Application of overexpression of GmGAI gene in improving aluminum stress tolerance of soybean

Abstract

The application discloses overexpression GmGAI The application belongs to the technical field of plant stress resistance and aims to improve the aluminum stress resistance of soybeans. The application provides an application of GmGAI protein in improving the aluminum stress resistance of soybeans, and the amino acid sequence of the GmGAI protein is shown as SEQ ID NO. 1. The application provides a theoretical basis for the aluminum stress resistance of soybeans.

Description

Technical Field

[0001] This invention belongs to the field of plant stress resistance technology, specifically involving overexpression GmGAI Application of genes in improving the aluminum stress tolerance of soybeans. Background Technology

[0002] Approximately 30% of the world's land area and 50% of its potential arable land are composed of acidic soils. In acidic soils (pH < 5), aluminum ions are activated into a free state, which enters the roots through passive absorption by plants, damaging the internal structure of root cells, hindering the absorption of water and nutrients by the roots, producing strong toxicity to the roots, and thus causing yield reduction.

[0003] Soybeans Glycine max Soybean roots (L.) are particularly sensitive to aluminum stress; even micromolar levels of aluminum can inhibit root growth, making it one of the key abiotic stresses limiting soybean production. While applying quicklime to the soil can neutralize soil acidity and reduce aluminum activation, this approach presents challenges such as increased costs and the inability to alter the pH of deeper soil layers. Therefore, identifying aluminum-tolerant genes in soybeans and cultivating new aluminum-tolerant germplasm is crucial. Summary of the Invention

[0004] The purpose of this invention is to improve the resistance of soybeans to aluminum stress.

[0005] This invention provides an application of GmGAI protein in improving the aluminum stress resistance of soybeans. The amino acid sequence of GmGAI protein is shown in SEQ ID NO.1.

[0006] This invention provides an overexpression GmGAI Application of genes in improving the aluminum stress tolerance of soybeans GmGAI The gene is shown in SEQ ID NO.2.

[0007] This invention provides a product containing GmGAI The application of recombinant gene vectors in improving the aluminum stress tolerance of soybeans is characterized by, GmGAI The gene is shown in SEQ ID NO.2.

[0008] Further specifying, the launch vehicle is pB7WG2.

[0009] This invention provides a product containing GmGAI Application of recombinant microbial cells in improving the aluminum stress tolerance of soybeans. GmGAI The gene is shown in SEQ ID NO.2.

[0010] To further specify, the originating microbial cells are Escherichia coli or Agrobacterium.

[0011] Further, the aluminum stress condition is specified as 25 µM AlCl3.

[0012] This invention provides a breeding method for improving the aluminum stress tolerance of soybeans, the specific steps of which are as follows: Step 1: Amplification GmGAI Genes, inserting gene sequences into expression vectors; GmGAI The gene is shown in SEQ ID NO. 2; Step 2: Transform the vector obtained in step (1) into Agrobacterium, infect soybean cotyledon node explants, and obtain transgenic soybeans; Step 3: Identify the genetically modified soybeans obtained in step (2) to obtain positive genetically modified soybeans.

[0013] This invention provides a soybean resistant to aluminum stress, by overexpressing [a specific gene] in soybean. GmGAI Genes, the ones mentioned GmGAI The gene is shown in SEQ ID NO.2.

[0014] This invention provides an overexpression GmGAI The application of transgenic soybean plants with the gene in breeding to improve soybean's tolerance to aluminum stress, the aforementioned GmGAI The gene is shown in SEQ ID NO.2.

[0015] Beneficial effects: This invention obtained T5 generation transgenic soybeans by introducing the recombinant plasmid pB7WG2-GmGAI into the soybean variety Dongnong 50. GmGAI Positive plants of the genetically modified soybean were obtained, and the GmGAI protein was found to enhance plant stress resistance: under aluminum stress conditions, compared with the recipient control Dongnong 50, the T5 generation of genetically modified soybeans showed improved resistance. GmGAI The genetically modified soybean lines exhibited good growth, increased relative taproot elongation, higher aboveground fresh weight, and reduced reactive oxygen species accumulation. Therefore, GmGAI protein holds significant theoretical and practical value in cultivating soybean plants with enhanced aluminum tolerance, and has broad application potential and market prospects in the agricultural field. Attached Figure Description

[0016] Picture 1 This is a diagram illustrating the construction process of the recombinant plasmid pB7WG2-GmGAI.

[0017] The receptor control Dongnong 50 will be referred to as WT below. GmGAI-OE-2 , GmGAI-OE-3 and GmGAI-OE-4 Representative transfer GmGAI Three strains of genetically modified soybeans.

[0018] Picture 2 For DNA-level screening of T5 generation transgenic cells GmGAI Genetically modified soybeans, one of which is GmGAI-OE-2 ,2 is GmGAI-OE-3 3-5 GmGAI-OE-4 6 represents WT.

[0019] Picture 3 To detect T5 generation using colloidal gold test strips GmGAI Genetically modified soybeans, of which 1 is WT and 2 is GmGAI-OE-2 ,3 is GmGAI-OE-4 .

[0020] Picture 4 Transitioning to T5 GmGAI Screening for glufosinate resistance in genetically modified soybeans, where 1 is WT and 2 is... GmGAI-OE-2 ,3 is GmGAI-OE-4 Apply glufosinate to the upper half of the leaves, but leave the lower half uncoated.

[0021] Picture 5 For RNA-level screening of T5 generation transgenic cells GmGAI Genetically modified soybeans. * indicates significant differences ( p <0.05), ** indicates a highly significant difference ( p <0.01).

[0022] Picture 6 Transitioning to T5 GmGAI Statistical results of taproot elongation and relative taproot elongation rate in soybean seedlings of gene-modified soybean and recipient control Dongnong 50 under aluminum stress. Control represents no aluminum treatment, 25 μM AlCl3 represents aluminum treatment with 25 μM AlCl3, and ** represents highly significant differences. p <0.01), scale = 2 cm.

[0023] Picture 7 Transitioning to T5 GmGAI Statistical results of aboveground phenotype and aboveground fresh weight of soybean seedlings (gene-treated and recipient control, Dongnong 50) under prolonged aluminum stress. * indicates significant difference. p <0.05), ** indicates a highly significant difference ( p <0.01).

[0024] Picture 8 Transitioning to T5 GmGAI Results of root tip staining with nitrotetrazole blue (NBT) and diaminobenzidine (DAB) on genetically modified soybean and recipient control Dongnong 50 seedlings after aluminum stress. Scale bar = 1 cm. Detailed Implementation

[0025] The following examples are provided to help better understand the present invention, but are not intended to limit the invention.

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0027] Unless otherwise specified, the test materials used in the following examples can be purchased from regular biochemical reagent stores.

[0028] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0029] The soybean variety Dongnong 50 is described in the following literature: Jin Y, Qu JJ, Ren GM, Dong L. Effects of transgenic DREB Soybean Dongnong50 on the diversity of soil ammonia-oxidizing bacteria. Agricultural Science & Technology. 2013, 14(7): 988 - 992. In the following text, soybean variety Dongnong50 is referred to as Dongnong50 and is available to the public from Northeast Agricultural University (i.e., the applicant) to replicate the experiments in this application.

[0030] The GV3101 Agrobacterium strain is a product of Tiangen Biotech Co., Ltd.; the pB7WG2 vector is a product of Invitrogen.

[0031] Example 1. Contains GmGAI Gene vector construction 1. GmGAI Cloning of gene sequences (1) Total RNA was extracted from the leaves of Dongnong 50 seedlings using the Trizo1 method and then reverse transcribed to generate cDNA.

[0032] (2) Using the above cDNA as a template, PCR amplification was performed using the primer pair consisting of the sequences SEQ ID NO. 3 and SEQ ID NO. 4 to obtain a 1569-bp sample as shown in SEQ ID NO. 2. GmGAI Gene coding region.

[0033] SEQ ID NO. 1: MKRERQQLGSNAGTSSCGYSSGKSNLWEEEGGMDELLAVVGYKVRSSDMAEVAQKLERLEEAMGNVQDDLTDLSNDAVHYNPSDISNWLQTMLSNFDPLPSEEPEKDSASSDYDLKAIPGKAIYGGGSDALPNPKRVRTDESTRAVVVVDLQENGIRLVHSLMACAEAVENNNLAVAEALVKQIGFLALSQVGAMRKVATYFAEALARRIYRVFPQQHSLSDSLQIHFYETCPYLKFAHFTANQAILEAFQGKNRVHVIDFGINQGMQWPALMQALALRNDGPPVFRLTGIGPPAADNSDHLQEVGWKLAQLAERIHVQFEYRGFVANSLADLDASMLDLREDESVAVNSVFEFHKLLARPGAVEKVLSVVRQIRPEILTVVEQEANHNGLSFVDRFTESLHYYSTLFDSLEGSPVNPNDKAMSEVYLGKQICNVVACEGMDRVERHETLNQWRNRFGSTGFSPVHLGSNAYKQASMLLSLFGGGDGYRVEENNGCLMLGWHTRPLIATSVWQLATKSVVAAH; SEQ ID NO. 2 SEQ ID NO. 3: 5'-TGGAGCTCGGTACCCATGAAGAGAGAGCGCCAGCAGC-3'; SEQ ID NO. 4: 5'-GATCCTGGGATCCCCGAGTGAGCAGCAACCACCGATTTT-3' The reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 2 min, 35 cycles; 72℃ for 10 min.

[0034] 2. Construction of recombinant plasmid pB7WG2-GmGAI. The construction process diagram of recombinant plasmid pB7WG2-GmGAI is shown in [link to diagram]. Picture 1 .

[0035] (1) The above GmGAI Recombination of gene coding region fragments into... Sma The recombinant plasmid pENTRY-FLAG-GmGAI was obtained by digesting the linearized vector pENTRY-FLAG with restriction endonuclease I.

[0036] (2) The recombinant plasmid pENTRY-FLAG-GmGAI and the pB7WG2 vector were subjected to LR reaction (Logistic Regression) to obtain the recombinant plasmid pB7WG2-GmGAI.

[0037] The recombinant plasmid pB7WG2-GmGAI was sequenced. The positive recombinant plasmid pB7WG2-GmGAI contained the DNA sequence shown in SEQ ID NO. 2 of the sequence listing.

[0038] Example 2. (Transfer) GmGAI Obtaining and Identifying Genetically Modified Soybeans Cultured under white light with an intensity of 250 μmol / m² -2 s -1 .

[0039] 1. The recombinant plasmid pB7WG2-GmGAI was introduced into Agrobacterium strain GV3101 to obtain a strain containing... GmGAI Recombinant Agrobacterium GV3101 / pB7WG2-GmGAI gene.

[0040] 2. T0 generation proposed transfer GmGAI Obtaining genetically modified soybeans Agrobacterium-mediated genetic transformation of soybean cotyledonary nodes (described in: Olhoft PM, Donovan CM, Somers DA. Soybean...). Glycine maxTransformation using mature cotyledonary node explants. Methods in Molecular Biology. 2006, 343(12): 385). The GV3101 / pB7WG2-GmGAI obtained in step 1 was introduced into Dongnong 50 to obtain T0 generation. GmGAI Genetically modified soybeans. The specific steps are as follows: (1) Take the seeds of Dongnong 50, sterilize them with chlorine for 16 hours, and then soak them in sterile water for 12 hours.

[0041] (2) After completing step (1), peel the soybeans, separate the two cotyledons, make a light cut near the cotyledon node with a blade, and then put them into a container containing... GmGAI Infect the gene in Agrobacterium resuspension for 30 min.

[0042] The preparation method of Agrobacterium resuspension is as follows: Take a single clone of recombinant Agrobacterium obtained in step 1, add 20 mL of YEP liquid medium, and incubate at 28℃ and 200 rpm until OD. 600 The bacterial suspension (1) was approximately 0.5 mg / L; this suspension was then inoculated into 200 mL of YEP liquid medium and incubated at 28°C and 200 rpm until the OD value reached 0.5%. 600 Bacterial suspension 2 was prepared with a concentration of 1.6-2.0. Bacterial suspension 2 was centrifuged at 5000 rpm for 10 min at room temperature, and the precipitate was collected. The precipitate was resuspended in liquid staining medium to obtain the OD. 600 It is an Agrobacterium resuspension at a concentration of approximately 0.8.

[0043] The solutes and their concentrations in the liquid inoculation medium were 0.321 g / L Gamborg B-5 BasalMedium, 30 g / L sucrose, 3.9 g / L 2-morpholinoethanesulfonic acid (MES), 1.67 mg / L 6-benzylaminopurine (6-BA), 0.25 mg / L gibberellin (GA3), and 0.04 g / L acetylsyringone (AS); the solvent was water; and the pH was 5.4.

[0044] (3) After completing step (2), take soybean cotyledons, first use filter paper to absorb the resuspension, and then place them in a solid co-culture medium and culture at 25°C for 5 days.

[0045] The solutes and their concentrations in the solid co-culture medium were 0.321 g / L B5 medium, 30 g / L sucrose, 3.9 g / L LMES, 1.67 mg / L 6-BA, 0.25 mg / L GA3, 0.04 g / L AS, 0.4 g / L L-cysteine ​​(L-Cys), 0.15 g / L dithiothreitol (DTT), and 0.5% (w / v) agar powder; the solvent was water; and the pH was 5.4.

[0046] (4) After co-culture, the soybean cotyledons were transferred to the shoot induction medium and cultured at 25°C with alternating light and dark conditions (16 h light / 8 h dark) for 28 days.

[0047] The solutes and concentrations of the shoot induction medium were as follows: 0.321 g / L B5 medium, 30 g / L sucrose, 0.59 g / L MES, 1.67 mg / L 6-BA, 200 mg / L cephalosporin (Cef), 200 mg / L termethin (Tim), 5 mg / L glufosinate and 0.75% (w / v) agar powder; the solvent was water; the pH was 5.4.

[0048] (5) Cut off the clustered buds from the soybean cotyledons, transfer the clustered buds to the clustered bud elongation medium, and culture at 25°C with alternating light and dark conditions (16 h light / 8 h dark) until the regenerated plants in the clustered buds elongate to 2 cm.

[0049] The solutes and concentrations of the shoot elongation medium were 4.43 g / L MS, 30 g / L sucrose, 0.59 g / L MES, 0.5 mg / L GA3, 1 mg / L zeatin nucleoside (ZR), 1 mg / L L-asparagine (Asp), 0.1 mg / L auxin (IAA), 200 mg / L Cef, 200 mg / L Tim, 3 mg / L glufosinate and 0.75% (w / v) agar powder; the solvent was water; the pH was 5.6.

[0050] (6) Plantlets regenerated from the clustered buds were cut off from the base and transferred to rooting medium. They were cultured at 25°C with alternating light and dark conditions (16 h light / 8 h dark) to obtain the T0 generation of pseudotransformers. GmGAI Genetically modified soybeans.

[0051] The solutes and their concentrations in the rooting medium were 0.321 g / L B5 medium, 20 g / L sucrose, 3.9 g / L MES, 1 mg / L IBA and 0.8% (w / v) agar powder; the solvent was water; and the pH was 5.6.

[0052] 3. T5 transfer GmGAI Obtaining and Identifying Genetically Modified Soybeans Awaiting T0 conversion GmGAI After the genetically modified soybeans mature, they are harvested, self-pollinated, and the T1 generation of genetically modified soybeans is obtained. GmGAI Genetically modified soybean seeds; soybeans were grown in a greenhouse, and positive plants were identified by the following methods. They were then self-pollinated to the T5 generation for aluminum tolerance testing.

[0053] (1) Extract Dongnong 50 and T5 for transfer GmGAI DNA from the gene line. PCR amplification was performed using a primer pair consisting of SEQ ID NO. 5 and SEQ ID NO. 6 sequences. PCR products were detected by agarose gel electrophoresis, where SEQ ID NO. 5 sequence was derived from... GmGAI The gene sequence, SEQ ID NO. 6, is derived from the pB7WG2 vector. (Transfer / Transfer) GmGAI A band can be detected in soybean lines that are positive for the gene ( Picture 2 ). SEQ ID NO. 5:5'-ACAATGGGTGTCTCATGTTGGG-3; SEQ ID NO. 6: 5'-TGTAGTGGTTGACGATGGTGCA-3'; Detection results are shown below Picture 2 (1: GmGAI-OE-2 ;2: GmGAI-OE-3 3-5: GmGAI-OE-4 ;6: WT receptor control Dongnong 50).

[0054] (2) Immunogold immunochromatographic assay strips (EnviroLogix products) were used to test WT Dongnong 50 and T5 transgenic cells, respectively. GmGAI Genetically modified soybean plants were tested to observe whether Bar protein was expressed (refer to the instructions for the immunogold test strip for specific steps). GmGAI Bar protein expression can be detected in soybean plants that are positive for the gene. Picture 3 The test results are shown below. Picture 3 (1: WT; 2 and 3: GmGAI-OE-2 and GmGAI-OE-4 ).

[0055] (3) Cultivate WT Dongnong 50 and T5 gene transfers GmGAI From seed to full development of trifoliate compound leaves, resistance was assessed by applying a 130 mg / L glufosinate solution to fresh leaves. Plants resistant to glufosinate were designated as the T5 generation transgenic plants. GmGAI Positive plants of genetically modified soybeans ( Picture 4 (1: WT; 2 and 3: WT) GmGAI-OE-2 and GmGAI-OE-4 ).

[0056] (4) Extract WT Dongnong 50 and T5 for transfer GmGAIRNA from the gene line was reverse transcribed into cDNA. A primer pair consisting of SEQ ID NO. 7 and SEQ ID NO. 8 sequences was used for detection. GmGAI Expression of [characteristics]. In transgenic positive lines... GmGAI The expression level was significantly higher than that of Dongnong 50 ( Picture 5 ). SEQ ID NO. 7: 5'-GCGTCGTTTGAAAGGTCCGTGAG-3'; SEQ ID NO. 8: 5'-AGTGGTGGGTTACAAGGTGAGGTCA-3'.

[0057] Example 3: Application of GmGAI protein in improving plant aluminum tolerance 1. Aluminum stress treatment The plant to be tested was obtained in Example 2. GmGAI-OE-2 , GmGAI-OE-4 Compared with the WT receptor control Dongnong 50.

[0058] Aluminum stress treatment group: Soybean seeds were evenly sown on vermiculite and cultured under the following conditions: 16 h light / 8 h dark, temperature 25℃. After 3 days of growth, seedlings with uniform growth were selected and transferred to a 0.5 mM CaCl2 (pH=5.8) solution for pretreatment. After 24 h of pretreatment, the seedlings were transferred to a 0.5 mM CaCl2 (pH=4.5) solution containing 25 µM AlCl3 for aluminum stress treatment.

[0059] Aluminum-free control group: The test plants were parallel to the aluminum stress treatment group and were transferred to 0.5 mM CaCl2 (pH=4.5) solution for further culture after pretreatment.

[0060] Three replicate experiments were set up, and 15 plants of each type were selected for observation and statistical analysis in each replicate experiment.

[0061] 2. Identification of aluminum resistance (1) Main root elongation After 24 h of aluminum treatment, the taproot length of seedlings in the aluminum stress treatment group and the aluminum-free control group was measured, and the relative taproot elongation rate was calculated: Relative taproot elongation rate = [(Taproot length after 24 h of aluminum treatment - Taproot length after 0 h of aluminum treatment) / (Taproot length after 24 h of no treatment - Taproot length after 0 h of no treatment)] * 100%. Results are as follows: Picture 6 As shown, in the aluminum-free control group, WT and transfer GmGAI There was no significant difference in the relative elongation of the taproots among the genetic lines; after treatment with 25 µM AlCl3 for aluminum... GmGAI-OE-2 and GmGAI-OE-4 The relative elongation of the main root is significantly higher than that of Dongnong 50, which indicates that GmGAIOverexpression of the gene can alleviate the inhibition of soybean taproot growth by aluminum stress and improve aluminum tolerance.

[0062] (2) Aboveground phenotype and aboveground fresh weight after long-term aluminum stress treatment WT receptor control Dongnong 50, transgenic GmGAI Gene line ( GmGAI-OE-2 and GmGAI-OE-4 The culture was subjected to aluminum stress treatment (25 µM AlCl3), with the culture medium changed every 3 days. After 30 days of treatment, the aerial phenotype was observed, and the fresh weight of the aerial parts was recorded. Under aluminum-free conditions, the cells were transformed... GmGAI There was no significant difference in aboveground growth between the genetically modified strain and the WT strain; after prolonged aluminum stress, soybean plants in both strains exhibited slow growth and development, and yellowing of true leaves, but the modified strain... GmGAI The leaves of genetically modified soybean lines turn yellow much slower than those of WT soybeans, and the transgenic soybean lines... GmGAI The genetically modified soybean lines have intact trifoliate compound leaves, while the WT lines' trifoliate compound leaves are wilted and brittle. In addition, the genetically modified... GmGAI The aboveground fresh weight of the genetically modified strain was significantly higher than the total weight (WT). This further illustrates... GmGAI Overexpression of this gene can enhance the aluminum tolerance of soybean plants. Picture 7 ) (3) Nitrotetrazole blue (NBT) staining and diaminobenzidine (DAB) staining NBT staining can identify the accumulation of superoxide anion free radicals in plant tissues, while DAB staining can preliminarily reflect the accumulation of hydrogen peroxide (H2O2) in soybean root tips. (The text then abruptly shifts to a completely unrelated topic: WT receptor control Dongnong 50, transgenic...) GmGAI Gene line ( GmGAI-OE-2 and GmGAI-OE-4 After aluminum treatment (25 µM AlCl3) for 24 h, the root tips were cut off and washed three times with distilled water for 5 min each time. The root tips were then stained in 50 mM Tris-HCl (pH=6.5) containing 0.1% NBT for 20 min, followed by destaining with alcohol, and the results were photographed and recorded. The root tips were then stained in 50 mM Tris-HCl (pH=3.8) buffer containing 1 mg / mL DAB for 3 h, followed by destaining using the same method, and the staining results were photographed and recorded.

[0063] Staining results as follows Picture 8 As shown, under aluminum-free conditions, the root tips of the samples could be stained by both NBT and DAB, and the staining degree was basically the same; after aluminum stress treatment, the staining deepened overall, but the transition... GmGAI The NBT and DAB staining intensity of the root tips of the genetically modified strains was lighter than that of the WT strains, indicating that... GmGAI Overexpression of the substance reduced the accumulation of superoxide anion radicals and H2O2, thus alleviating the damage caused by aluminum stress.

Claims

1. The application of GmGAI protein in improving the aluminum stress tolerance of soybeans, characterized in that, The amino acid sequence of the GmGAI protein is shown in SEQ ID NO.

1.

2. Overexpression GmGAI The application of genes in improving the aluminum stress tolerance of soybeans is characterized by, GmGAI The gene is shown in SEQ ID NO.

2.

3. Contains GmGAI The application of recombinant gene vectors in improving the aluminum stress tolerance of soybeans is characterized by, GmGAI The gene is shown in SEQ ID NO.

2.

4. The application according to claim 3, characterized in that, The launch vehicle is pB7WG2.

5. Contains GmGAI The application of recombinant microbial cells in improving the aluminum stress tolerance of soybeans is characterized by, GmGAI The gene is shown in SEQ ID NO.

2.

6. The application according to claim 5, characterized in that, The starting microbial cells are Escherichia coli or Agrobacterium.

7. The application according to any one of claims 1-6, characterized in that, The aluminum stress condition was 25 µM AlCl3.

8. A breeding method for improving the aluminum stress tolerance of soybeans, characterized in that, The specific steps are as follows: Step 1: Amplification GmGAI Genes, inserting gene sequences into expression vectors; GmGAI The gene is shown in SEQ ID NO.2; Step 2: Transform the vector obtained in step (1) into Agrobacterium, infect soybean cotyledon node explants, and obtain transgenic soybeans; Step 3: Identify the genetically modified soybeans obtained in step (2) to obtain positive genetically modified soybeans.

9. A soybean resistant to aluminum stress, characterized in that, Overexpression in soybeans GmGAI Genes, the ones mentioned GmGAI The gene is shown in SEQ ID NO.

2.

10. Overexpression GmGAI The application of transgenic soybean plants with the gene in breeding to improve soybean's tolerance to aluminum stress is characterized by, The GmGAI The gene is shown in SEQ ID NO.2.

Images