Application of GmUGT72E1 gene in regulation and control of soybean plant height and enhancement of drought and alkali stress resistance

Abstract

The invention belongs to the technical field of plant genetic engineering and molecular breeding, and particularly relates to application of a soybean GmUGT72E1 gene in regulation and control of soybean plant height and enhancement of resistance of soybeans to drought and alkali stress. Plant height is a key agronomic character influencing lodging resistance and photosynthetic efficiency of soybeans, drought and alkali stress are core abiotic stress factors restricting yield and quality of the soybeans, and the three factors jointly lead to remarkable global soybean yield reduction loss. Gene cloning, vector construction and transgenic function verification prove that overexpression of the GmUGT72E1 gene can reduce the plant height of soybeans by 15%-25%, meanwhile, the tolerance of the soybeans to drought and alkali stress is remarkably improved, the relative water content of leaves under the drought stress is increased by 25%-30% compared with that of wild type leaves, and chlorophyll under the alkali stress is increased by 40%-50% compared with that of the wild type leaves. According to the application disclosed by the invention, the dual regulation and control functions of the GmUGT72E1 gene are defined, key gene resources and technical support are provided for soybean dwarf stress-resistant collaborative improvement, dwarf stress-resistant soybean varieties can be cultivated by means of transgenosis, gene editing and the like, and the application has important agricultural application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, and particularly relates to soybeans. GmUGT72E1 New uses for the gene include its application in regulating soybean plant height and improving soybean resistance to drought and alkali stress. Background Technology

[0002] Soybeans Glycine max As one of the world's most important dual-purpose crops (food and oil), soybean holds an irreplaceable strategic position in food processing, feed production, and industrial raw material supply. However, soybean production faces the dual challenges of abnormal plant height and abiotic stress: on the one hand, excessive plant height easily leads to lodging in the later stages of soybean growth, causing difficulties in mechanical harvesting, grain shedding, and an average yield reduction of 20%-30%; on the other hand, abiotic stresses such as drought and soil alkalization are widespread. Globally, drought causes a 30%-50% reduction in soybean yield annually, while alkali stress can damage soybean roots and hinder photosynthesis, resulting in a yield reduction of 25%-40%. The combination of these two factors seriously threatens the safety of soybean production. Plant secondary metabolites play a crucial role in important physiological processes such as plant growth and development, innate immunity, disease and pest defense, and environmental stress response. Plant glycosyltransferases are key enzymes that catalyze the glycosylation modification of secondary metabolites and play a central role in regulating plant growth, development, and environmental adaptability. The UGT72 family of glycosyltransferases, an important branch of the glycosyltransferase family, has been shown to participate in the glycosylation modification of substances such as lignin monools and flavonoids. These substances not only affect plant cell wall structure but also participate in antioxidant defense, suggesting that UGT72 family genes may simultaneously regulate plant growth traits and stress resistance. However, to date, information regarding the members of the soybean UGT72 family remains limited. GmUGT72E1 Research on this gene remains incomplete; its involvement in soybean plant height regulation, its responsiveness to drought and alkali stress, and its potential for synergistic regulation of dwarfing and stress resistance are all unclear. In current soybean breeding practices, dwarfing and stress-resistance genes are mostly discovered independently, lacking key genes that can simultaneously improve plant height and stress resistance. This results in long development cycles and low efficiency in breeding soybean varieties with synergistic dwarfing and stress resistance. Therefore, identifying genes with dual regulatory functions is crucial. GmUGT72E1 To elucidate the role of genes in plant height and responses to drought and alkali stress is of urgent need and great significance for overcoming the bottleneck of synergistic breeding of soybean dwarfing and stress resistance. Summary of the Invention

[0003] The purpose of this invention is to disclose GmUGT72E1 This gene plays a dual role in regulating soybean plant height and enhancing resistance to drought and alkali stress. Research on the function of this gene provides genetic resources and technical support for breeding new soybean varieties resistant to lodging and stress. The present invention provides the following technical solution:

[0004] First aspect

[0005] This invention provides GmUGT72E1 The application of genes in regulating soybean plant height and enhancing resistance to drought and alkali stress, including:

[0006] 1. The application is achieved through overexpression GmUGT72E1 This is achieved by increasing the expression level of the protein in the gene;

[0007] 2. The aforementioned GmUGT72E1 The genome sequence of the gene is shown in SEQ ID NO.1;

[0008] 3. The aforementioned GmUGT72E1 The coding sequence of the gene is shown in SEQ ID NO.2;

[0009] 4. The aforementioned GmUGT72E1 The amino acid sequence of the gene is shown in SEQ ID NO.3;

[0010] 5. The plant mentioned is selected from: soybean;

[0011] Second aspect

[0012] This invention provides a portable GmUGT72E1 The application of gene plasmids in plant dwarfing and stress resistance, including:

[0013] The plasmid is pBIB-BSTAT-35S-GWR-FLAG Used to drive GmUGT72E1 The gene promoter is a strong 35S promoter;

[0014] Third aspect

[0015] This invention provides pBIB-BASTA-35S-FLAG The application of recombinant vectors in plant dwarfing and stress resistance, including:

[0016] 1. The recombinant vector contains GmUGT72E1 The encoded sequence is shown in SEQ ID NO.2;

[0017] 2. The carrier is pBIB-BSTAT-35S-FLAG ;

[0018] Fourth aspect

[0019] This invention provides GmUGT72E1 The application of genes in plant dwarfing and stress-resistance co-breeding, including:

[0020] 1. The application is achieved by regulating plant height and improving plant tolerance to abiotic stress;

[0021] 2. Applicable plants are the same as in the first aspect;

[0022] Fifthly, the present invention provides GmUGT72E1The method for constructing gene-overexpressing plants includes the following steps:

[0023] Step 1: Search on Phytozome (https: / / phytozome-next.jgi.doe.gov / ) GmUGT72E1 CDS sequence of the (Glyma.01G177900) gene;

[0024] Step 2: Primer design;

[0025] Step 3: Using soybean William 82 as a template, amplification GmUGT72E1 CDS sequence;

[0026] Step 4: Homologous recombination of the target fragment from Step 3 into... pBIB-BASTA-35S-GWR-FLAG On the carrier;

[0027] Step 5: Transform the recombinant plasmid from Step 4 into DH5α for propagation and expression;

[0028] Step 6: Select single colonies for PCR and sequencing identification;

[0029] Step 7: Transform the recombinant plasmid from Step 6 into EHA105 competent cells for expression;

[0030] Step 8: Obtain soybean overexpression material by using Agrobacterium-mediated genetic transformation of soybean cotyledonary nodes, based on the Agrobacterium-mediated genetic transformation method identified in Step 7.

[0031] Step 9: The transgenic material obtained in Step 8 was subjected to 0.1% Basta, PCR and transcriptional identification. After screening for homozygous positive materials, phenotypic experiments were carried out in the laboratory.

[0032] The beneficial effects of this invention are:

[0033] This invention is the first to demonstrate GmUGT72E1 This gene possesses a dual function: regulating soybean plant height and enhancing resistance to drought and alkali stress. However, even a single resistance phenotype has not been previously observed. This study fills a gap in the functional research of this gene, expands our understanding of the regulatory roles of the UGT72 family of genes, and provides new gene targets and theoretical basis for research on the molecular mechanisms of soybean lodging and stress resistance. Through transgenic technology, it can be... GmUGT72E1 Genes are applied to the breeding of new soybean varieties that are resistant to lodging and stress. The gene resources and application methods involved in this invention have broad application prospects and can be promoted and applied in different soybean ecological regions, thus promoting the sustainable development of the soybean industry. Attached Figure Description

[0034] Figure 1 The image shows the conditions under abiotic stress. GmUGT72E1 Gene expression levels, where A represents drought stress and B represents alkaline stress;

[0035] Figure 2 As shown GmUGT72E1 Positive identification results of overexpressing soybean plants (A), and the results of overexpressing soybean plants... GmUGT72E1 The expression level (B). GmUGT72E1 -OE-2 and GmUGT72E1 -OE-13 indicates two lines that overexpress soybean ( GmUGT72E1 -OE-2-1、 GmUGT72E1 -OE-2-2、 GmUGT72E1 -OE-2-3 indicates that sample number 2 is a duplicate. GmUGT72E1 (Similarly for OE-13);

[0036] Figure 3 The figure shows the whole plant phenotype of soybean under normal conditions (A) and the plant height statistics (B).

[0037] Figure 4 The image shows the whole plant phenotype (A) and relative water content (B) of soybean under drought stress.

[0038] Figure 5 The image shows the whole plant phenotype (A) and chlorophyll content (B) of soybean under alkali stress. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0040] The experimental materials used in the embodiments of this invention are as follows:

[0041] Plant material: DN50;

[0042] bacterial strain: Agrobacterium ( Agrobacterium EHA105, Escherichia coli ( Escherichia coli DH5α;

[0043] plasmid: pBIB-BSTAT-35S-GWR-FLAG Used for gene cloning and overexpression vector construction;

[0044] The experimental culture medium is as follows:

[0045] LB liquid (solid) medium contains 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and (10 g / L agar).

[0046] YEP liquid (solid) medium is a medium containing 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, (10 g / L agar), and pH=7.0.

[0047] COC co-culture medium (0.321 g / L G398, 3.9 g / L MES, 30 g / L sucrose, 40 mg / L AS acetylsyringone, 0.25 mg / L GA3, 1.67 mg / L BAP, 0.4 g / L L-cysteine, 0.154 g / L DTT, 0.158 g / L Sodium thiosulfate);

[0048] SI bud induction medium (3.21 g / L G398, 0.6 g / L MES, 30 g / L sucrose, 5 g / L agar powder, 2 g / L plant gel, 1.7 mg / L BAP, 50 mg / L Timetin, 50 mg / L Vancimycin, 100 mg / L Cefotaxime, 6 mg / L Glufosinate);

[0049] SE shoot elongation medium (4.43 g / L M519, 0.6 g / L MES, 30 g / L sucrose, 50 mg / L Timetin, 50 mg / L Vancimycin, 100 mg / L Cefotaxime, 1 mg / mL GA3, 0.1 mg / 500ul IAA, 50 mg / mL LAPsp, 100 mg / mL L-Pyroglutamic acid, 6 mg / L Glufosinate);

[0050] RM root induction medium (4.43 g / L M519, 0.6 g / L MES, 20 g / L sucrose, 5 g / L plant gel, 50 mg / L Timetin, 50 mg / L Vancimycin, 100 mg / L Cefotaxime, 50 mg / mL Asp, 1 mg / mL LIBA);

[0051] Example 1

[0052] GmUGT72E1 Gene expression under drought and alkaline stress

[0053] (1) DN50 seeds were wrapped in gauze and soaked in water for 16-48 hours. Seeds with good growth and uniform vigor were selected and cultured in Pinscher potting mix until stage V1. RNA was extracted from DN50 seeds after 0, 6, 12, and 24 hours of treatment with white light and 10% PEG6000, respectively, from both treated and untreated DN50 seeds. Actin was used as an internal reference gene, and the expression of the target gene was analyzed by RT-qPCR. (See attached table). Figure 1 (A).

[0054] (2) After treatment with white light and 150 mM NaHCO3 for 0, 6, 12, and 24 h respectively, RNA was extracted from DN50 cells subjected to different stress durations and those not treated. Actin was used as an internal reference gene, and the expression of the target gene was analyzed by RT-qPCR. See [link to RT-qPCR analysis]. Figure 1 (B).

[0055] Example 2

[0056] GmUGT72E1 Gene cloning and construction of overexpression materials

[0057] (1) Query using the soybean database Soybase (https: / / soybase.org / GlycineBlastPages / ). GmUGT72E1 CDS sequence of the gene.

[0058] (2) Using cultivated soybean DN50 as material, total RNA was extracted using the Fuji Biotechnology Co., Ltd. RNA mini-extraction kit (RE-04013), and reverse transcription was performed. The obtained cDNA was stored at -20℃ for later use. The specific implementation method is as follows: Take 1 g of soybean root tissue, put it in a pre-cooled mortar, add liquid nitrogen, and grind the root tissue into a fine and uniform powder using a mortar and pestle. Then, transfer 50 mg of powder into a 1.5 mL centrifuge tube. Extract total RNA using the RNA mini-extraction kit. Take 1 μg of total RNA for reverse transcription. The reaction system is as follows: 2 μL 5×gDNA Clean Reation Mix, 1 μg RNA, and RNase-free water to 10 μL. React at 42℃ for 2 min to remove genomic DNA. Add 4 μL 5×Evo M-MLVRT Reation Mix and 6 μL RNase-free water to a PCR tube containing 10 μL of reaction solution. Incubate in a metal bath at 37℃ for 15 min, then at 85℃ for 5 s. The obtained cDNA was stored at -20℃ for later use.

[0059] (3) Design cloning primers

[0060] primer1- GmUGT72E1-F:5'-ATTTACGAACGATAGCCGGTACCATGGTAACATCAAAGCCTCATGCAGC-3'

[0061] primer2- GmUGT72E1 -R:3'-GTAGTCCACCACTTTGTACAGAGAGCGAACGACGTCGCTGAGCACA-5'

[0062] (4) PCR amplification

[0063] The reaction was performed using the Novizan Biotech kit 2×Phanta Max Master Mix (P510-01), and the reaction system is shown in Table 1 below:

[0064] Table 1 PCR amplification reaction system

[0065] Reagent System / μL cDNA 1 primer1 0.4 Primer2 0.4 2 × Phanta Max Master Mix 5 ddH2O 3.2

[0066] PCR program: 98℃, 3 min pre-denaturation; 98℃, 10 s denaturation; 55-60℃, 15 s annealing; 72℃, 2 kb / min extension, 32 cycles; 72℃, 5 min amplification extension. Store at 4℃.

[0067] (5) Collection and purification of PCR products

[0068] The target fragment was collected and purified using the SanPrep Column DNA Gel Recovery Kit (B518131-0050) from Sangon Biotech. The specific steps are described in the kit instructions.

[0069] (6) Homologous recombination

[0070] The recovered fragments are attached to the vector via an infusion reaction. pBIB-BSTAT-35S-GWR-FLAG Using Bsp1407Ⅰ and KpnⅠ restriction endonucleases, first... pBIB-BSTAT-35S-GWR-FLAG The vector was linearized by enzyme digestion and then recombinantly cloned using the Novozymes ClonExpress II One Step Cloning Kit (C112). GmUGT72E1 Build to pBIB-BASTA-35S-FLAG On the carrier. The reaction system is shown in Table 2:

[0071] Table 2 Homologous recombination reaction system

[0072] Reagent System / μL Linearized vector pBIB-BSTAT-35S-GWR-FLAG DNA fragment GmUGT72E1 5×CEII Buffer 2 ExnaseII 1 ddH2O Up to 10

[0073] (7) Escherichia coli transformation (DH5α)

[0074] The recombinant reaction product was added to competent cells, gently mixed, and incubated on ice for 30 min; then incubated in a water bath at 42°C for 45 s, followed by an ice bath for 5 min. 800 μL of LB broth was added; the cells were incubated at 37°C and 200 rpm for 1 h; after centrifugation, all cells were plated onto plates containing 50 mg / mL AMP or 50 mg / mL Kan, and incubated overnight at 37°C. Once colonies had grown, positive clones were detected by PCR, and subsequently, positive clones were sequenced for verification.

[0075] (8) Identification and sequencing

[0076] The primers were designed as follows:

[0077] Primer1- GmUGT72E1 -F:5'-ATTTACGAACGATAGCCGGTACCATGGTAACATCAAAGCCTCATGCAGC-3'

[0078] Primer3-BF-FLAG-R:3'-TGCTACTGCGTTTACTGCCA-5'

[0079] Table 3 Colony PCR Reaction System

[0080] Reagent System / μL Template 1 Primer1 0.4 Primer3 0.4 Vazyme P112-AA 5 ddH2O 3.2

[0081] White single colonies were identified by PCR (see Table 3 above). After identification as positive clones, positive clones were picked and cultured in 50 μg / mL Kan liquid LB medium (5 g / L Yeast extract, 10 g / L Tryptone, 10 g / L NaCl) at 37°C overnight with shaking. Plasmids were then extracted using the TSP501-50 plasmid extraction kit and sequenced.

[0082] (9) Agrobacterium transformation

[0083] Transform 1 μg of recombinant plasmid into Agrobacterium competent cells, incubate on ice for 5 min; flash freeze in liquid nitrogen for 5 min; incubate in water at 37°C for 5 min; incubate on ice for 5 min. Add 800 μL of LB liquid medium, incubate at 28°C and 200 rpm for 2 h, spread on plates containing 50 mg / mL Rif or other antibiotics, and incubate at 28°C for 2 days. After colonies have grown, detect positive clones by PCR.

[0084] (10) GmUGT72E1 Gene overexpression in soybean

[0085] This invention uses Agrobacterium-mediated genetic transformation of cotyledonary nodes to... GmUGT72E1Two lines were obtained by transferring the soybean into DN50. The soybean was transformed into a conventional experimental method in this field, which will not be described in detail here.

[0086] (11) GmUGT72E1 Identification of positive seedlings with overexpression material

[0087] PCR identification

[0088] Primer4-BF-35S-F:5'-CAAACGAATCTCAAGCAATC-3'

[0089] Primer3-BF-FLAG-R:3'-TGCTACTGCGTTTACTGCCA-5'

[0090] Based on the primer pair above, DNA-level identification of genetically modified soybeans was performed, and the results are shown below. Figure 2 (A).

[0091] Identification of transcriptional levels in genetically modified soybeans

[0092] Using GmACTIN3 (Glyma.09g17040) as an internal reference gene, quantitative real-time PCR was performed on the Novozymes SYBR kit (Q711-02 / 03) to determine the expression level of the GmUGT72E1 gene in wild-type and positive transgenic soybeans. The results are shown in the figure. Figure 2 (B)

[0093] Example 3

[0094] Phenotypic identification of genetically modified soybeans

[0095] (1) Plant height phenotypic identification

[0096] Twelve soybean GmUGT72E1 overexpression lines (line 2 and line 13) and DN50 seeds were planted in a greenhouse (conditions: 25℃, 16h light / 8h dark, relative humidity 60%). Plant height (from cotyledon node to plant tip) was measured periodically. Results are shown below. Figure 3 The plant height of the overexpression lines was significantly reduced.

[0097] (2) Identification of drought stress phenotypes

[0098] 12 soybeans GmUGT72E1 Seeds from overexpressing lines (line 2, line 13) and DN50 were planted in white culture pots. The culture pots were placed under white light for 24 days of growth culture. After reaching stage V1, watering was stopped for 7 days, and the phenotype was observed and recorded; then watering was resumed for 3 days, and the phenotype was observed and recorded again (results are shown in...). Figure 4As shown, the drought tolerance of the overexpression line was significantly higher than that of the wild type. Fresh weight (Wf) of compound leaves was measured before and after drought. The weighed leaves were placed in a petri dish containing 20 mL of deionized water and soaked for 12 h. The leaves were then removed, dried with paper, and weighed again for saturated fresh weight (Wt). The weighed leaves were placed in a clean envelope and dried in a drying oven at 80℃ for 48 h. The dry weight after drying was measured and recorded as Wd. The relative water content of the leaves was calculated using the following formula (see results). Figure 4 ).

[0099]

[0100] (3) Identification of alkali stress phenotype

[0101] Twelve soybean GmUGT72E1 overexpression lines (line 2 and line 13) and DN50 seeds were planted in white culture pots. The culture pots were placed under white light for 24 days of growth culture. After a 10-day growth cycle, the plants were treated with 150 mM NaHCO3 via rhizosphere irrigation. Phenotypic characteristics were observed and photographed 8 days later (results are shown in [link to results]). Figure 5 As shown, the overexpression strain exhibited significantly higher alkali tolerance than the wild type. Simultaneously, 0.1 g of single leaves were taken and tripled, and placed in 15 mL stoppered graduated test tubes. 15 mL of extraction buffer (acetone:water = 4:1) was added, and the tubes were soaked in the dark for approximately 24 hours, until the leaves completely lost their green color. The mixture was inverted several times during this period to ensure a uniform color of the extract. The mixed extract (acetone:water = 4:1) served as a blank control. The absorbance of the extract was measured colorimetrically at wavelengths of 663.2 nm and 646.8 nm using a UV-Vis spectrophotometer. The concentrations of chlorophyll and total carotenoids per unit area were calculated using the following formulas (results are shown in [link to results]). Figure 5 ).

[0102] Calculate the total chlorophyll content (Chla+b) and the ratio of chlorophyll a to chlorophyll b (Chla / b).

[0103] In the above formula, V refers to the total volume of the extract; W refers to the fresh weight of the material.

[0104] In summary, GmUGT72E1 Genes play an important role in regulating soybean plant height and responding to drought and alkali stress. This invention provides information on... GmUGT72E1 Preliminary research was conducted on the dwarfing, drought-resistant, and alkali-resistant functions of this gene, laying a solid foundation for a deeper understanding of its functions and mechanisms in regulating soybean plant height and responding to drought and alkali stress.

[0105] Furthermore, GmUGT72E1 Gene overexpression can be applied to crops such as rapeseed and corn to regulate their plant height and enhance their tolerance to drought and alkaline stress.

[0106] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the plant height, drought and alkali stress and specific details described in the above embodiments. All simple modifications to the technical solutions of the present invention within the scope of the technical concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. Soybeans GmUGT72E1 Application of genes in regulating soybean plant height and enhancing soybean resistance to drought and alkali stress.

2. The application according to claim 1, characterized in that: The plant is selected from soybeans.

3. The application according to claim 2, characterized in that: The GmUGT72E1 It is overexpressed in the plant.

4. The application according to claim 2, characterized in that: The GmUGT72E1 The genome sequence is shown in SEQ ID NO.

1.

5. The application according to claim 2, characterized in that: The GmUGT72E1 The encoded sequence is shown in SEQ ID NO.

2.

6. The application according to claim 2, characterized in that: The GmUGT72E1 The protein sequence is shown in SEQ ID NO.

3.

7. The application of a plasmid in the synergistic effect of plant dwarfing and stress resistance, characterized in that: The plasmid carries GmUGT72E1 Genes, and the GmUGT72E1 The gene was overexpressed in the plant.

8. The application of a recombinant vector in the synergistic effect of plant dwarfing and stress resistance, characterized in that: The recombinant vector carries GmUGT72E1 Genes, and the GmUGT72E1 The gene was overexpressed in the plant.

9. GmUGT72E1 The application of genes in plant dwarfing and stress-resistance synergistic breeding is characterized by: The purpose of the molecular breeding is to dwarf plants and improve their tolerance to drought and alkali stress.

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