mg / ml hygromycin (Hyg) for screening. Each time the green seedlings screened out are transplanted into a small black square filled with nutrient soil (vermiculite: soil = 2:1), and placed in the Arabidopsis culture room for growth. During this period, 1 / 8 Hogland nutrient solution is irrigated every 3d. After growing for about a week, take a rosette leaf and perform PCR positive detection (as shown in 2) until the T3 generation homozygous strain is obtained.

[0111] Example 5. Application of GmRPN11d gene

[0112] 1. Determination of chlorophyll content:

[0113] (1) Pigment extraction: Take 0.1 g of leaves from the same position after treatment, put them into 12.5 ml of 95% ethanol, extract them in the dark at 4°C until the leaves turn white, let them stand at room temperature for 3-5 min, transfer them to a new 20 ml centrifuge tube, centrifuge them at 4000 rpm for 5 min, and collect the supernatant.

[0114] (2) Determination of absorbance: Pour the chlorophyll extract into a cuvette with a light path of 1 cm, use the 95% ethanol extract as a blank, and use an ultraviolet photometer to measure the absorbance of the sample at wavelengths of 665 nm, 649 nm, and 470 nm, and calculate the chlorophyll a, chlorophyll b, and total chlorophyll contents.

[0115] (3) Calculation of results:

[0116] Ca=13.95A 665 -6.88A 649

[0117] Cb=24.96A 649 -7.32A 665

[0118] Cc=(1000A 470 -2.05Ca-114.8Cb) / 245

[0119] Substitute the absorbance value into the formula to calculate the concentration of chlorophyll a, b and carotenoids (mg L -1 The total chlorophyll concentration (mg L) is obtained by adding the concentrations of chlorophyll a and b. -1 )

[0120] Substituting the concentration of the pigment into the following formula, the content of various chlorophyll per unit fresh weight of tissue can be calculated:

[0121] Chlorophyll content (mg g -1 )=CVNW -1

[0122] Where: C——pigment concentration (mg g-1);

[0123] V——extraction volume (ml);

[0124] W——fresh weight of plant tissue (g);

[0125] N——dilution multiple.

[0126] 2. Detection of antioxidant enzyme activity: For Arabidopsis thaliana grown for about 21 days, 0.1g of leaves from the same leaf position of the treatment group and the control group were taken and placed in a 2ml EP tube containing a small steel ball at 3 days after salt treatment. The EP tube containing the material was placed in liquid nitrogen, fully broken by a crusher, and the extraction buffer was added. Centrifugation was performed and the supernatant was collected for the determination of SOD, POD and CAT enzyme activities. Each sample was repeated at least 8 times. For specific determination methods, please refer to the superoxide dismutase (SOD) test kit (WST-8 method) (SOD-1-W), peroxidase (POD) test kit (POD-1-Y) and catalase (CAT) kit (CAT-1-Y) of Suzhou Keming Biotechnology Co., Ltd.

[0127] 3. Determination of malondialdehyde content

[0128] (1) Sample extraction: The sampling method and time are consistent with those for measuring SOD, POD, and CAT. Take 1.0 g of leaves and put them into a 2 ml EP tube filled with small steel balls. Put the EP tube containing the material into liquid nitrogen and fully break it with a crusher. Add 2 ml 10% TCA and then add 8 ml TCA and mix by inverting. Centrifuge at 4000 rpm for 10 min. Transfer the supernatant to a new EP tube. The resulting supernatant is the sample extract.

[0129] (2) Color reaction and determination: Take 2 ml of supernatant (add 2 ml of distilled water for control), add 2 ml of 0.6% TBA solution, mix well, react in a boiling water bath for 15 min, cool quickly and centrifuge. Take the supernatant and measure the absorbance at 450 nm, 532 nm and 600 nm.

[0130] (3) Calculation of results:

[0131] C=6.45(A 532 -A 600 )-0.56A 450

[0132] MDA content (μmol g -1 )=CVW -1

[0133] Where: C——MDA concentration (μmol L -1 )

[0134] V——extraction volume (mL)

[0135] W——Fresh weight of plant tissue (g)

[0136] (4) Determination of H2O2 content: The sample collection method is the same as that of SOD, POD, and CAT, and the detection method uses the hydrogen peroxide content (H2O2) kit (H2O2-1-Y)

[0137] 4. DAB staining and NBT staining: Take the same part of Arabidopsis leaves, put them into DAB and NBT dye solution respectively, dye for about 4 hours, and decolorize them in 95% ethanol boiling water bath until the part without oxidative damage turns white, stop decolorizing, observe the staining results and take pictures. The sampling time is the same as above.

[0138] 5. Determination of stress-related gene expression

[0139] (1) Detection of salt stress response gene expression: Using Columbia wild-type Arabidopsis as a control, first sterilize and vernalize seeds inoculate on 1 / 2MS solid culture medium in a clean bench. When the Arabidopsis seedlings just grow the sixth leaf, transplant them into a small black square filled with nutrient soil (vermiculite: soil = 2:1) (soaked with 1 / 8 Hogland nutrient solution), put them into the Arabidopsis culture room and grow for about 10 days, irrigate with 200mMNaCl, treat for 3 days, take leaves from the same part of the treatment and control groups, extract RNA and reverse transcribe. RT-qPCR detects the expression of the relevant stress genes in the table below. The detection primers are shown in Table 8 below:

[0140] Table 8 RT-qPCR primers for salt stress response genes

[0141]

[0142] (2) Determination of ABA stress-related gene expression

[0143] Using Columbia wild-type Arabidopsis as the control, first sterilized and vernalized seeds were inoculated into 1 / 2MS solid culture medium in a clean bench, and placed flat in an Arabidopsis constant temperature incubator for 4 days. Arabidopsis seedlings with consistent growth were selected and transplanted onto solid culture medium of 1 / 2MS, 1 / 2MS+1μM ABA and 1 / 2MS+3μM ABA. Four strains of each line were placed vertically in an Arabidopsis constant temperature incubator. Their growth status was observed and photographed every day. When the leaves of the same parts of the control group and the treatment group were taken at 15 days of growth, RNA was extracted and reverse transcribed, and RT-qPCR was used to detect the expression of the relevant stress genes in the following table. The primers for ABA stress-related genes are shown in Table 9:

[0144] Table 9 Primers for ABA stress-related genes

[0145]

[0146]

[0147] 6. Measurement of Stomatal Aperture

[0148] The sterilized and vernalized seeds were inoculated on 1 / 2MS solid culture medium, and the Columbia wild type was used as the control. Arabidopsis was grown in a constant temperature incubator for 16 days. The second rosette leaf from the top of the Arabidopsis seedling was taken, and the lower epidermis of Arabidopsis was torn off with tweezers and placed on a transparent tape. The tape and the material were placed in a stomatal opening solution (50mM KCl, 0.2mM CaCl2, 10mM (MES)-KOH, pH 5.8) and irradiated with strong light at 24°C. The degree of stomatal opening was observed under a fluorescent inverted microscope. After full opening, the leaves were moved to a MES-KCl buffer containing 30μM ABA and cultured at room temperature. The stomatal opening was observed, and the aspect ratio of each stoma was measured using the software Image-J.

[0149] 7. Observation of chloroplast ultrastructure

[0150] The leaves of seedlings grown for 15 days on 1 / 2MS vertical medium and ABA vertical medium were cut into strips of 3 mm long and 1 mm wide (avoiding the veins), and after fixation, dehydration, infiltration, embedding, polymerization, block cutting, ultrathin sectioning and uranyl acetate-lead citrate staining, the ultrastructure of chloroplasts in the leaves was observed using a transmission electron microscope. Each sample was repeated three times.

[0151] 8.Na + , K + , Determination of content

[0152] (1)Na + , K + Determination of content

[0153] The sampling time was consistent with the growth time of the material for measuring antioxidant enzyme activity. Each sample was placed in an oven at 60°C for drying. The dry weight of each sample was 0.5 g, and there were 3 biological replicates.

[0154] First, the pressure tank digestion method was used to fully digest the sample. The specific experimental steps are as follows:

[0155] Weigh 0.1-0.5g of dry sample or 0.5-2mL of liquid into a polytetrafluoroethylene digestion inner tank, add 5mL of nitric acid and soak overnight. Cover the inner cover, tighten the stainless steel jacket, put it into a constant temperature drying oven, keep it at 80℃ for 2h, keep it at 120℃ for 2h, and then increase it to 160℃ for 4h. Cool it naturally to room temperature in the oven, open it and heat it to drive out the acid until it is almost dry, wash the digestion liquid into a 25mL volumetric flask, wash the inner tank and inner cover with a small amount of nitric acid solution (1%) 3 times, combine the washing liquid into the volumetric flask and dilute it to the scale with 1% nitric acid, mix well and set aside. At the same time, do a reagent blank test. The test solution is used to determine the content of each element in plant tissue using the ICP-MS method (inductively coupled plasma mass spectrometer).

[0156] Result calculation

[0157] Element content

[0158] Where:

[0159] c——concentration of the element in the solution (mg L-1);

[0160] V——extraction volume (ml);

[0161] D——dilution multiple;

[0162] m——Sample mass (g)

[0163] 2.1. Treatment methods for soybean abiotic stress

[0164] Pick out soybean seeds of the variety "Dongnong 50" with uniform particle size and full shape, place them on filter paper soaked with tap water, put them in a 90mm culture dish, dark-treat them for germination at room temperature for 2 days, move them into a small black square containing pure vermiculite, and culture them under light / dark conditions for 16h / 8h, 27℃ / 25℃ until the soybean seedlings grow the second trifoliate leaf. During this period, water them with 1 / 8 Hoagland nutrient solution to keep the vermiculite moist. Select seedlings with consistent growth status, take them out, wash the vermiculite on the roots of the seedlings, and place them in a tissue culture bottle containing 1 / 8 Hoagland nutrient solution for 1 day to adapt them. When the second trifoliate leaf just unfolds, move the seedlings into 200mM NaCl, 50mM NaHCO3, 100μM ABA, 1mM SA, 100μM Salt, alkali, ABA, SA and MeJA were treated in 1 / 8 Hoagland nutrient solution containing MeJA. The second trifoliate leaf samples of soybean were removed at five time points: 0h, 1h, 6h, 12h and 24h of treatment. Three replicates were taken at the same leaf position and state at each time point. The obtained materials were placed in a -80℃ refrigerator for later use.

[0165] 2. Heterologous expression of GmRPN11d enhances Arabidopsis resistance to salt stress

[0166] The T3 generation positive homozygous lines OE#14-1, OE#15-1 and OE#18-1 were subjected to RT-PCR detection, using the Actin gene as the internal reference gene and GmRPN11d specific primers for RT-PCR detection. Figure 3As shown in A, when the Actin internal reference is amplified, the amount of cDNA template sample is adjusted until the brightness of the Actin internal reference band on the agarose gel is basically consistent; then the specific primers of the GmRPN11d gene are selected for RT-PCR detection. Since GmRPN11d is not an endogenous gene of Arabidopsis, when the specific detection primers of the gene are used for RT-PCR detection, WT does not amplify the band, while OE#14, OE#15, and OE#18 are detected as positive strains. The expression levels of strains OE#15 and OE#18 are higher at the transcriptional level. Then, using the WT plant as a control, the GFP tag antibody fused with the target gene on the vector is used to detect the expression of GmRPN11d in strains OE#15 and OE#18 at the protein level. The results show that in transgenic strains 15 and 18, GmRPN11d protein can be normally expressed. (As shown in Figure 3 (As shown in B) Therefore, strains OE#15 and OE#18 were selected for subsequent experimental analysis.

[0167] From the expression pattern of GmRPN11d gene, it can be seen that GmRPN11d gene responds to salt stress. In order to further detect the response of GmRPN11d gene to salt stress, the homozygous seeds of Columbia wild type and GmRPN11d overexpressing Arabidopsis thaliana T3 generation were sterilized and vernalized in a 4℃ refrigerator for 2 days, and then inoculated into 1 / 2MS solid medium, 1 / 2MS+100mM NaCl and 1 / 2MS+125mM NaCl solid medium, respectively, to conduct germination experiments under salt stress, and take pictures to record the germination of each strain of Arabidopsis thaliana, with three biological replicates each time. Figure 3 As shown in C, F, G and Tables 10 and 11, the growth status of WT and the three overexpression lines OE#14, OE#15 and OE#18 on the basal medium was consistent, while on the 100mM NaCl and 125mM NaCl stress treatment medium, it can be clearly seen that the growth status of the three overexpression lines was significantly better than that of the wild type. The germination rate and green leaf rate were statistically found that on the salt stress treatment medium, the germination rate of the three overexpression lines was slightly later than that of the wild type, while the green leaf rate of the three overexpression lines was significantly higher than that of the wild type, and the wild type almost did not expand its leaves on the 125mM NaCl medium. Therefore, overexpression of the GmRPN11d gene in Arabidopsis enhances seed tolerance to salt stress.

[0168] Table 10 Statistics of germination rate, green leaf rate, root length and lateral root number under salt stress treatment

[0169]

[0170] Table 11 Green leaf rate under salt stress treatment

[0171]

[0172]

[0173] In order to further explore the reasons why GmRPN11d gene overexpression enhances Arabidopsis seeds' tolerance to salt stress, root length experiments under salt stress were conducted on transgenic GmRPN11d Arabidopsis seeds. Root phenotype experiments under salt stress were conducted on the T3 generation of transgenic GmRPN11d Arabidopsis. First, WT, OE#14, OE#15 and OE#18 were sterilized and vernalized and inoculated on 1 / 2MS solid culture medium, and placed in a constant temperature and light incubator for germination for 3 days. Seedlings with consistent growth conditions were selected and inoculated into 1 / 2MS vertical solid culture medium containing 100mM NaCl and 125mM NaCl for salt stress treatment. 1 / 2MS solid culture medium was used as blank control. Three biological replicates were performed in each group. The root growth of each strain of Arabidopsis was observed and photographed. It was found that in 1 / 2MS solid medium, there was no significant difference in root length between WT and the three overexpression lines OE#14, OE#15 and OE#18, while in the medium treated with 125mM NaCl salt stress, the root lengths of the three lines OE#14, OE#15 and OE#18 were significantly shorter than those of the wild type (e.g. Figure 3 D in and Table 12), by counting the number of lateral roots, it was found that the number of lateral roots of the three overexpression lines increased compared with that of WT on 100 mM NaCl solid medium (as shown in Figure 3 E, H and Table 13); Overexpression of the GmRPN11d gene may enhance the plant's tolerance to salt stress by increasing the number of lateral roots in Arabidopsis seedlings.

[0174] Table 12 Statistics of root length under salt stress treatment

[0175]

[0176] Table 13 Statistics of roots under salt stress treatment

[0177]

[0178] To verify that heterologous expression of GmRPN11d can enhance Arabidopsis resistance to salt stress, three Arabidopsis strains, WT, OE#15, and OE#18, were sterilized and inoculated onto 1 / 2MS solid culture medium and cultured in a constant temperature and light incubator for about 10 days. Then, Arabidopsis seedlings with consistent growth were selected and moved into small black squares containing nutrient soil (vermiculite: soil = 2:1). Four Arabidopsis seedlings were moved into each small black square and cultured for 13 days. During this period, 1 / 8 Hoagland nutrient solution was watered twice. After the second watering of Hoagland nutrient solution, the nutrient soil in the small black square was dried, and then treated with 200mM NaCl solution for 6 days. Water treatment was used as the control. The results showed that under water treatment, the growth status of the two overexpression lines was not significantly different from that of WT, but under 200mM NaCl treatment, the leaves of WT Arabidopsis thaliana were wrinkled and wilted, and yellowed seriously, indicating that the plants were seriously damaged by stress; while the leaves of Arabidopsis thaliana of the two lines OE#15 and OE#18 were upright and had no obvious yellowing phenomenon (such as Figure 3 As shown in I in the figure, it shows that GmRPN11d overexpressing Arabidopsis suffers less stress damage; chlorophyll content is an important indicator for measuring plant photosynthesis. To this end, we further counted the fresh weight and chlorophyll content of wild-type and GmRPN11d overexpressing Arabidopsis, and found that the aboveground fresh weight and chlorophyll content of the two overexpressing strains were higher than those of wild-type Arabidopsis (as shown in Figure 3 J, K and Tables 14 and 15), which further supports the phenotypic differences under salt stress.

[0179] Table 14 Fresh weight

[0180]

[0181] Table 15 Chlorophyll

[0182]

[0183] Excessive salt concentration in the soil can damage the ion homeostasis and redox system of plants, thereby inhibiting the normal growth of plants. Salt-resistant plants regulate ion homeostasis by excreting excessive Na+ or isolating Na+ into vacuoles and absorbing K+ to make plants salt-resistant. For this purpose, the Na+ and K+ contents in the leaves and roots of various Arabidopsis were tested. By calculating the Na+ / K+ ratio, it was found that under normal circumstances, the Na+ / K+ value in the leaves was about 0.03, and the Na+ / K+ value in the roots was about 0.2. There was no obvious difference between the overexpression plants and the wild type; after salt treatment, the Na+ / K+ values ​​in both leaves and roots of OE#15 and OE#18 were significantly lower than those of the wild type (such as Figure 3 -L and Table 16), this result indicates that overexpression of GmRPN11d in Arabidopsis can make plants salt-tolerant by inhibiting the accumulation of Na+.

[0184] Table 16 Na+ / K+

[0185]

[0186] To further analyze the reasons for the enhanced salt resistance of transgenic GmRPN11d Arabidopsis, the accumulation of ROS in transgenic GmRPN11d Arabidopsis and control before and after salt stress was detected by staining with 3,3′-diaminobenzidine (DAB) and tetrazolium blue chloride (NBT). For Arabidopsis treated with salt stress for 3 days, the 6th leaf from the bottom was immersed in DAB and NBT staining solution respectively, and the sample was placed in a vacuum instrument to extract the air in the instrument so that the leaf was completely immersed in the staining solution for 6 hours, with three replicates for each sample. The staining showed that in the control group, the WT and the two overexpression lines had lighter staining colors and no obvious difference; but under salt stress treatment, it can be clearly seen that the color of the wild-type Arabidopsis leaves increased and the color deepened compared with the leaves of the control group, while the color of the two transgenic GmRPN11d lines OE#15 and OE#18 changed little compared with before treatment, and the color was obviously lighter than the wild type (such as Figure 4 (A and B in Figure 3) Heterologous GmRPN11d expression in Arabidopsis enhanced the antioxidant capacity of Arabidopsis under salt stress, resulting in reduced ROS accumulation in Arabidopsis, reduced oxidative damage in Arabidopsis leaves, and enhanced Arabidopsis resistance to salt stress.

[0187] When plants are in adverse conditions, they often evolve a series of protective mechanisms to adapt to the living environment. Under adverse conditions, the reactive oxygen ROS in plants tends to increase, and plants will remove ROS by increasing the activity of antioxidant enzymes in the body. For example, POD and SOD can effectively prevent reactive oxygen and other peroxide free radicals from damaging the membrane system, and CAT can decompose H2O2 produced by adverse stress in plants, thereby protecting plants. Therefore, the enzyme activities of SOD, POD, and CAT and the contents of MDA and H2O2 in WT and two transgenic lines OE#15 and OE#18 in the treatment group and the control group were determined and analyzed. Figure 4It can be seen from C in and Tables 17, 18, and 19 that under water treatment conditions, the enzyme activities of POD, SOD, and CAT and the contents of MDA and H2O2 of WT and the two transgenic lines of the GmRPN11d gene remained at normal levels and were relatively close. In the salt treatment group, the activities of the three oxidative scavenging enzymes SOD, POD, and CAT and the MDA content in Arabidopsis thaliana increased, but the enzyme activities and MDA contents of the two transgenic lines of the GmRPN11d gene OE#15 and OE#18 were higher than those of WT. Moreover, the trend of the change in H2O2 content under salt treatment conditions was exactly opposite to that of catalase CAT, which was consistent with the above phenotypes and indicated that under salt stress, transgenic Arabidopsis thaliana with the GmRPN11d gene reduced the degree of oxidative damage of plants by increasing the activity of antioxidant enzymes, thereby enhancing the ability of plants to resist salt stress.

[0188] Table 17 Changes in SOD, POD and CAT activities in seedlings under salt stress

[0189]

[0190] Table 18 Changes in MDA and H2O2 contents in seedlings under salt stress

[0191]

[0192]

[0193] Table 19 Changes in H2O2 content in seedlings under salt stress

[0194]

[0195] To further analyze the reasons for the enhanced salt resistance of transgenic GmRPN11d Arabidopsis, the ABA signaling pathway genes AtNCED3, AtABA3, AtABI5 and stress-related genes AtRD29A, AtSOS1 and AtSOS2 were analyzed at the transcriptional level under salt stress. Arabidopsis leaves treated with salt stress for 3 days at the same leaf position were taken, RNA was extracted and reverse transcribed, and RT-qPCR was used to detect the expression levels of ABA signaling pathway genes AtNCED3, AtABA3, AtABI5 and stress-related genes AtRD29A, AtSOS1 and AtSOS2. Compared with the control group, the expression levels of ABA signaling pathway genes AtNCED3, AtABA3, AtABI5 and stress-related genes AtRD29A, AtSOS1 and AtSOS2 were increased under salt stress (e.g. Figure 5 It is possible that the GmRPN11d gene in Arabidopsis thaliana resists salt stress by increasing the expression levels of AtNCED3, AtABA3, AtABI5, AtSOS1 and AtSOS2 in the plant under salt stress.

[0196] 3. Heterologous expression of GmRPN11d enhances Arabidopsis thaliana's sensitivity to ABA

[0197] From the above results, it can be seen that heterologous overexpression of GmRPN11d can enhance the expression of ABA biosynthesis genes and improve the salt resistance of Arabidopsis. The salt resistance of transgenic GmRPN11d Arabidopsis may be regulated by the ABA pathway. First, whether GmRPN11d overexpression affects the sensitivity of Arabidopsis to ABA was detected during the seed germination period. The three strains of WT, OE#15 and OE#18 were sterilized and vernalized, and inoculated into 1 / 2MS, 1 / 2MS+1μM ABA and 3μM ABA solid culture media for germination experiments. 1 / 2MS solid culture medium was used as the control, and photos were taken every day to record the germination of each strain of Arabidopsis, with three biological replicates in each group. It was found that the germination conditions of the control group and the treatment group, WT and OE#15, OE#18 were basically the same, and WT, E#15 and OE#18 did not unfold their leaves on the ABA-treated culture medium (such as Figure 6 A BC and shown in Tables 20, 21, and 22).

[0198] Table 20 Statistics of germination rate under ABA stress treatment

[0199]

[0200]

[0201] Table 21 Leaf expansion rate under ABA stress treatment

[0202]

[0203] Table 22 Statistics of root length under ABA stress treatment

[0204]

[0205] The germinated Arabidopsis seeds were inoculated in vertical culture plates to observe root growth, and 1 / 2MS medium was used as a blank control. It was found that in the control group, there was no significant difference in the growth of the three strains of WT, OE#15 and OE#18; in the ABA treatment group, OE#15 and OE#18 showed obvious yellowing in the aboveground parts, and the length of the underground roots was significantly shorter than that of WT; through the determination of chlorophyll content, it was found that the chlorophyll content of the overexpression line in the treatment group was also significantly lower than that of WT. As shown in Table 23, overexpression of GmRPN11d enhanced the sensitivity of Arabidopsis to ABA.

[0206] Table 23 Statistics of chlorophyll content

[0207]

[0208] The sensitivity of Arabidopsis thaliana overexpressing GmRPN11d to ABA was further verified by the in vitro leaf chlorosis experiment. The first rosette leaf of 20-day-old WT, OE#15 and OE#18 seedlings was placed in solutions containing 30μM ABA and 60μM ABA for in vitro treatment, with water treatment as the control and cultured at 24°C for 48h. Compared with WT, OE#15 and OE#18 leaves turned yellow significantly, and the change was more obvious under the 30μM ABA treatment condition (e.g. Figure 7 By measuring the chlorophyll and MDA contents, it was found that in the treatment group, the chlorophyll content of the two overexpression lines was significantly lower than that of WT, and the MDA content was significantly higher than that of WT (as shown in Figure 7 B and Table 24). This indicates that exogenous ABA treatment can accelerate the damage of Arabidopsis leaves overexpressing GmRPN11d and accelerate the senescence of Arabidopsis.

[0209] Table 24 Statistics of chlorophyll and MDA content in detached leaves before and after ABA treatment

[0210]

[0211] Leaf yellowing is the most obvious feature of plant aging. During the aging process, the ABA content in the plant body will increase significantly, and ABA will promote stomatal closure. In order to explore whether overexpression of GmRPN11d affects stomatal movement under ABA treatment, the epidermis of 23-day-old Arabidopsis rosette leaves was torn off and placed in a stomatal opening buffer containing 5mM KCl, 50mM CaCl2, and 10mM MES-Tris (pH=5.8). It was placed in a light incubator for 2h to fully open the stomata, and then treated with 30μM ABA to observe the stomatal movement. The aspect ratio of WT, OE#15 and OE#18 was counted using Digimizer software. It can be seen that under ABA stress treatment, compared with WT, the stomata of the two overexpression lines were significantly closed (such as Figure 8 shown).

[0212] Leaf yellowing is the most obvious characteristic of plant senescence, and the most important reason for leaf yellowing is the destruction of chloroplast structure. To further explore the cause of leaf yellowing in Arabidopsis thaliana overexpressing GmRPN11d under exogenous ABA treatment, the expression patterns of chlorophyll degradation-related genes (AtNYE1, AtNYC1, AtPAO, AtPPH) and senescence-related genes (AtSAG12 and AtSAG15) were detected. The 3-day-old seedlings of WT, OE#15 and OE#18 were transferred to 1 / 2MS medium containing 1 μM ABA, and 1 / 2MS solid medium was used as the control group. After culturing for 15 days, leaves from the same part were taken to extract RNA and reverse transcribed into cDNA. RT-qPCR was used to detect the expression levels of AtNYE1, AtNYC1, AtPAO, AtPPH, AtSAG12 and AtSAG15 at the transcriptional level. It was found that the expression levels of these marker genes were upregulated in the treatment groups, and compared with WT, the expression levels of these genes in OE#15 and OE#18 were higher than those in WT (e.g. Fig. 9 shown).

[0213] In order to verify whether the chloroplast structure of the leaves of overexpressing Arabidopsis was damaged under exogenous ABA treatment, the second and third rosette leaves of Arabidopsis cultured on ABA medium for 15 days were cut into strips of 3 mm long and 1 mm wide (avoiding the veins). After fixation, dehydration, infiltration, embedding, polymerization, block cutting, ultrathin sectioning and uranyl acetate-lead citrate staining, the ultrastructure of chloroplasts in leaves of WT, OE#15 and OE#18 were observed by transmission electron microscopy. The materials grown on 1 / 2MS medium were used as the control group, and each sample was repeated 3 times. Under the control conditions, the chloroplast structure of WT was complete and oval, the grana lamellae were arranged neatly, the starch grains were small and the number of chloroplasts in the whole cell was large; after ABA treatment, larger starch grains appeared in the WT strain, and the grana lamellae in the chloroplast began to thin. Compared with WT, the entire chloroplast structure in the GmRPN11d overexpression line began to dissolve and become irregular, the grana lamellae also broke, and the number of chloroplasts in the whole cell decreased (such as Fig.10 This is consistent with the increase in transcriptional expression of chlorophyll degradation genes, thus preliminarily suggesting that heterologous expression of GmRPN11d is involved in ABA-induced plant senescence.

[0214] Example 4. Identification and acquisition of GmRPN11d transgenic soybean hairy roots

[0215] 1. The recombinant vector obtained in Example 1 was transformed into competent Agrobacterium rhizogenes K599, and complex soybean plants containing the GmRPN11d gene were generated by cutting the hypocotyls of soybean seedlings.

[0216] 2. Screening of GmRPN11d soybean hairy root chimeras

[0217] The main root of 7-day-old healthy soybean seedlings was removed and K599-GmRPN11d was applied to the wound. The explants coated with bacterial blocks were then planted in wet sterile vermiculite and watered with K599-GmRPN11d bacterial suspension. The plants were covered with plastic film and placed in a soybean culture room for normal growth. During this period, 1 / 5 B5 salt culture solution was watered to induce the production of hairy roots.

[0218] Results: Detection and acquisition of GmRPN11d transgenic soybean hairy roots: The pCAM35S-GmRPN11d-GFP plasmid was transformed into the competent Agrobacterium rhizogenes strain K599 using the conventional freeze-thaw method. The obtained transformants were spread on solid LB medium containing kanamycin and streptomycin, cultured at 28°C overnight, single colonies were picked, and the bacterial liquid was PCR positive identified using GmRPN11d gene-specific primers. The positive bacterial liquid was activated once and twice (1 ml of the once activated bacterial liquid was applied to the solid LB medium containing kanamycin and streptomycin, cultured at 28°C for two days, and kept for backup). When the OD of the secondary activated bacterial liquid reached 0.04, the OD of the secondary activated bacterial liquid reached 0.04. 600nm =0.6, centrifuge at 4℃5000rpm for 10min, resuspend the bacterial precipitate with 1 / 5B5 salt resuspension solution, and keep the resuspension solution for later use. Cut the main root of healthy soybean seedlings, apply K599-GmRPN11d bacterial blocks on the wound and transplant them into wet sterile vermiculite for cultivation, and water 5ml K599-GmRPN11d bacterial suspension around each explant, cover with plastic film and let it grow normally in the culture room, and water 1 / 5B5 salt nutrient solution during the period to induce it to produce hairy roots. The fluorescence of the grown hairy roots is observed under a fluorescent inverted microscope. Those with fluorescence are hairy root chimeras containing the GmRPN11d gene.

[0219] Example 4. Application of GmRPN11d gene

[0220] 1. Determination of physiological indicators 1. Antioxidant enzyme activity detection

[0221] For the composite soybean plants grown for about 15 days, 0.1g of hairy roots of the treatment group and the control group were taken and placed in 2ml EP tubes containing small steel balls at 12h after salt treatment. The EP tubes containing the materials were placed in liquid nitrogen, fully broken by a crusher, and extraction buffer was added. After centrifugation, the supernatant was collected for the determination of SOD, POD and CAT enzyme activities, with at least 8 replicates for each sample. For specific determination methods, please refer to the superoxide dismutase (SOD) test kit (WST-8 method) (SOD-1-W) and peroxidase (POD) test kit (POD-1-Y) of Suzhou Keming Biotechnology Co., Ltd.

[0222] 2. Malondialdehyde content detection

[0223] The samples were collected after 12 hours of treatment with 200 mM NaCl, and the detection method was the same as the malondialdehyde content detection method for the GmRPN11d gene in Example 3.

[0224] 2. Overexpression of the GmRPN11d gene can enhance soybean resistance to salt stress

[0225] The hairy roots of the composite soybean plants grown for 15 days were observed under a fluorescent inverted microscope for positive detection of hairy root chimeras. Fig.11 As shown in A, the positive hairy roots can be observed under a fluorescence microscope because the GmRPN11d gene is fused with a GFP tag. Next, the complex plants infected with K559 empty bacteria were used as blank controls, and the complex soybean plants were treated with 200mM NaCl. It can be seen that after 12 hours of salt treatment, the leaves of the K599 plants showed obvious wilting and curling, while the leaves of the K599-GmRPN11d complex plants did not wilt significantly. Fig.11 As shown in B and C in the figure. At the same time, the activities of three enzymes, SOD, POD, and CAT, and the content of MDA in the complex soybean plants were detected after 12 hours of salt treatment. It was found that the activities of the three enzymes, SOD, POD, and CAT, were significantly enhanced under salt treatment conditions, and the activities in the K599-GmRPN11d complex soybean plants were higher, indicating that the overexpression of GmRPN11d in soybeans enhanced the activity of antioxidant enzymes, thereby reducing the damage of ion accumulation to soybeans under salt stress, which is consistent with the results of MDA content detection. Fig.11 C in and Table 25. Therefore, overexpression of the GmRPN11d gene in soybean may also enhance the resistance of soybean to salt stress.

[0226] Table 25 MDA content and (SOD, POD, CAT) enzyme activity detection in salt-treated transgenic soybeans

[0227]

Claims

1. GmRPN11d protein or GmRPN11d The use of a gene in improving plant resistance to salt stress or improving plant sensitivity to ABA is characterized in that: The amino acid sequence of GmRPN11d protein is shown in SEQ ID NO.2; GmRPN11d The nucleotide sequence of the gene is shown in SEQ ID NO.1; the improving the salt stress resistance of plants is to improve the salt stress resistance of soybean or Arabidopsis; the improving the ABA sensitivity of plants is to improve the ABA sensitivity of Arabidopsis.

2. The use according to claim 1, characterized in that: The concentration of salt applied in the salt stress resistance is 100 mM-200 mM; the concentration of ABA in the improvement of plant sensitivity to ABA is 1 µM-3 µM.

3. A method for cultivating soybean plants resistant to salt stress, characterized in that: The method comprises the following steps: (1) Amplification GmRPN11d Gene, insert the gene sequence into the plant overexpression vector; GmRPN11d The nucleotide sequence of the gene is shown in SEQ ID NO.1 (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transfer the vector into soybean to obtain transgenic plants; (3) Identify the transgenic soybeans obtained in step (2) to obtain positive transgenic plants.

4. A method for cultivating Arabidopsis plants with increased ABA sensitivity, characterized in that: The method comprises the following steps: (1) Amplification GmRPN11d Gene, insert the gene sequence into the plant overexpression vector; GmRPN11d The nucleotide sequence of the gene is shown in SEQ ID NO.1 (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transfer into Arabidopsis thaliana to obtain transgenic plants; (3) Identify the transgenic Arabidopsis thaliana obtained in step (2) to obtain positive transgenic plants.

5. A method for increasing the antioxidant enzyme activity of soybean or Arabidopsis plants under salt stress, characterized in that: The method is to overexpress GmRPN11d After the transgenic soybean or Arabidopsis plants with the gene were placed in a 100 mM NaCl environment for stress treatment, the activities of antioxidant enzymes in the transgenic soybean or Arabidopsis plants were detected; GmRPN11d The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that The antioxidant enzyme is SOD, POD or CAT.

7. A method for increasing the number of lateral roots, fresh weight of aboveground parts and chlorophyll content of soybean or Arabidopsis plants under salt stress, characterized in that: The method is to overexpress GmRPN11d Gene-transgenic soybean or Arabidopsis plants were placed in a 100 mM NaCl environment for stress treatment; GmRPN11d The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. Overexpression GmRPN11d The use of a plant containing a gene for improving the resistance of soybean plants to salt stress or improving the sensitivity of Arabidopsis plants to ABA is characterized in that: GmRPN11d The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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