Soybean acyl-coenzyme A oxidase and application of coding gene thereof in regulation and control of plant salt tolerance

Abstract

The invention provides soybean acyl-coenzyme A oxidase and application of a coding gene thereof in regulation and control of plant salt tolerance, and belongs to the technical field of plant breeding. The invention provides soybean acyl coenzyme A oxidase and a coding gene thereof. Experiments prove that the soybean acyl coenzyme A oxidase or the coding gene thereof can improve the salt tolerance of plants through positive regulation. Therefore, the invention provides the application of a reagent for improving soybean acyl-coenzyme A oxidase or gene expression or activity or a reagent for increasing or enhancing gene expression in increasing the salt tolerance of plants, preparing a product for increasing the salt tolerance of plants, cultivating plant varieties with salt tolerance advantages, preparing a product for cultivating plants with increased salt tolerance and breeding plants with salt tolerance.

Description

Technical Field

[0001] This application belongs to the field of plant breeding technology, specifically relating to the application of soybean acyl-CoA oxidase and its encoding gene in regulating plant salt tolerance. Background Technology

[0002] Acyl-CoA oxidase (EC 1.3.3.6, Acyl-CoA Oxidase, ACX) is a peroxisome flavoprotein belonging to the redox enzyme class. Its active site contains a flavin adenine dinucleotide (FAD) cofactor. ACX from different plant sources exhibits some conservation in its amino acid sequence and three-dimensional structure. Taking Arabidopsis thaliana as an example, its ACX protein contains multiple domains. The N-terminal domain is responsible for substrate-specific binding, and different ACX isoforms show varying affinities for short-chain, medium-chain, or long-chain acyl-CoA. The C-terminal domain participates in protein oligomerization, affecting ACX activity and stability. Fatty acid β-oxidation is an important metabolic pathway in plants. Plant ACX, as the initiating enzyme of this pathway, catalyzes the conversion of acyl-CoA to trans-2-enoyl-CoA and produces hydrogen peroxide, initiating the gradual degradation of fatty acids and providing energy for plant physiological activities. It occupies a central position in the plant's material and energy metabolism network. Because this reaction is considered an important pathway for controlling carbon flux, ACXs are also considered rate-limiting enzymes for β-oxidation.

[0003] The ACX gene is located in the peroxisome in plants, and the peroxisome β-oxidation pathway is one of the main pathways of fatty acid metabolism. Many studies have shown that this pathway is closely related to biological processes such as lipid reserves during seed germination, senescence, starvation, membrane lipid turnover, and the biosynthesis of plant hormones (such as jasmonic acid and indoleacetic acid). It participates in plant responses to biotic and abiotic stresses, regulates the expression of related genes, and coordinates metabolic and defense responses within plants. However, there are currently no studies on the application of ACX or the ACX gene in regulating plant salt tolerance. Summary of the Invention

[0004] The purpose of this invention is to provide a soybean acyl-CoA oxidase protein that has the characteristics of regulating plant salt tolerance, thus providing a new means for salt-tolerant plant breeding.

[0005] This invention provides a soybean acyl-CoA oxidase, comprising at least one of the following proteins: A. A protein with an amino acid sequence as shown in SEQ ID NO:1; B. Proteins that share more than 80% identity and the same function as the proteins shown in item A; C. Proteins with the same function formed by substituting, deleting, or adding one or more amino acid residues based on the protein shown in item A; D. A fusion protein with the same function formed by attaching a tag to the end of at least one of the proteins shown in options A, B, and C.

[0006] This invention provides a gene encoding the soybean acyl-CoA oxidase.

[0007] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO:2 or SEQ ID NO:3.

[0008] The present invention provides a derivative product containing the gene, including at least one of the following: expression cassette, recombinant vector, recombinant microbial strain, recombinant plant cell line, and recombinant plant tissue or organ.

[0009] This invention provides the application of the soybean acyl-CoA oxidase or the gene in regulating plant salt tolerance.

[0010] Preferably, the soybean acyl-CoA oxidase or gene is used in the positive regulation of plant salt tolerance.

[0011] This invention provides the application of the soybean acyl-CoA oxidase, the gene, reagents for increasing the expression level or enzyme activity of soybean acyl-CoA oxidase, or reagents for upregulating or enhancing the expression of the gene, in at least one of the following: 1) Increase the salt tolerance of plants; 2) Prepare products that increase the salt tolerance of plants; 3) Cultivate plant varieties with salt tolerance advantages; 4) Prepare products that cultivate plants with increased salt tolerance; and 5) Salt-tolerant plant breeding.

[0012] Preferably, the plant includes dicotyledonous plants or monocotyledonous plants; Preferably, the dicotyledonous plant includes legumes.

[0013] This invention provides a method for increasing the salt tolerance of plants, comprising the following steps: Overexpression of the soybean acyl-CoA oxidase or the gene in plants, or application of reagents that promote the increase of the enzyme activity of the soybean acyl-CoA oxidase or the expression level of the gene.

[0014] Preferably, the plant includes dicotyledonous plants or monocotyledonous plants; Preferably, the dicotyledonous plant includes legumes.

[0015] This invention provides a soybean acyl-CoA oxidase and its encoding gene. Experiments demonstrate that under salt stress conditions, this invention... GmACX2The transgenic soybeans exhibited significantly higher salt-related enzyme activity than the control, and the transgenic lines showed significantly better growth than the control. These results indicate that... GmACX2 It can significantly improve the salt tolerance of transgenic plants. GmACX2 Salt tolerance genes can be used to breed stress-resistant soybean varieties. Attached Figure Description

[0016] Figure 1 For genetically modified soybean plants GmACX2 Expression level analysis results; Note: P <0.01; Figure 2 for GmACX2 Results of gene expression pattern analysis under different NaCl stress time; P <0.01; Figure 3 for GmACX2 Results of enzyme activity assay in transgenic plants; P <0.01; Figure 4 Under salt stress GmACX2 Morphological results of transgenic plants; Figure 5 Under salt stress GmACX2 Results of measurements on root length, plant height, and above-ground parts of transgenic plants; P <0.05; P <0.01. Detailed Implementation

[0017] This invention provides a soybean acyl-CoA oxidase, comprising at least one of the following proteins: A. A protein with an amino acid sequence as shown in SEQ ID NO:1 ( ); B. Proteins that share more than 80% identity and the same function as the proteins shown in item A; C. Proteins with the same function formed by substituting, deleting, or adding one or more amino acid residues based on the protein shown in item A; D. A fusion protein with the same function formed by attaching a tag to the end of at least one of the proteins shown in options A, B, and C.

[0018] In this invention, the protein described in item B is a protein whose identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher than that of the protein described in item A. The protein described in item C meets the requirements of the protein described in item B. The identity refers to the identity of the amino acid sequence or nucleotide sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of the amino acid sequence, the identity value (%) can then be obtained.

[0019] In this invention, the amino acids used to replace or add amino acids in the protein described in item C can be common types of amino acids, as long as the requirement of not changing the function of the protein is met.

[0020] In this invention, the tag is preferably attached to the N-terminus and / or C-terminus of the protein. The tagged protein preferably includes, but is not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein. The tag can be used for protein purification, reporting protein locations, and labeling proteins for detection.

[0021] In this invention, the soybean transcription factor SPL6a can be transgenic into plant cells via gene encoding, and protein overexpression can be achieved through gene expression.

[0022] This invention provides a gene encoding the soybean acyl-CoA oxidase. GmACX2 .

[0023]

[0024] The present invention provides a derivative product containing the gene, including at least one of the following: expression cassette, recombinant vector, recombinant microbial strain, recombinant plant cell line, and recombinant plant tissue or organ.

[0025] In this invention, the expression cassette refers to DNA capable of expressing the soybean acyl-CoA oxidase in a host cell. The expression cassette preferably further includes single-stranded or double-stranded gene elements for regulating the gene. The gene elements preferably include a leader sequence, a polyadenylated sequence, a propeptide sequence, an enhancer sequence, a promoter, a signal sequence, and a transcription terminator. The promoter contains a transcriptional regulatory sequence mediating the expression of the soybean acyl-CoA oxidase or the DNA of the RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that can function in the selected host cell can be used in this invention. The leader sequence is an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively linked to the 5' end of a nucleic acid sequence encoding a protein or the DNA of the RNA molecule. Any leader sequence that can function in a selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein, capable of guiding the expression of the DNA encoding the protein or RNA molecule into the cellular secretory pathway. Signal peptide coding regions capable of guiding the expressed protein or RNA molecule's DNA into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate the expression of the protein or RNA molecule's DNA according to the growth status of the host cell may also be necessary. Examples of transcriptional regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that enable gene amplification. The backbone vector of the recombinant vector preferably includes plasmids, granules, bacteriophage vectors, or viral vectors. The host microorganism in the recombinant microbial strain preferably includes a eukaryotic expression system, a prokaryotic expression system, or algae. The eukaryotic expression system preferably includes yeast. The prokaryotic expression vector preferably includes bacteria. The bacteria preferably include Agrobacterium. The Agrobacterium species preferably include Agrobacterium rhizogenes. The transgenic plant cell lines preferably do not include propagation material and can be somatic cells derived from roots, stems, or leaves.

[0026] This invention provides the application of the soybean acyl-CoA oxidase or the gene in regulating plant salt tolerance.

[0027] In this invention, the soybean acyl-CoA oxidase or gene is preferably used in the positive regulation of plant salt tolerance, that is, increasing the expression level or enzyme activity of soybean acyl-CoA oxidase, or increasing the gene expression level or transcription level, is beneficial to improving the plant's tolerance to salt stress. The salt stress is preferably simulated by applying a sodium chloride solution to mimic the effect of saline-alkali soil on plant growth under natural conditions. The stress concentration of the sodium chloride solution is preferably above 100 mM, and can be 120-150 mM.

[0028] In this invention, the plant preferably includes dicotyledonous or monocotyledonous plants. The dicotyledonous plants preferably include legumes. The legumes preferably include plants of the genus *Glycine*. The *Glycine* species preferably include soybeans.

[0029] This invention provides the application of the soybean acyl-CoA oxidase, the gene, reagents for increasing the expression level or enzyme activity of soybean acyl-CoA oxidase, or reagents for upregulating or enhancing the expression of the gene, in at least one of the following: 1) Increase the salt tolerance of plants; 2) Prepare products that increase the salt tolerance of plants; 3) Cultivate plant varieties with salt tolerance advantages; 4) Prepare products that cultivate plants with increased salt tolerance; and 5) Salt-tolerant plant breeding.

[0030] In this invention, the enhancement of plant salt tolerance preferably includes improving the plant's tolerance to growth in saline soil, manifested as unaffected growth, including abundant root systems and vigorous above-ground growth.

[0031] In this invention, the products that increase the salt tolerance of plants preferably include growth promoters, stress-resistant agents, etc. The cultivation of plant varieties with superior salt tolerance preferably involves using transgenic technology to transfer reagents that enhance the expression or activity of soybean acyl-CoA oxidase or its gene, or reagents that upregulate or enhance the expression of said gene, into plant cells to promote the expression of soybean acyl-CoA oxidase and its gene, thereby increasing the activity of soybean acyl-CoA oxidase and thus improving the plant's tolerance to salt stress. The plant salt tolerance breeding involves constructing transgenic plants that overexpress soybean acyl-CoA oxidase and using them as breeding materials in plant breeding.

[0032] In this invention, the reagent that upregulates the expression level of the soybean transcription factor SPL6a or the reagent that upregulates or enhances the gene expression preferably includes the gene-derived products described in the above technical solution.

[0033] This invention provides a method for increasing the salt tolerance of plants, comprising the following steps: Overexpression of the soybean acyl-CoA oxidase or the gene in plants, or application of reagents that promote the increase of the enzyme activity of the soybean acyl-CoA oxidase or the expression level of the gene.

[0034] In this invention, the method preferably involves introducing a recombinant vector overexpressing the gene into plants for expression, or using an activator that enhances the activity of soybean acyl-CoA oxidase in plants. This invention does not impose any particular limitation on the method of introducing the recombinant vector into plants, and uses Agrobacterium-mediated transformation, which is well-known in the art. The Agrobacterium preferably includes Agrobacterium rhizogenes.

[0035] In one embodiment of the present invention, the effect of soybean acyl-CoA oxidase or the gene on plant salt tolerance was evaluated using a recombinant vector introduced into soybean. The results showed that the present invention successfully constructed an overexpression of... GmACX2 The transgenic soybean lines (OE1 and OE2) were modified to respond to salt stress, with expression levels reaching their peak after 12 hours of salt stress. The activity of acyl-CoA oxidase GmACX2 in OE1 and OE2 was significantly higher than that in wild-type soybean (WT). Under NaCl stress, the transgenic lines OE1 and OE2 showed significantly better growth than the wild-type, indicating that GmACX2 can improve soybean's tolerance to salt stress.

[0036] The following examples illustrate the application of soybean acyl-CoA oxidase and its encoding gene provided by the present invention in regulating plant salt tolerance. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] The Fu28-GFP and pSOY1 vectors used in this embodiment are described in the non-patent literature “Kim Yunchol. Molecular mechanism of GmTCP and GmNLP response to nitrate nitrogen concentration on soybean nodulation [D]. Northeast Agricultural University, 2023. DOI:10.27010 / d.cnki.gdbnu.2023.000037”. This biomaterial is available to the public from the Heilongjiang Provincial Academy of Green Food Sciences (National Soybean Engineering Technology Research Center, National Dairy Engineering Technology Research Center).

[0039] The data obtained in the examples were processed using GraphPad Prism 8 statistical software. Experimental results are expressed as mean ± standard deviation, and one-way ANOVA was used. P <0.05 ( () indicates a significant difference.

[0040] Example 1 GmACX2 Methods for preparing overexpression plants Using the genomic DNA of Dongnong 50 soybean as a template, the primer pairs shown in Table 1 were used for... GmACX2 The CDS fragment of the gene was cloned, and the reaction system and procedure are shown in Tables 2 and 3. It was then ligated into the entry vector Fu28-GFP via homologous recombination to construct the Fu28-GmACX2 vector (entry vector plasmid). The constructed... Fu28-GmACX2 The vector and empty vector plasmid pSOY1 were used to prepare the reaction system shown in Table 4 for Gateway cloning reaction. The reaction program was 5 h in a metal bath at 25 °C. The GmACX2-Fu28 entry vector was then used. attL1 and attL2 Intermediate segments (including promoters and) GmACX2 (Gene fragment) Replacement in pSOY1 empty vector attR1 and attR2 The fragment was used to obtain the final pSOY1-GmACX2 expression vector. pSOY1-GmACX2 contains... GmACX2 Genes (DNA molecules with the nucleotide sequence SEQ ID NO:2) can express GmACX2 Gene.

[0041] Table 1 Cloning GmACX2 Primer sequence

[0042] Table 2 GmACX2 amplification reaction system

[0043] Table 3 GmACX2 amplification reaction procedure

[0044] Table 4 Gateway cloning reaction system

[0045] Will get pSOYI-GmACX2Recombinant Agrobacterium was obtained by transferring Agrobacterium rhizogenes strain K599 (purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number: AC1080S). Selected plump soybean seeds were sterilized with Cl2 for 12 hours, then removed and placed in a clean bench to remove chlorine. The seeds were then soaked in sterile ultrapure water for 32 hours to activate the recombinant Agrobacterium. The culture dishes and filter paper were sterilized. The soaked soybean radicles were cut off, leaving the hypocotyl near the cotyledons and the explants. The explants were immersed in a resuspended Agrobacterium solution for at least 30 minutes, the solution was wiped dry on sterile paper, and the explants were placed flat on LCCM medium lined with filter paper and cultured in the dark for 3 days. The co-cultured soybean explants (with swollen hypocotyls) were then vertically inserted into the rooting induction medium and cultured for two weeks to obtain complete plants.

[0046] Example 2 GmACX2 Detection of expression levels in overexpressing plants Total RNA was extracted from leaves of transgenic soybeans (OE-1, OE-2, and OE-3) and DN50 using TRIzol reagent (Invitrogen). Reverse transcription was performed using HiScript® II Q RT SuperMix for qPCR (+gDNA wiper). Quantitative PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) kit. The reaction mixture contained 10 μl of 2×ChamQ Universal SYBR qPCR, 0.5 μM forward and reverse primers, and 1 μl of cDNA template (equivalent to 100 ng of RNA), for a total volume of 20 μl. Primers and housekeeping gene amplification primers are shown in Table 5. Real-time quantitative PCR was performed using a Roche fluorescence quantitative PCR instrument. The real-time quantitative reaction program was: 95 ℃ for 5 min; 95 ℃ for 10 s, 62 ℃ for 30 s, 72 ℃ for 30 s, for 40 cycles; melting curve was performed: 95 ℃ for 5 s, 65 ℃ for 1 min, 97 ℃ for 30 s; cooling at 4 ℃. The obtained CT values ​​were analyzed using 2... -△△CT Method calculation, comparison GmACX2 Gene expression differed between transgenic lines and DN50. Each sample underwent three independent biological replicates and three technical replicates per sample.

[0047] Table 5 Primer Sequences for Real-Time Quantitative PCR

[0048] qRT-PCR results are as follows Figure 1 As shown, GmACX2 The genes were expressed in soybean leaves (OE-1, OE-2, and OE-3), and showed varying degrees of increase compared to the control DN50 (WT).P <0.01). This indicates that the present invention has successfully constructed an overexpression... GmACX2 Soybean plants with genetically modified genes.

[0049] Example 3 Soybeans under different salinity treatments GmACX2 Gene expression pattern analysis The experiment was repeated three times, with each repetition as follows: Group OE1: OE1 seeds were germinated in plastic pots (25 cm in diameter, 6 cm in height) filled with vermiculite. After germination, they were transplanted into 6-well black hydroponic boxes for hydroponics. Hoagland solution was used as the culture medium. After 2 weeks of hydroponics, soybean leaves were treated with 120 mM NaCl. Samples were taken from soybean leaves at different time points (0, 1 h, 3 h, 6 h, 12 h, and 24 h), flash-frozen in liquid nitrogen, and stored at -80℃ for total RNA extraction. qRT-PCR was used for detection. GmACX2 Response patterns to abiotic stress. Real-time quantitative PCR was performed using a Bio-Rad Chromo4 real-time PCR system. The reaction mixture contained 12.5 μl of 2 × SYBR Green Real-time PCR Master Mix (Toyobo), 0.5 μM upstream and downstream primers, and 2 μl of cDNA template (equivalent to 100 ng of RNA), for a total volume of 25 μl. The reaction conditions were: 94℃, 30 s; 45 cycles: 94℃, 12 s; 58℃, 30 s; 72℃, 30 s; and finally 80℃, 1 s. Relative expression levels were determined using 2... -△△CT Method calculation. GmACX2 Expression levels in the 0 h root were set to 1, and other treatments were calculated accordingly. Each sample underwent three independent biological replicates, with each replicate undergoing three technical replicates. All primers are shown in Table 1. The experiment was repeated three times.

[0050] OE2 group: The difference between this group and OE1 group is that OE2 seed is used instead of OE1 seed, and the rest of the operation is the same as OE1 group.

[0051] WT group: The difference between this group and the OE1 group is that the DN50 seed is used instead of the OE1 seed. The rest of the operation is the same as the OE1 group.

[0052] Using real-time quantitative PCR analysis GmACX2 Gene expression patterns under salt stress, results as follows Figure 2 As shown, after 1 hour of NaCl stress treatment, GmACX2 Gene expression was significantly upregulated in plant leaves. After 12 hours of NaCl treatment, GmACX2Gene expression intensity reached its maximum. These results indicate that NaCl can promote the expression of genes in soybean leaves to varying degrees. GmACX2 The expression. Therefore, it can be seen that... GmACX2 It is an important regulatory factor in the process of soybean adapting to abiotic stress.

[0053] Example 4 GmACX2 Enzyme activity assay of overexpression plants The experiment was repeated three times, with each repetition as follows: OE1 group: 1 g of leaves from transgenic soybean OE-1 were ground into powder using liquid nitrogen. 9 ml of pre-cooled homogenate was added, and the filtrate was centrifuged at 12000 rpm for 15 min at 4℃. The supernatant was collected as the crude enzyme extract. The acyl-CoA oxidase activity of the transgenic lines was determined using a plant acyl-CoA oxidase (ACX) enzyme-linked immunosorbent assay (ELISA) kit (purchased from Huijia Biotechnology). The specific operating procedure is as follows: 1. Dilution and addition of standards: Set up 10 wells for standards on an enzyme-labeled plate. Add 100 μl of standard (the standard from the aforementioned plant acyl-CoA oxidase (ACX) enzyme-linked immunosorbent assay (ELISA) kit) to the first and second wells respectively. Then add 50 μl of standard diluent to the first and second wells and mix well. Next, take 100 μl from each of the first and second wells and add it to the third and fourth wells respectively. Then add 50 μl of standard diluent to each of the third and fourth wells and mix well. Next, take 50 μl from each of the third and fourth wells and discard it. Then take 50 μl from each of the third and fourth wells and add it to the fifth and sixth wells respectively. Then add 50 μl of standard diluent to each of the fifth and sixth wells and mix well. After mixing, take 50 μl from each of the fifth and sixth wells and add it to the seventh and eighth wells respectively. Then add 50 μl of standard diluent to each of the seventh and eighth wells respectively. After mixing, take 50 μl from wells 7 and 8 and add it to wells 9 and 10 respectively. Then add 50 μl of standard dilution to wells 9 and 10 respectively, mix well, and discard 50 μl from wells 9 and 10 (the amount of sample added to each well after dilution is 50 μl, with concentrations of 240 ng / L, 160 ng / L, 80 ng / L, 40 ng / L and 20 ng / L respectively).

[0054] 2. Sample Addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. First, add 40 μl of sample diluent to the sample wells on the enzyme-labeled plate, then add 10 μl of the sample to be tested (the final sample dilution is 5-fold). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix.

[0055] 3. Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0056] 4. Solution preparation: Dilute the 30-fold (20 times that of 48T) concentrated washing solution with distilled water at a ratio of 30-fold (20 times that of 48T) and set aside.

[0057] 5. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing solution, let stand for 30 seconds and then discard. Repeat this process 5 times, then pat dry.

[0058] 6. Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for blank wells.

[0059] 7. Incubation: Same procedure as 3.

[0060] 8. Washing: Same as step 5.

[0061] 9. Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0062] 10. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0063] 11. Measurement: Zero the instrument using the blank well, and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0064] OE2 group: The difference between this group and OE1 group is that OE2 blades are used instead of OE1 blades, and the rest of the operation is the same as OE1 group.

[0065] WT Group: The difference between this group and the OE1 group is that the OE1 blades are replaced with DN50 blades, and the rest of the operation is the same as the OE1 group.

[0066] The results are as follows Figure 3 As shown, the activity of acyl-CoA oxidase GmACX2 in OE1 and OE2 was significantly higher than that in wild-type soybean (WT).

[0067] Example 5 GmACX2 Salt tolerance identification of overexpression plants The experiment was repeated three times, with each repetition as follows: OE1 group: OE1 seeds were germinated in plastic pots (25 cm in diameter, 6 cm in height) filled with vermiculite. After germination, they were transplanted into 6-cell black hydroponic boxes for hydroponics. Hoagland nutrient solution was used for hydroponics. Sponge blocks were used to fix the lower part of the cotyledons. Each pot was filled with distilled water, and 6 plants were planted in each pot. After transplanting, the plants were cultured in a greenhouse with a diurnal temperature range of (26±2)℃ / (24±2)℃, a photoperiod of 12 h, and a relative humidity of 75%. When the first trifoliate leaf unfolded, 1 L of 120 mM NaCl was added to the hydroponic box. Photos were taken at 0d, 1d, 2d, and 3d of stress treatment.

[0068] OE2 group: The difference between this group and OE1 group is that OE2 seed is used instead of OE1 seed, and the rest of the operation is the same as OE1 group.

[0069] WT group: The difference between this group and the OE1 group is that the DN50 seed is used instead of the OE1 seed. The rest of the operation is the same as the OE1 group.

[0070] The results are as follows Figure 4 and Figure 5 As shown, under NaCl stress, the transgenic lines OE1 and OE2 exhibited significantly better growth than the wild type, with significantly superior root length, plant height, and underground dry weight. This indicates... GmACX2 It can improve the tolerance of soybeans to salt stress.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A soybean acyl-CoA oxidase, characterized in that, Includes at least one of the following proteins: A. A protein with an amino acid sequence as shown in SEQ ID NO:1; B. Proteins that share more than 80% identity and the same function as the proteins shown in item A; C. Proteins with the same function formed by substituting, deleting, or adding one or more amino acid residues based on the protein shown in item A; D. A fusion protein with the same function formed by attaching a tag to the end of at least one of the proteins shown in options A, B, and C.

2. A gene encoding the soybean acyl-CoA oxidase of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:2 or SEQ ID NO:

3.

4. A derivative product comprising the gene of claim 2 or 3, characterized in that, It includes at least one of the following: expression cassette, recombinant vector, recombinant microbial strain, recombinant plant cell line, and recombinant plant tissue or organ.

5. The application of the soybean acyl-CoA oxidase of claim 1 or the gene of claim 2 in regulating plant salt tolerance.

6. The application according to claim 5, characterized in that, The application of soybean acyl-CoA oxidase or gene in the positive regulation of plant salt tolerance.

7. The use of at least one of the following: the soybean acyl-CoA oxidase of claim 1, the gene of claim 2, a reagent for increasing the expression level or enzyme activity of soybean acyl-CoA oxidase, or a reagent for upregulating or enhancing the expression of said gene. 1) Increase the salt tolerance of plants; 2) Prepare products that increase the salt tolerance of plants; 3) Cultivate plant varieties with salt tolerance advantages; 4) Prepare products that cultivate plants with increased salt tolerance; and 5) Salt-tolerant plant breeding.

8. The application according to claim 7, characterized in that, The plants include dicotyledonous plants; Preferably, the dicotyledonous plant includes legumes.

9. A method for increasing the salt tolerance of plants, characterized in that, Includes the following steps: Overexpression of the soybean acyl-CoA oxidase of claim 1 or the gene of claim 2 in plants, or application of a reagent that promotes the increase of enzyme activity of the soybean acyl-CoA oxidase of claim 1 or the expression level of the gene of claim 2.

10. The method according to claim 9, characterized in that, The plants include dicotyledonous plants or monocotyledonous plants; Preferably, the dicotyledonous plant includes legumes.

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