Method for improving soybean alkali stress resistance based on autophagy key gene and application

Abstract

The invention relates to a method for improving soybean alkali stress resistance based on an autophagy key gene and application of the method, and belongs to the technical field of plant genetic engineering and crop genetic breeding. An autophagy related gene GmATG8c is overexpressed in the soybean and is combined with specific mononucleotide mutation located on a 20th chromosome, so that the alkali resistance of the soybean is synergistically enhanced. Experiments prove that the germination rate, the plant survival rate and the biomass of a transgenic soybean strain (NK-Al) simultaneously carrying GmATG8c gene overexpression and mononucleotide mutation under an alkali stress condition are obviously superior to those of a wild type strain, a strain independently carrying GmATG8c overexpression and a strain independently carrying the mutation. The invention discloses a new mechanism for synergistically regulating and controlling the alkali resistance of the plant by a cell autophagy pathway and a specific transcription factor, and provides a new gene resource and a technical strategy for cultivating alkali-resistant soybeans and other new important crop varieties.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and crop genetic breeding technology, specifically a method and application for improving the tolerance of soybeans and other crops to alkaline stress by synergistically regulating autophagy-related genes and single nucleotide mutations at specific gene loci. Background Technology

[0002] Global soil salinization is becoming increasingly serious. According to the first global assessment report on saline soils in 50 years, released by the Food and Agriculture Organization of the United Nations (FAO) at the end of 2024, approximately 1.4 billion hectares of land worldwide (more than 10% of the total land area) are affected by salinity. About 10% of irrigated farmland and 10% of rainfed farmland are also affected by salinization. With the increasing frequency of extreme weather events, such as heat waves and droughts, coupled with improper agricultural land management and excessive fertilization, soil salinization continues to worsen.

[0003] Saline-alkali soils include saline soils and alkaline soils (Miao et al., 2023). Soils with a salt content of 0.6%–1.0% or higher are called saline soils, while alkaline soils refer to soils with an alkalinity index (ESP) exceeding 15% and a pH above 8.0, with surface salt content generally below 0.5%. Currently, globally, saline soils and alkaline soils account for 40% and 60% of saline-alkali soils, respectively. Severe soil salinization severely restricts the normal growth and development of crops, leading to reduced yields. High salt content lowers soil water potential, limits plant water absorption, affects seed germination and seedling survival rates, and triggers sodium degradation. + Salt-alkali soils cause ion toxicity, Na⁺ / K⁺ imbalance, osmotic stress, stomatal dysfunction, and decreased enzyme activity. High salt also induces the accumulation of reactive oxygen species (ROS) in cells, damaging cell membranes, DNA, and proteins, reducing photosynthetic efficiency and chlorophyll content, ultimately leading to stunted crop growth, reduced biomass, lower yield, and deteriorated quality. The high pH of alkaline soils leads to soil colloid swelling and compaction, reducing soil permeability and aeration, and increasing the risk of flooding and erosion. Simultaneously, it inhibits the absorption and utilization of elements such as Fe, Zn, and Pi by plants, leading to iron deficiency, yellowing leaves, and Pi starvation. It also disrupts root cell metabolism and membrane function, hindering water and nutrient absorption, resulting in stunted root development, growth retardation, and yield loss. Therefore, identifying important salt-alkali tolerance genes and improving crop salt tolerance through genetic engineering has significant application value.

[0004] Autophagy in plant cells is a conserved degradation mechanism that involves forming double-membrane autophagosomes to encapsulate damaged organelles and macromolecules such as proteins in the cytoplasm and transport them to the vacuole for hydrolysis and recycling. This process is regulated by core autophagy-related genes, such as ATG1, ATG5, and ATG8. In plants, autophagy is involved in plant growth and development, programmed cell death, and stress responses. Current research indicates that salt stress induces the expression of ATG genes, promoting autophagosome formation, which helps degrade damaged organelles and denatured proteins, mitigate oxidative damage, maintain ion balance, and isolate sodium ions into the vacuole, thereby improving the plant's high salt tolerance. Regarding salt tolerance research, there are currently few reports on the application of key autophagy genes in improving crop resistance to alkaline stress, and no such reports have been found in soybeans. Therefore, it is speculated that regulating key autophagy genes individually may present certain technical challenges in improving crop resistance to alkaline stress.

[0005] This invention successfully created a new soybean material with significant tolerance to alkali stress by synergistically regulating autophagy and a specific gene locus. Summary of the Invention

[0006] The purpose of this invention is to address the lack of effective technical solutions for improving soybean's resistance to alkali stress in existing technologies. It provides a new and highly efficient method for enhancing the alkali tolerance of soybean plants, and its application has been effectively verified. Through a combination of "overexpression of GmATG8c + single nucleotide substitution of a specific gene," soybean plants exhibit significantly enhanced alkali stress tolerance, far exceeding that of any single modification. The specific solution is as follows: The application of a soybean autophagy-related gene, GmATG8c, in improving soybean tolerance to alkali stress involves overexpressing the GmATG8c gene in soybeans to enhance their tolerance to alkali stress or alkali resistance.

[0007] Furthermore, the present invention also provides an application of the soybean autophagy-related gene GmATG8c in improving the tolerance of soybean to alkali stress. By overexpressing the GmATG8c gene in soybean and combining it with a recessive mutation of the C2H2 zinc finger protein transcription factor, the alkali stress tolerance or alkali resistance of soybean is synergistically enhanced.

[0008] Furthermore, the recessive mutation of the C2H2 zinc finger protein transcription factor refers to a single nucleotide substitution at nucleotide position 38947578 on soybean chromosome 20, where guanine (G) is mutated into cytosine (C), resulting in the 9th amino acid of the protein encoded by the gene in this region being mutated from aspartic acid (Asp) to histidine (His).

[0009] This invention also provides a method for improving soybean tolerance to alkali stress based on the soybean autophagy-related gene GmATG8c, comprising synergistic regulation of the following two genetic factors in soybean plants: (a) overexpression of the autophagy-related gene GmATG8c; (b) introduction of a single nucleotide mutation that changes the 9th amino acid of the C2H2 zinc finger protein from aspartic acid (Asp) to histidine (His).

[0010] Furthermore, the method can be applied in the study of cultivating alkali-tolerant plants or improving the tolerance of plants to alkali stress.

[0011] Furthermore, the present invention also provides a propagation material for transgenic soybeans cultivated using the method described above, wherein the propagation material is selected from seeds, embryos, callus tissue, cells, or plants.

[0012] Compared with the prior art, the beneficial effects of the present invention are: (1) it reveals and verifies that the autophagy pathway plays a certain role in regulating the alkali resistance of soybeans; (2) For the first time, the synergistic effect of key autophagy genes and specific transcription factor mutations in improving crop alkali tolerance was revealed and confirmed. By jointly regulating the expression of the GmATG8c gene and the mutation at this specific site, the alkali tolerance of soybean plants was further improved. This also provides a new idea and valuable gene resources for the breeding of alkali-tolerant soybeans and other important new crop varieties, and has important application prospects. Attached Figure Description

[0013] Figure 1 Genomic PCR insertion detection results of exogenous GmATG8c gene in NK-Al strains: M: Trans 2Kplus DNA marker; TL-1 is non-transgenic soybean “Tianlong No. 1”; the lanes shown in NK-Al represent two GmATG8c overexpression gene lines carrying single nucleotide mutations in the genome obtained through screening; “-” is the negative control (using ddH2O as a template); “+” is the positive control (using the overexpression GmATG8c binary expression vector plasmid as a template).

[0014] Figure 2 RT-PCR transcriptional detection results of exogenous GmATG8c gene in NK-Al lines: TL-1 is non-transgenic cultivated soybean 'Tianlong No. 1'; lane NK-Al is NK-Al line; GmATG8c-ox is GmATG8c overexpression line without single nucleotide mutation in the genome.

[0015] Figure 3 Southern Blot hybridization analysis results of the NK-Al strain: The red horizontal lines mark the specific hybridization bands between the NK-Al and GmATG8c-ox strains.

[0016] Figure 4 Results of seed germination experiments of different strains under alkaline stress.

[0017] Figure 5 Phenotypic observation results of different strains after hydroponic alkali stress treatment.

[0018] Figure 6 Observation results of biomass phenotypes of different strains after alkali stress treatment. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] Example 1: Cloning of the GmATG8c gene and construction of a plant expression vector Target gene amplification: Soybean RNA was extracted using the Trizol method and reverse transcribed into cDNA. Using soybean cultivar cDNA as a template, PCR amplification was performed using the following primer pairs to obtain the 360 ​​bp coding region sequence of the GmATG8c gene, as shown in SEQ ID NO.1. Upstream primer sequence: 5′-ATTTTCGCCATGGCCAAAACCTCCTTCAAGCTTC -3′ (introducing Nco = 1*ROMAN I restriction site); Downstream primer sequence: 5′-GATTTGGGTCACCTATTTACAGAAGTTAACATGCATTATGC-3′ (Introducing BstE = 1 *ROMAN II restriction site). The PCR reaction system in this example is shown in the table below: The PCR reaction procedure is as follows: reaction temperature reaction time Loop count 94 ℃ 5min 1 time 94 ℃ 30s 30 times 58℃ 30s 72 ℃ 10min 1 time TA Cloning and Identification: A. Recovery of the target gene fragment: The target DNA fragment was recovered by agarose gel electrophoresis. The recovery method used was the DNA agarose gel recovery kit from TaKaRa Biotechnology Co., Ltd. For specific operating procedures, please refer to the product instructions.

[0021] B. Ligation: Add the reagents of the following reaction system and react overnight at 16 °C to achieve ligation of the target fragment with the pMD-18T vector (purchased from Takara).

[0022] C. Transformation and Identification of Positive Clones: *E. coli* DH5α competent cells were prepared using standard CaCl2 induction and transformation methods. The competent cells were transformed with 10 mL of ligation product and then evenly spread onto LB agar plates containing Amp. The plates were incubated upside down at 37°C for 12–14 hours. Single colonies on the transformation plates were selected, and plasmids were extracted using standard methods. The plasmids were double-digested with NcoI and BstE II to produce a 2.7 kb pMD-18 vector fragment and a 360 bp fragment of the GmATG8c gene. Using the plasmid extract as a template, PCR amplification was performed using the primers and amplification conditions described above. Agarose gel electrophoresis was used to detect the 360 ​​bp GmATG8c gene fragment, indicating a positive clone containing the GmATG8c gene sequence.

[0023] D. Sequencing verification: The identified positive clones were sent to Sangon Biotech (Shanghai Sangon Biotech Co., Ltd.) for DNA sequencing, and their nucleic acid sequences are shown in SEQ ID NO.1.

[0024] (3) Construction of plant binary expression vector: A. First, extract the pCAMBIA 3301 plasmid from Escherichia coli (this strain can be purchased from a biotechnology company or CAMBIA), and recover the large vector fragment (containing the CaMV 35S promoter sequence) after double digestion with Nco I / BstE II; B. Extract the plasmid from the prepared TA clone, double digest it with Nco I / BstE II, and recover the GmATG8c gene fragment by agarose gel electrophoresis; C. Ligate the above two fragments overnight at 16 ℃ under the catalysis of ligase to complete the construction of the binary expression vector pCAMBIA 3301 vector driven by the CaMV35S promoter to express the GmATG8c gene.

[0025] The connection system is shown in the table below: D. Transform E. coli DH5α competent cells using a ligation mixture, following the same method as the TA cloning process; E. Select single colonies on the transformation plate (Kan resistant), extract plasmids using conventional methods, and digest the plasmid DNA with Nco I and BstE II to produce two fragments: one is the pCAMBIA 3301 vector fragment, and the other is the GmATG8c gene fragment. F. Perform PCR reaction using plasmid as a template, following the same method as TA cloning; G. Positive clones identified by enzyme digestion and PCR are sent to a sequencing company for sequencing. H. Plasmids were extracted from positive clones and transformed into Agrobacterium LBA4404 using conventional methods to obtain engineered Agrobacterium for plant transformation.

[0026] Example 2: Obtaining transgenic soybeans and screening for the mutant NK-A1 (1) Soybean genetic transformation: Using the soybean cultivar “Tianlong No. 1” (TL-1) as the recipient, the expression vector pCAMBIA3301-GmATG8c was introduced into soybean using Agrobacterium-mediated cotyledon node transformation. Resistant shoots were screened in a medium containing glufosinate-ammonium (Basta) herbicide, and the resistant shoots were further verified using PAT / bar transgenic test strips. The confirmed resistant shoots were grafted and cultured to obtain transgenic positive plants (T0 generation).

[0027] (2) Identification of transgenic lines: After the seeds harvested from the grafted seedlings are sown, when the transgenic soybean candidate lines (T1 generation) have grown 3-4 true leaves, about 100 mg of soybean leaves are taken and plant genomic DNA is extracted by SDS method. PCR amplification is performed using primers located in the 35S promoter and inside the GmATG8c gene (sequences shown as F35S and RA8) to detect whether the GmATG8c gene has been introduced into the soybean genome. PCR amplification is also performed using primers located in the plant resistance gene BAR (sequences shown as FB and RB) to detect whether the resistance gene BAR has been inserted into the genome.

[0028] F35S: 5′-GAGGACCTAACAGAACTCGCCG-3′; RA8: 5′-TCAGCTTTTCTCCACAATCACAGG-3′; FB: 5′-TCAAATCTCGGTGACGGGC-3′; RB: 5′-TACATCGAGACAAGCACGGT-3′; PCR reaction system:

[0029] The PCR reaction procedure is as follows: reaction temperature reaction time Loop count 94 ℃ 5min 1 time 94 ℃ 30s 32 times 58℃ 30s 72 ℃ 40s 72 ℃ 10min 1 time For lines that tested positive by genomic PCR, approximately 100 mg of leaves were collected, and total RNA was extracted using the Trizol method. cDNA was then synthesized using 1 μg of RNA via reverse transcription. RT-PCR primers (sequences shown as RT-A8-F and RT-A8-R) were designed based on the GmATG8c gene sequence and the NOS terminator sequence. The transcriptional level of the exogenous GmATG8c gene was detected by RT-PCR, and transcriptionally positive lines were screened.

[0030] RT-A8-F: 5′-GAAGTGACATTCCAGACATTGATAAG-3′; RT-A8-R: 5′-ATAATCATCGCAAGACCGGCAACAG-3′.

[0031] The PCR reaction procedure is as follows: reaction temperature reaction time Loop count 94 ℃ 5min 1 time 94 ℃ 30s 28 times 58℃ 30s 72 ℃ 40s 72 ℃ 10min 1 time Single-copy lines were screened using Southern blotting. T1 generation lines with normal GmATG8c gene expression were selected, and genomic DNA was extracted. Hybridization was performed using the GmATG8c probe and the Nos terminator sequence, respectively. Genomic DNA was digested with restriction endonucleases EcoR V, Hind III, Pst I, and Sac I. Single-copy lines were screened based on the results, and subsequent homozygous line screening was conducted, naming the resulting line GmATG8c-ox.

[0032] (3) Screening and identification of single nucleotide mutants: During the field propagation of homozygous GmATG8c-ox transgenic soybean, a line with a single nucleotide substitution in the genome was screened. Sequencing confirmed that this transgenic line had a single nucleotide substitution at nucleotide position 38947578 on chromosome 20, where guanine (G) was mutated to cytosine (C), resulting in a mutation at position 9 (Asp) of the protein encoded by the Glyma.20G116200 gene to His (the mutant nucleotide sequence and the encoded mutant protein sequence are shown in SEQ ID NO.2 and SEQ ID NO.3). This line was named NK-Al. Genomic PCR was performed on this line according to the aforementioned methods. Figure 1 ) and RT-PCR detection ( Figure 2 The results showed that the exogenous GmATG8c gene was inserted and expressed normally in all cases. Southern blot results also showed that this strain was a single-copy insertion strain of the exogenous gene, and the insertion site was the same as that of the GmATG8c-ox strain without a single nucleotide mutation. Figure 3 ).

[0033] Example 3: Alkali tolerance identification of the NK-Al strain To verify the synergistic effect of GmATG8c overexpression and this single nucleotide mutation, four experimental plants were set up: wild-type TL-1, the mutant TL-1m containing only the single nucleotide substitution of this specific gene (obtained by backcrossing), the line GmATG8c-ox which overexpresses only GmATG8c, and the line NK-Al which has both modifications.

[0034] (1) Seed germination experiment: Seeds of wild-type TL-1 and the NK-Al line with both modifications were sown on 1 / 2 MS medium containing 40 mM NaHCO3 (pH 8.4) and bromocresol purple as a pH indicator. Germination was observed after 72 hours. Figure 4 Under control conditions, there was no difference in germination among the different lines. Under alkaline treatment, TL-1 germination was severely inhibited, while NK-Al showed the best germination. Notably, the purple color of the medium around the rhizosphere of NK-Al significantly faded, indicating that its roots have a stronger acidification capacity, which is beneficial for nutrient absorption in an alkaline environment.

[0035] (2) Hydroponic stress experiment: Thirty-five plump and uniform soybean seeds were selected from the reference variety TL-1, the single nucleotide mutant TL-lm under the TL-1 background, the transgenic line GmATG8c-ox overexpressing GmATG8c, and the transgenic mutant NK-Al. These seeds were sown in plastic cups containing a 1:1 ratio of nutrient soil and vermiculite. When the soybean seeds germinated and the first trifoliate leaf emerged, 24 plants with identical developmental stages were selected from each variety. The roots were washed with clean water and then transferred to a 40mM NaHCO3 aqueous solution at pH 8.3. The light duration and temperature were controlled to be the same for all four varieties. After 48 hours of treatment, the plants were restored using Hoagland's nutrient solution. After 10 days of recovery, photographs were taken to observe the plant phenotype. Figure 5 As shown. Figure 5 The comparison of four soybean plants (TL-1, TL-lm, GmATG8c-ox, and NK-Al) before and after NaHCO3 treatment shows that before treatment, the leaves of all plants were green and healthy. After alkali treatment, the leaves of TL-1 plants were significantly withered and yellowed, showing obvious damage. TL-lm and GmATG8c-ox lines also showed varying degrees of damage. Although the leaves of NK-Al plants also changed slightly, the degree of damage was significantly less than that of the other three control plants.

[0036] (3) Biomass analysis: Plants that had recovered from hydroponic stress were collected, and their aboveground and underground phenotypes were observed, such as... Figure 6 As shown, the NK-Al strain exhibited significantly better alkali tolerance than the other three control strains in terms of aboveground and underground fresh weight and root length. The TL-1m and GmATG8c-ox strains also showed some improvement in alkali tolerance compared to TL-1, but the effect was far less than that of the NK-Al strain.

[0037] The overall analysis results are shown in the table below: strain Genetic background GmATG8c overexpression Single nucleotide mutation on chromosome 20 Alkali stress tolerance TL-1 Wild-type control none none Weakest TL-lm TL-1 background none <![CDATA[Having (C2H2 zinc finger protein mutation)]]> Weak GmATG8c-ox TL-1 background have none Weak NK-Al GmATG8c-ox background have <![CDATA[Having (C2H2 zinc finger protein mutation)]]> strongest Based on the above experimental results, it is demonstrated that overexpression of GmATG8c and the G38947578C single nucleotide mutation on chromosome 20 have a significant synergistic effect in improving the alkali tolerance of soybeans. This invention provides a new solution for the genetic improvement of alkali-tolerant crops.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Application of soybean autophagy-related gene GmATG8c in improving alkali stress tolerance of soybean, characterized in that, The GmATG8c gene is overexpressed in soybean to improve the alkali stress tolerance or alkali tolerance of the soybean.

2. The application of soybean autophagy-related gene GmATG8c in improving the alkali stress tolerance of soybean, characterized in that, The alkali stress tolerance or alkali tolerance of soybean is synergistically improved by overexpressing the GmATG8c gene in soybean and combining a recessive mutation of a C2H2 zinc finger protein transcription factor.

3. Use according to claim 2, characterized in that, The recessive mutation of the C2H2 zinc finger protein transcription factor refers to a single nucleotide substitution of guanine (G) to cytosine (C) at the 38947578th nucleotide site on chromosome 20 of soybean, which causes the 9th amino acid of the protein encoded by the gene in the region to mutate from aspartic acid (Asp) to histidine (His).

4. A method for improving alkali stress tolerance of soybean based on autophagy-related gene GmATG8c, characterized in that, The method comprises synergistically regulating the following two genetic factors in a soybean plant: (a) overexpressing the autophagy-related gene GmATG8c; and (b) introducing a single nucleotide mutation that causes the 9th amino acid of the C2H2 zinc finger protein to mutate from aspartic acid (Asp) to histidine (His).

5. The method of claim 4 is used in the research of breeding alkali-tolerant plants or improving the alkali stress tolerance of plants.

6. Propagation material of a transgenic soybean bred by the method of claim 4 or 5, wherein the propagation material is selected from seeds, embryos, calli, cells or plants.

Images