A method to improve soybean salt tolerance by overexpressing the GmMORF3 gene
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-18
- Publication Date
- 2026-08-14
AI Technical Summary
[0013]本发明的有益效果如下：本发明通过构建GmMORF3过表达表达盒，并采用发根农杆菌介导的毛状根转化方式获得GmMORF3过表达大豆嵌合材料，可在不进行稳定遗传转化植株再生和后代遗传鉴定的条件下，评价GmMORF3基因过表达对大豆耐盐性的影响。
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Abstract
Description
Technical Field
[0001] This invention belongs to the fields of plant genetic engineering and crop stress resistance breeding technology, and in particular relates to a method for improving the salt tolerance of soybeans by overexpressing the GmMORF3 gene. Background Technology
[0002] Soybean is an important dual-purpose crop for both grain and oil, with high application value in plant protein, edible oil, feed processing, and crop rotation. Salt stress is a significant environmental factor affecting soybean growth. When salt enters the rhizosphere, changes occur in soybean root water absorption, ion uptake, leaf photosynthesis, and dry matter accumulation. Salt stress also affects chloroplast and mitochondrial function, leading to the accumulation of reactive oxygen species (ROS) and oxidative damage. Soybeans possess an antioxidant defense system; superoxide dismutase (SOD), peroxidase, and catalase participate in ROS scavenging, while malondialdehyde (MDA), hydrogen peroxide, and superoxide anion are commonly used as indicators of the degree of salt stress damage. Existing soybean salt tolerance improvement technologies include salt-tolerant germplasm screening, hybridization breeding, molecular marker-assisted selection, exogenous regulator treatment, and genetic engineering breeding. Research on salt tolerance-related genes mainly focuses on ion transport, osmotic regulation, hormone responses, transcriptional regulation, and redox balance. MORF proteins are multi-organelle RNA editing factors involved in chloroplast and mitochondrial RNA editing processes. MORF proteins are associated with organelle gene expression, energy metabolism, and stress responses. There are multiple GmMORF family members in the soybean genome, and salt stress can induce the upregulation of GmMORF gene expression in roots and leaves.
[0003] Existing technologies have not yet established a salt tolerance improvement scheme based on the GmMORF3 gene, and the application of organelle RNA editing factors in soybean salt tolerance breeding lacks clear disclosure, which limits the utilization of GmMORF3 gene resources in the creation of salt-tolerant soybean materials. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a method for improving soybean salt tolerance by overexpressing the GmMORF3 gene. The technical problem this invention aims to solve is: how to construct a GmMORF3 overexpression cassette, obtain GmMORF3 overexpressing soybean chimeric materials, and conduct NaCl salt stress screening, thereby solving the problem that existing technologies have not established a soybean salt tolerance improvement scheme centered on the GmMORF3 gene.
[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for improving soybean salt tolerance by overexpressing the GmMORF3 gene, comprising: S1. Provides a soybean GmMORF3 gene coding sequence, wherein the soybean GmMORF3 gene coding sequence is derived from the soybean GmMORF3 gene and encodes a multi-organelle RNA editing factor containing a conserved MORF domain, wherein the soybean GmMORF3 gene is a salt stress-induced expression gene; S2. The soybean GmMORF3 gene coding sequence is operatively linked to a plant promoter to construct a GmMORF3 overexpression cassette, and the GmMORF3 overexpression cassette is ligated to a plant expression vector to obtain a recombinant overexpression vector; S3. The recombinant overexpression vector is introduced into Agrobacterium rhizogenes, and soybean recipient material is treated with Agrobacterium rhizogenes-mediated hairy root transformation to induce the formation of hairy roots carrying the GmMORF3 overexpression cassette, thereby obtaining GmMORF3-transformed soybean chimeric material. The hairy roots of the GmMORF3-transformed soybean chimeric material are the transformed tissue, and the aboveground part is the non-transgenic tissue. S4. Using the empty vector-transformed Agrobacterium rhizogenes as a control material, the transcript level of the soybean GmMORF3 gene in the hairy roots of the GmMORF3-transformed soybean chimeric material was detected, and GmMORF3-overexpressing soybean chimeric materials with a higher transcript level of the soybean GmMORF3 gene in the hairy roots than the corresponding transcript level of the control material were screened. S5. The GmMORF3-overexpressing soybean chimeric material and the control material were cultured under the same NaCl-containing salt stress conditions, and the root length, biomass, reactive oxygen species accumulation, membrane lipid peroxidation characterization value, and antioxidant enzyme activity were detected respectively. When the corresponding detection values of root length, biomass, and antioxidant enzyme activity of the GmMORF3-overexpressing soybean chimeric material were greater than those of the control material, and the corresponding detection values of reactive oxygen species accumulation and membrane lipid peroxidation characterization value were less than those of the control material, it was determined that the salt tolerance of the GmMORF3-overexpressing soybean chimeric material was improved.
[0006] The present invention is further configured such that the soybean GmMORF3 gene coding sequence is the open reading frame sequence of GmMORF3, a member of the soybean MORF family, and the protein encoded by the open reading frame sequence contains a conserved MORF domain and belongs to the plant multicellular organelle RNA editing factor family of proteins.
[0007] The present invention is further configured such that the plant promoter is a constitutive plant promoter or a salt stress-responsive plant promoter, the GmMORF3 overexpression cassette further includes a transcription termination sequence, and the plant promoter, the soybean GmMORF3 gene coding sequence and the transcription termination sequence are connected in the direction from the 5' end to the 3' end.
[0008] The present invention is further configured such that the recombinant overexpression vector further includes a screening marker gene or a reporter gene, the screening marker gene or reporter gene being used to identify hairy roots carrying the GmMORF3 overexpression cassette in the GmMORF3-transformed soybean chimeric material.
[0009] The present invention is further configured such that the soybean acceptor material is a soybean hypocotyl.
[0010] The present invention is further configured such that the transcript level of the soybean GmMORF3 gene is detected by reverse transcription PCR or real-time quantitative PCR, and the soybean internal reference gene is used as a correction gene during detection.
[0011] The present invention is further configured such that the salt stress conditions containing NaCl include a culture medium, nutrient solution or cultivation substrate containing NaCl, and the GmMORF3 overexpressing soybean chimeric material and the control material are cultured simultaneously under the same NaCl conditions.
[0012] The present invention is further configured such that the salt tolerance evaluation index of the GmMORF3 overexpressing soybean chimeric material includes growth index, oxidative damage index and antioxidant enzyme activity. The growth index includes root length and biomass. The oxidative damage index includes reactive oxygen species accumulation and membrane lipid peroxidation characterization value. The reactive oxygen species accumulation is characterized by superoxide anion accumulation or hydrogen peroxide content. The membrane lipid peroxidation characterization value is malondialdehyde content. The antioxidant enzyme activity includes superoxide dismutase activity, peroxidase activity or catalase activity.
[0013] The beneficial effects of this invention are as follows: By constructing a GmMORF3 overexpression cassette and using Agrobacterium rhizogenes-mediated hairy root transformation to obtain GmMORF3 overexpressing soybean chimeric materials, the effects of GmMORF3 gene overexpression on soybean salt tolerance can be evaluated without performing stable genetic transformation plant regeneration and offspring genetic identification.
[0014] This invention uses GmMORF3 transcript level detection and NaCl salt stress screening evaluation, combined with indicators such as root length, biomass, reactive oxygen species accumulation, membrane lipid peroxidation characterization value and antioxidant enzyme activity, to screen out salt-tolerant soybean materials with good growth status and low oxidative damage. Attached Figure Description
[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.
[0016] Figure 1 This is a flowchart of the overall process of the method of the present invention.
[0017] Figure 2 This is a flowchart illustrating the process of obtaining and identifying GmMORF3-overexpressing soybean materials according to the present invention.
[0018] Figure 3 This is a flowchart illustrating the evaluation process of the tolerance of GmMORF3-overexpressing soybean materials under NaCl salt stress according to the present invention. Detailed Implementation
[0019] The technical solutions of the present invention will be described below with reference to the accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments.
[0020] Example 1 Please see Figures 1-3 This invention relates to a method for improving the salt tolerance of soybeans by overexpressing the GmMORF3 gene, comprising: S1. Provides the coding sequence of the soybean GmMORF3 gene. The soybean GmMORF3 gene coding sequence is derived from the soybean GmMORF3 gene and encodes a multi-organelle RNA editing factor containing a conserved MORF domain. The soybean GmMORF3 gene is a salt stress-induced expression gene. The soybean GmMORF3 gene coding sequence is the open reading frame sequence of GmMORF3, a member of the soybean MORF family. The protein encoded by the open reading frame sequence contains a conserved MORF domain and belongs to the plant multi-organelle RNA editing factor family of proteins.
[0021] S2. The soybean GmMORF3 gene coding sequence was operatively linked to a plant promoter to construct a GmMORF3 overexpression cassette. This cassette was then ligated into a plant expression vector to obtain a recombinant overexpression vector. The plant promoter was either a constitutive plant promoter or a salt-stress-responsive plant promoter. The GmMORF3 overexpression cassette also included a transcription termination sequence. The plant promoter, the soybean GmMORF3 gene coding sequence, and the transcription termination sequence were linked from the 5' to the 3' end. The recombinant overexpression vector also included a selection marker gene or a reporter gene, which was used to identify hairy roots carrying the GmMORF3 overexpression cassette in soybean chimeric materials transformed with GmMORF3.
[0022] S3. The recombinant overexpression vector was introduced into *Agrobacterium rhizogenes*, and soybean recipient material was treated with *Agrobacterium rhizogenes*-mediated hairy root transformation to induce the formation of hairy roots carrying the GmMORF3 overexpression cassette, thus obtaining GmMORF3-transformed soybean chimeric material. The hairy roots of the GmMORF3-transformed soybean chimeric material were the transformed tissue, and the aboveground parts were the non-transgenic tissue. The soybean recipient material was the soybean hypocotyl.
[0023] In the resulting soybean chimeric material, the hairy roots were transformed tissues carrying GmMORF3 overexpression cassettes, while the aboveground parts were non-transgenic tissues. In this embodiment, regeneration of stable genetically transformed plants and genetic identification of progeny were not performed.
[0024] S4. Using empty vector-transformed Agrobacterium rhizogenes as a control soybean chimeric material, prepared by hairy root transformation, the transcript level of the soybean GmMORF3 gene in the hairy roots of the GmMORF3-transformed soybean chimeric material was detected. Soybean chimeric materials overexpressing GmMORF3 with higher transcript levels in hairy roots than the corresponding transcript levels in the control material were screened. The transcript level of the soybean GmMORF3 gene was detected by reverse transcription PCR or real-time quantitative PCR, using a soybean internal reference gene as a correction gene.
[0025] S5. The GmMORF3-overexpressing soybean chimeric material and the control material were cultured under the same NaCl-containing salt stress conditions. Root length, biomass, reactive oxygen species (ROS) accumulation, membrane lipid peroxidation characterization value, and antioxidant enzyme activity were measured. When the corresponding values for root length, biomass, and antioxidant enzyme activity of the GmMORF3-overexpressing soybean chimeric material were greater than those of the control material, and the corresponding values for ROS accumulation and membrane lipid peroxidation characterization value were less than those of the control material, the salt tolerance of the GmMORF3-overexpressing soybean chimeric material was determined to be improved. NaCl-containing salt stress conditions included NaCl-containing culture media, nutrient solutions, or cultivation substrates. The GmMORF3-overexpressing soybean chimeric material and the control material were cultured simultaneously under the same NaCl conditions. The salt tolerance evaluation indicators for GmMORF3 overexpressing soybean chimeric materials include growth indicators, oxidative damage indicators, and antioxidant enzyme activities. Growth indicators include root length and biomass. Oxidative damage indicators include reactive oxygen species (ROS) accumulation and membrane lipid peroxidation. ROS accumulation is characterized by superoxide anion accumulation or hydrogen peroxide content, while membrane lipid peroxidation is characterized by malondialdehyde content. Antioxidant enzyme activities include superoxide dismutase activity, peroxidase activity, or catalase activity.
[0026] This invention employs Agrobacterium rhizogenes-mediated hairy root transformation to achieve overexpression of the GmMORF3 gene in soybean hairy roots. This eliminates the need for stable genetic transformation plant regeneration and offspring genetic identification, thus enabling the verification of the salt tolerance function of the GmMORF3 gene. This shortens the functional identification cycle and ensures that gene expression detection corresponds to salt stress phenotype evaluation.
[0027] Under NaCl salt stress, by comparing the root length, biomass, reactive oxygen species accumulation, membrane lipid peroxidation characterization value, and antioxidant enzyme activity of GmMORF3-overexpressing soybean chimeric materials and empty vector control materials, the effects of the GmMORF3 gene on soybean salt tolerance can be evaluated from three aspects: growth status, degree of oxidative damage, and antioxidant capacity. This provides experimental evidence for the application of the GmMORF3 gene in the study of soybean salt tolerance function.
[0028] Example 2 Please see Figure 2 Based on Example 1, it was demonstrated that soybean overexpression material with a higher GmMORF3 transcript level than the empty vector control could be obtained by constructing a GmMORF3 overexpression cassette and transforming it with Agrobacterium rhizogenes.
[0029] 1. Obtaining the coding sequence of the GmMORF3 gene Soybean seedlings of uniform growth were selected, and 0.10g of tender leaves were collected, ground in liquid nitrogen, and total RNA was extracted. 1.0μg of total RNA was used for reverse transcription to obtain first-strand cDNA. Using the first-strand cDNA as a template, the open reading frame sequence of the soybean GmMORF3 gene was amplified.
[0030] The PCR reaction mixture consisted of 50 μL of first-strand cDNA, 1 μL of forward primer, 1 μL of reverse primer, 25 μL of 2× high-fidelity PCR mixture, and 21 μL of sterile water. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 20 s, 72℃ extension for 60 s, 35 cycles, followed by a final extension at 72℃ for 5 min. The amplified products were recovered after detection by 1.0% agarose gel electrophoresis.
[0031] The recovered fragment was sequenced and verified to be the coding sequence of the soybean GmMORF3 gene. The soybean GmMORF3 gene coding sequence is an open reading frame sequence, encoding a protein containing a conserved MORF domain, belonging to the plant multicellular organelle RNA editing factor family. In the instruction manual, the soybean GmMORF3 gene coding sequence may be listed as SEQ ID NO:1, and the encoded protein sequence as SEQ ID NO:2.
[0032] 2. Detection of salt stress-induced expression of the GmMORF3 gene Ten-day-old soybean seedlings of uniform growth were selected and subjected to salt stress treatment with 120 mmol / L NaCl. Roots and leaves were collected at 0 h, 6 h, 12 h, and 24 h after treatment. The samples were flash-frozen in liquid nitrogen and then stored at -80 °C.
[0033] Total RNA was extracted and cDNA was synthesized. 1 μg of total RNA was used to synthesize the first-strand cDNA. Using GmACTIN11 as an internal reference gene, the transcript level of the soybean GmMORF3 gene was detected by real-time quantitative PCR. 2 -ΔΔCt Relative transcript levels were calculated using a method. Each treatment was configured with three biological replicates and three technical replicates.
[0034] The results showed that after treatment with 120 mmol / L NaCl, the transcript level of the GmMORF3 gene was upregulated in soybean roots and leaves, indicating that the GmMORF3 gene is a salt stress-induced gene.
[0035] 3. Construction of GmMORF3 overexpression cassette and recombinant overexpression vector The soybean GmMORF3 gene coding sequence was ligated into a plant expression vector. The recombinant overexpression vector contained a plant constitutive promoter, the soybean GmMORF3 gene coding sequence, a transcription termination sequence, and a reporter gene. The plant constitutive promoter, the soybean GmMORF3 gene coding sequence, and the transcription termination sequence were aligned from the 5' end to the 3' end to form a GmMORF3 overexpression cassette.
[0036] The ligation product was transformed into competent *E. coli* cells. Single clones were picked for colony PCR detection. Positive clones were validated by sequencing, and plasmids were extracted. Sequencing results showed that the insert orientation was correct and no frameshift mutations occurred in the open reading frame, thus obtaining the recombinant overexpression vector.
[0037] 4. Agrobacterium rhizogenes transformation and positive material screening The GmMORF3 recombinant overexpression vector was introduced into Agrobacterium rhizogenes K599. The OD of the culture medium was... 600 The value is 0.5-0.6.
[0038] Williams82 soybean seedlings with uniform growth were selected, and hypocotyls were cut off as recipient materials. The hypocotyls were immersed in Agrobacterium rhizogenes solution for 10-15 minutes and cultured at 25°C under 16 hours of light and 8 hours of darkness to induce the formation of hairy roots.
[0039] Agrobacterium rhizogenes with an introduced empty vector was treated in the same manner to obtain EV hairy root empty vector control material. Hairy roots with consistent growth status were screened to obtain OE-MORF3 hairy root overexpression material. The resulting soybean material was a soybean chimeric material in which the hairy roots belonged to the transformed tissue and the aboveground parts belonged to the non-transgenic tissue.
[0040] Hairy roots of OE-MORF3 and EV materials were collected separately, and the transcript level of the GmMORF3 gene was detected by real-time quantitative PCR. The results showed that the transcript level of the GmMORF3 gene in OE-MORF3 hairy roots was 12.7 times that in EV hairy roots, indicating that the GmMORF3 gene was overexpressed in soybean hairy roots.
[0041] Example 3 Please see Figure 3 Based on Examples 1 and 2, it was demonstrated that GmMORF3 overexpressing soybean materials have an advantage in salt tolerance evaluation under NaCl salt stress conditions.
[0042] 1. Salt stress treatment The OE-MORF3 hairy root overexpressing soybean chimeric material obtained in Example 2 was used as a control material, with the EV hairy root empty vector control soybean chimeric material as a control material.
[0043] OE-MORF3 and EV materials were set up as normal culture group and salt stress group, respectively. The salt stress group was treated with 120 mmol / L NaCl, while the normal culture group was not treated with NaCl. Each group had 10-15 biological replicates, the culture temperature was 25℃, the photocycle was 16h light and 8h dark, and the treatment time was 5 days.
[0044] 2. Measurement of growth indicators and oxidative damage indicators After the salt stress treatment was completed, root length, aboveground fresh weight, root fresh weight, and total dry weight were measured. Fresh weight was measured immediately after sampling. Dry weight was measured after drying at 65℃ to constant weight.
[0045] 0.10 g of root tissue from each group was ground with liquid nitrogen. After adding pre-cooled extraction solution, the mixture was centrifuged, and the supernatant was collected to determine the superoxide anion generation rate, hydrogen peroxide content, and malondialdehyde content. The superoxide anion generation rate and hydrogen peroxide content were used to characterize the amount of reactive oxygen species accumulated, while the malondialdehyde content was used to characterize the level of membrane lipid peroxidation.
[0046] 3. Antioxidant enzyme activity assay Superoxide dismutase (SOD), peroxidase (PERO), and catalase (CAT) activities were determined using a colorimetric method. The samples used for these measurements were the same as those used for the oxidative damage indices in section 2.2. Growth indices were measured in groups of 12 plants, and physiological indices were measured in groups of 3 biological replicates. Data are expressed as mean ± standard deviation.
[0047] Under normal culture conditions, there were no significant differences between the OE-MORF3 material and the EV control in terms of plant height, root length, aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight (P<0.05).
[0048] After treatment with 120 mmol / L NaCl for 5 days, the root length, aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight of the OE-MORF3 material were all greater than those of the EV control, with differences between groups reaching P<0.01.
[0049] Superoxide anion accumulation was detected by NBT staining, and hydrogen peroxide accumulation was detected by DAB staining. After salt stress treatment, the NBT and DAB staining intensity of leaves from the OE-MORF3 material was weaker than that of the EV control.
[0050] The malondialdehyde content, superoxide anion generation rate, and hydrogen peroxide content of the OE-MORF3 material were all lower than those of the EV control, while the superoxide dismutase activity, peroxidase activity, and catalase activity were all higher than those of the EV control, with intergroup differences reaching P<0.01.
[0051] The above results indicate that overexpression of the GmMORF3 gene in soybean hairy roots can alleviate reactive oxygen species accumulation and membrane lipid peroxidation under NaCl salt stress, and maintain root growth and biomass accumulation in soybean chimeric materials.
[0052] The conclusions of this embodiment are limited to soybean chimeric materials overexpressing hairy roots and do not indicate that a stable genetically inherited transgenic salt-tolerant soybean line has been obtained.
[0053] 4. Salt-resistant material determination Root length and biomass were used as growth evaluation indicators, superoxide anion production rate, hydrogen peroxide content and malondialdehyde content were used as oxidative damage evaluation indicators, and superoxide dismutase activity, peroxidase activity and catalase activity were used as antioxidant capacity evaluation indicators.
[0054] OE-MORF3 hairy root overexpression soybean chimeric material and EV hairy root empty vector control soybean chimeric material were simultaneously cultured under the same NaCl salt stress conditions. The results showed that the root length, aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight of the OE-MORF3 material were all greater than those of the EV control. The superoxide anion production rate, hydrogen peroxide content, and malondialdehyde content were all lower in the OE-MORF3 material than in the EV control. The activities of superoxide dismutase, peroxidase, and catalase were all greater in the OE-MORF3 material than in the EV control.
[0055] Based on the above test results, the root growth and biomass accumulation of the OE-MORF3 material under NaCl salt stress conditions were better than those of the EV control, while the amount of reactive oxygen species accumulation and membrane lipid peroxidation were less than those of the EV control, and the activity of antioxidant enzymes was greater than that of the EV control. This confirms that the salt tolerance of soybean chimeric materials is improved after the GmMORF3 gene is overexpressed in soybean hairy roots.
[0056] The above conclusions are limited to soybean chimeric materials overexpressing *Agrobacterium rhizogenes* in hairy roots and do not indicate that stable genetically inherited transgenic salt-tolerant soybean plants or salt-tolerant soybean lines have been obtained.
[0057] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. A method for improving soybean salt tolerance by overexpressing the GmMORF3 gene, characterized in that, include: S1. Provides a soybean GmMORF3 gene coding sequence, wherein the soybean GmMORF3 gene coding sequence is derived from the soybean GmMORF3 gene and encodes a multi-organelle RNA editing factor containing a conserved MORF domain, wherein the soybean GmMORF3 gene is a salt stress-induced expression gene; S2. The soybean GmMORF3 gene coding sequence is operatively linked to a plant promoter to construct a GmMORF3 overexpression cassette, and the GmMORF3 overexpression cassette is ligated to a plant expression vector to obtain a recombinant overexpression vector; S3. The recombinant overexpression vector is introduced into Agrobacterium rhizogenes, and soybean recipient material is treated with Agrobacterium rhizogenes-mediated hairy root transformation to induce the formation of hairy roots carrying the GmMORF3 overexpression cassette, thereby obtaining GmMORF3-transformed soybean chimeric material. The hairy roots of the GmMORF3-transformed soybean chimeric material are the transformed tissue, and the aboveground part is the non-transgenic tissue. S4. Using the empty vector-transformed Agrobacterium rhizogenes as a control material, the transcript level of the soybean GmMORF3 gene in the hairy roots of the GmMORF3-transformed soybean chimeric material was detected, and GmMORF3-overexpressing soybean chimeric materials with a higher transcript level of the soybean GmMORF3 gene in the hairy roots than the corresponding transcript level of the control material were screened. S5. The GmMORF3-overexpressing soybean chimeric material and the control material were cultured under the same NaCl-containing salt stress conditions, and the root length, biomass, reactive oxygen species accumulation, membrane lipid peroxidation characterization value, and antioxidant enzyme activity were detected respectively. When the corresponding detection values of root length, biomass, and antioxidant enzyme activity of the GmMORF3-overexpressing soybean chimeric material were greater than those of the control material, and the corresponding detection values of reactive oxygen species accumulation and membrane lipid peroxidation characterization value were less than those of the control material, it was determined that the salt tolerance of the GmMORF3-overexpressing soybean chimeric material was improved.
2. The method for improving soybean salt tolerance by overexpressing the GmMORF3 gene according to claim 1, characterized in that: The soybean GmMORF3 gene encoding sequence is the open reading frame sequence of GmMORF3, a member of the soybean MORF family. The protein encoded by the open reading frame sequence contains a conserved MORF domain and belongs to the plant multicellular organelle RNA editing factor family of proteins.
3. The method for improving soybean salt tolerance by overexpressing the GmMORF3 gene according to claim 1, characterized in that: The plant promoter is a constitutive plant promoter or a salt stress-responsive plant promoter. The GmMORF3 overexpression cassette also includes a transcription termination sequence. The plant promoter, the soybean GmMORF3 gene coding sequence, and the transcription termination sequence are connected in the 5' to 3' direction.
4. The method for improving soybean salt tolerance by overexpressing the GmMORF3 gene according to claim 1, characterized in that: The recombinant overexpression vector further includes a screening marker gene or a reporter gene, which is used to identify hairy roots carrying the GmMORF3 overexpression cassette in the GmMORF3-transformed soybean chimeric material.
5. The method for improving soybean salt tolerance by overexpressing the GmMORF3 gene according to claim 1, characterized in that: The soybean acceptor material is the soybean hypocotyl.
6. The method for improving soybean salt tolerance by overexpressing the GmMORF3 gene according to claim 1, characterized in that: The transcript level of the soybean GmMORF3 gene was detected by reverse transcription PCR or real-time quantitative PCR, with the soybean internal reference gene used as a correction gene during the detection.
7. The method for improving soybean salt tolerance by overexpressing the GmMORF3 gene according to claim 1, characterized in that: The NaCl-containing salt stress conditions include NaCl-containing culture media, nutrient solutions, or cultivation substrates, and the GmMORF3 overexpressing soybean chimeric material and the control material are cultured simultaneously under the same NaCl conditions.
8. The method for improving soybean salt tolerance by overexpressing the GmMORF3 gene according to claim 1, characterized in that: The salt tolerance evaluation indices of the GmMORF3 overexpressing soybean chimeric material include growth indices, oxidative damage indices, and antioxidant enzyme activities. The growth indices include root length and biomass. The oxidative damage indices include reactive oxygen species (ROS) accumulation and membrane lipid peroxidation characterization values. The ROS accumulation is characterized by superoxide anion accumulation or hydrogen peroxide content. The membrane lipid peroxidation characterization value is malondialdehyde content. The antioxidant enzyme activities include superoxide dismutase activity, peroxidase activity, or catalase activity.