Soybean salt tolerance promoting related protein GtCOBL9 as well as coding gene and application thereof
By overexpressing the GtCOBL9 gene in soybeans, coordinating ion homeostasis and enhancing cell wall structure, the problem of reduced soybean yield on saline-alkali land was solved, and the salt tolerance and stress resistance of soybeans were improved, which has commercial application value.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE SHENNONG LABORATORY
- Filing Date
- 2026-02-05
- Publication Date
- 2026-05-01
AI Technical Summary
Soybean yields drop sharply on saline-alkali land, and existing technologies have failed to effectively improve their salt tolerance, resulting in yield losses and economic risks.
By stably transferring the GtCOBL9 gene into soybeans, the salt tolerance and stress resistance of soybeans can be improved by coordinating ion homeostasis, reducing oxidative stress, and enhancing cell wall structure, and by utilizing the function of the GtCOBL9 protein.
It endows soybeans with excellent and stable resistance to stress, improves their survival rate and yield under salt stress, reduces economic risks, and provides a commercially viable breeding program.
Smart Images
Figure CN121950840A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a protein GtCOBL9 that promotes salt tolerance in soybeans, its encoding gene, and its applications. Background Technology
[0002] Soil salinization is a global problem leading to reduced crop yields. The accumulation of excessive sodium and chloride ions can cause a range of symptoms, including leaf scorching, growth inhibition, decreased photosynthesis, and even death. Soybeans, whose yields drop drastically in saline-alkali soils, are classified as moderately salt-sensitive crops. Salt stress induces osmotic stress, water loss, and oxidative imbalance.
[0003] COBRA-Like ( COBL The ) gene belongs to an important gene family, involved in the regulation of cell wall expansion, and is associated with some cellulase A ( CesA Members of the gene family are co-expressed. COBLs The gene encodes a glycosylphosphatidylinositol (GPI) anchoring protein, which is located on the outer surface of the plant plasma membrane. It receives signals from the cell wall, rapidly transmits them to the plasma membrane, and regulates the assembly and crystallization of cellulose chains. COBL The gene has been identified in a variety of dicotyledonous and monocotyledonous plants, such as tomatoes ( Solanum lycopersicum There are 16 of them, corn ( Zea maize There are 11 of them, rice ( Oryza sativa There are 9 of them, upland cotton ( Gossypium hirsutum There are 39 species in total, including Populus tomentosa (…). populus trichocarpa There are 14 of them, barley ( Hordeum vulgare There are 13 species in total, including Arabidopsis thaliana. Arabidopsis thaliana There are 12 in the group and 24 in the soybean. COBL Originally studied in Arabidopsis thaliana, it is essential for cellulose deposition. atcobl4 mutants (also known as mutants) irx6 It has reduced secondary cell wall (CW) thickness and collapsed xylem. Initially... COBL The gene was identified as a "brittle stalk" gene in rice and maize. The "Osbrittle culm 1" mutant and the "Zmbrittle stalk 2" mutant exhibited reduced cellulose content and cell wall thickness, resulting in significantly impaired stem mechanical strength. Furthermore, in poplar, COBL4 It regulates the rate of cellulose synthase and promotes cellulose deposition in the secondary cell wall. Although COBL Genes have been characterized in important crops, but COBL The function of salt tolerance in cultivated soybeans remains unexplored. Therefore, utilizing... GtCOBL9 As a salt-tolerant genetic resource, it can improve the salt stress resistance of soybeans and increase soybean yield. Summary of the Invention
[0004] The purpose of this invention is GtCOBL9 As a novel genetic tool for improving salt tolerance. Specifically: to... GtCOBL9 Stable genetic transformation into cultivated soybeans can confer salt tolerance. Furthermore, GtCOBL9 By coordinating ion homeostasis, mitigating oxidative stress, and enhancing cell wall structure, soybeans acquire excellent and stable stress resistance. This research specifically relates to GtCOBL9, a protein that promotes salt tolerance in soybeans, its encoding gene, and its applications.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a soybean gene GtCOBL9, as shown in SEQ ID NO.1.
[0006] The present invention also provides the protein encoded by the soybean gene GtCOBL9, as shown in SEQ ID NO.2.
[0007] This invention also provides the application of the soybean gene GtCOBL9 or the protein described herein in the preparation of products that improve plant salt tolerance, stress resistance, or cultivate salt-tolerant transgenic plants, wherein the products include any one of the following: (1) Recombinant expression vector; (2) Recombinant bacteria; The method for constructing the recombinant expression vector is as follows: the soybean gene GtCOBL9 is ligated into the pBC12 vector; The method for constructing the recombinant bacteria is as follows: the soybean gene GtCOBL9 or the recombinant expression vector described in (1) is transferred into Agrobacterium EHA105.
[0008] This invention also provides the application of the soybean gene GtCOBL9 in improving plant salt tolerance, stress resistance, or cultivating salt-tolerant transgenic plants, comprising the following steps: constructing a soybean gene containing the gene shown in SEQ ID NO.1. GtCOBL9 The recombinant expression vector was transformed into Agrobacterium and cultured to obtain recombinant bacteria carrying the recombinant expression vector. The recombinant bacteria carrying the recombinant expression vector were then used to infect plant plants. The empty vector used to construct the recombinant expression vector was pBC12; The Agrobacterium is EHA105.
[0009] The present invention also provides an expression cassette for improving plant salt tolerance, stress resistance, or cultivating salt-tolerant transgenic plants, comprising any one of the following: (1) Recombinant expression vector; (2) Recombinant bacteria; The method for constructing the recombinant expression vector is as follows: the soybean gene GtCOBL9 is ligated into the pBC12 vector; The method for constructing the recombinant bacteria is as follows: the soybean gene GtCOBL9 or the recombinant expression vector described in (1) is transferred into Agrobacterium EHA105.
[0010] The present invention also provides primer pairs for amplifying the soybean gene GtCOBL9 shown in SEQ ID NO.1, as shown in SEQ ID NO.3~4.
[0011] The present invention also provides primer pairs for detecting the expression level of the soybean gene GtCOBL9 shown in SEQ ID NO.1, as shown in SEQ ID NO.7~8.
[0012] This invention also provides the application of the primer pairs in screening soybean varieties with strong salt tolerance and stress resistance.
[0013] This invention also provides a method for improving the salt tolerance and stress resistance of plants, comprising the following steps: Overexpress the soybean gene GtCOBL9 shown in SEQ ID NO.1 in plants; or construct a recombinant expression vector containing the soybean gene GtCOBL9 shown in SEQ ID NO.1, transform the recombinant expression vector into Agrobacterium, culture it to obtain recombinant bacteria carrying the recombinant expression vector, and use the recombinant bacteria carrying the recombinant expression vector to infect plant plants. The Agrobacterium is EHA105.
[0014] This invention also provides a method for cultivating salt-tolerant transgenic plants, comprising the following steps: A recombinant expression vector containing the soybean gene GtCOBL9 shown in SEQ ID NO.1 was constructed. The recombinant expression vector was transformed into Agrobacterium and cultured to obtain recombinant bacteria carrying the recombinant expression vector. The recombinant bacteria carrying the recombinant expression vector were used to infect plant plants. The Agrobacterium is EHA105.
[0015] The solution of the present invention has the following advantages: Stable genetic transformation of the soybean gene GtCOBL9, shown in SEQ ID NO.1, into cultivated soybean confers salt tolerance. Furthermore, GtCOBL9 endows soybeans with excellent and stable stress resistance by coordinating ion homeostasis, mitigating oxidative stress, and enhancing cell wall structure. This invention also provides transgenic soybean plants expressing the soybean gene GtCOBL9, which exhibit higher salt tolerance than the wild type, providing a basis for constructing salt-tolerant soybean plants and having significant commercial implications for increasing soybean yield.
[0016] (1) Scientific and technological significance
[0017] Discovery of a new gene: This invention is the first to demonstrate that GtCOBL9 from perennial wild soybean can confer salt tolerance to cultivated soybeans, providing direct genetic evidence for the value of wild germplasm as a superior source of stress-resistant genetic modification.
[0018] Elucidating a novel mechanism: This invention establishes a causal link between GtCOBL9 and enhanced salt tolerance. It provides tools (overexpression lines, interacting factors such as GmAKT1 and GmCAX1) to dissect precise molecular pathways, possibly through targeting CESA genes to regulate osmotic and redox homeostasis and cellulose synthesis.
[0019] Creating research tools: The GtCOBL9-OE transgenic line is of great value and can be used for future research on plant cell wall biology, salt stress signal transduction, and the functional diversity of the COBL gene family.
[0020] (2) Agricultural and economic significance
[0021] Addressing a key production constraint: Soil salinization is a critical and increasingly severe abiotic stress that significantly limits global soybean yields. This invention provides a genetic solution to address salinization, offering a pathway to developing soybean varieties capable of maintaining productivity in saline-affected lands.
[0022] Improving yield stability and reducing risk: This invention helps stabilize yields in areas susceptible to salinization, thereby reducing economic risk for farmers, increasing arable land, and ensuring a balance between supply and demand.
[0023] (3) Practical and commercial significance
[0024] Shorten breeding time and cost: The ability to select traits at the seedling stage can shorten the breeding cycle by several generations and reduce the cost and space required for extensive field phenotypic identification under salt stress. Attached Figure Description
[0025] Picture 1 for GtCOBL9 Overexpression conferred salt tolerance on cultivated soybean (A represents the response of GtCOBL9-OE at the soybean seedling stage; B represents...). GtCOBL9 - The degree of salt damage in the OE strain; C is GtCOBL9 - Survival rate of OE strains); Picture 2 for GtCOBL9 Participating in ion steady state (A is) GmAKT1 The expression; B is GmCAX1 The expression; C~F is GtCOBL9 Na in the -OE strain + and K +Flux; G represents the in vitro interaction between GtCOBL9 and GmAKT1 and GmCAX1; H represents... GtCOBL9 and GmAKT1 and GmCAX1 (interactions within the body); Picture 3 for GtCOBL9 Protect soybeans from oxidative stress (A~B are) GtCOBL9 ROS accumulation in plant leaves; C~D are GmCAT and GmPOD E represents CAT enzyme activity; F represents POD activity; G represents proline production; H represents MDA production. Picture 4 for GtCOBL9 Regulation of cellulose biosynthesis (A: Conserved domain analysis of COBRA-encoded protein; B: Cellulose regulation of CESA1 gene expression; C: Cellulose regulation of CESA2 gene expression; D: Cellulose content in the roots of GtCOBL9-OE plants). Detailed Implementation
[0026]
[0027] The protein encoded by the soybean gene GtCOBL9 shown in SEQ ID NO.1 is shown in SEQ ID NO.2; SEQ ID NO.2: MEFESGTQKQKGTFACLKMVALIALCLILISPAESFDPLDPTGNVTIRWDIMSWTSDGYLATVTLFNFQLYRNIMNPGWTLGWTWAKKEIIWAMMGAQATEQGNCAKFKLKIPHSCKRNPEVVDLLPGAPFNMQFTNCCRGGVLTSWGQNPSGAVSAFQIGVGLSGTSNKTVKLPKNFKLLGPGPGYSCGPAKIVPSTAILTDDRRRKMQALMSWNVTCTYSQFLAS KNPSCCVSLSSFYSDKVTGCPPCACGCQNNNTCVTKDSKILQENVTSPHRKSDITLTPKPLLQCTHHLCHVRVHWHLKDNYKDYWRVKIAIINFNYRLNFTDWSLVVQHPNLNNVT QVYSFEYMPLLPYESINDTGMFYGLKYYNDLLMEAGPKGNVQSEVLMKKDKNIFTLKQGWAFPRRVYFNGDECMLPPPDSYPMLPNSGHKLPTTITLMATYVVFTLFFHLVVTLF.
[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0029] The primers used in the embodiments of this invention are shown in Table 1: Table 1 Primers
[0030] Example 1
[0031] 1. Method
[0032] 1.1 Identification and Cloning of Soybean Gene GtCOBL9
[0033] To identify salt-tolerant genes from perennial wild soybean germplasm resources, 24 germplasm resources were selected and treated twice with salt solutions of different concentrations (0, 200, 400, 500, 550, and 600 mM / L NaCl) for four weeks. Leaf salt damage was assessed according to the following criteria: Grade 1 = healthy, vibrant green leaves with no signs of salt damage; Grade 2 = mild salt damage, with slight yellowing of true leaves; Grade 3 = moderate salt damage, with yellowing of compound leaves with three leaves; Grade 4 = severe salt damage, with over 75% of leaves yellowing; Grade 5 = plant death and complete wilting. Grades 1 and 2 were considered salt-tolerant, while grades 3 to 5 were considered salt-sensitive. The control group required irrigation with only plain water in their pots.
[0034] After one month of treatment with a salt concentration of 500 mM / L, recovery culture was conducted. Two-thirds of the 24 germplasm resources were able to tolerate salt stress, and three of these resources were able to flower and bear fruit normally. Salt-tolerant varieties were selected from these. G. CHdetika and G. tabacina Transcriptome analysis was performed on two resources. Roots were treated with 200 mM NaCl during the VC stage, and samples were taken at 1, 6, 12, 24, and 48 hours after salt treatment, from both the control group (0 hours). Results revealed differentially expressed genes (DEGs) between the two resources, among which… G. tabacina Among them, 21 genes showed sustained upregulation of expression. GtCOBL9 This is one of them. It was synthesized from Sangon Biotech (Shanghai) Co., Ltd. GtCOBL9 The CDS sequence of the gene (as shown in SEQ ID NO.1) is then ligated with the target vector as described below, with specific steps in 1.3.
[0035] Primers for amplifying the CDS sequence of the soybean gene GtCOBL9 are shown in SEQ ID NO. 3~4.
[0036] The amino acid sequence encoded by the nucleotide shown in SEQ ID NO.1 is shown in SEQ ID NO.2.
[0037] 1.2 Creation and Salt Tolerance Verification of Transgenic Soybean Plants Overexpressing GtCOBL9
[0038] Will GtCOBL9 The full-length coding sequence (CDS) of the gene (SEQ ID NO:1) was inserted into the 35S Flag overexpression vector (pBC12) to obtain a recombinant expression vector. This recombinant expression vector was then transformed into Agrobacterium rhizogenes EHA105 via stable genetic transformation to obtain the recombinant bacteria. The recombinant bacteria were used to infect cultivated soybean variety Zhongji 602 to obtain transgenic soybeans (transgenic soybeans constructed by the Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences).
[0039] In order to detect GtCOBL9 Salt stress tolerance of overexpression materials, selected from transgenic soybeans GtCOBL9 -OE-1, genetically modified soybeans GtCOBL9 -OE-2 and wild-type Zhongji 602 were compared, with three replicates for each treatment. Each small pot (9.5cm × 10.8cm × 10.8cm) was filled with vermiculite, and three seeds were sown. The plants were grown in a greenhouse (26℃, 16h light / 8h darkness, 60% humidity). Nine small pots (each with three seedlings) were placed in a large blue box (46cm × 32cm × 10cm), and each large blue box was watered with 2 liters of water. Watering was then increased to 2 liters every three days. After the soybean true leaves had fully expanded (10 days), a salt treatment was applied, with 2 liters of 200 mM / L NaCl saline solution poured into the pots every two days until salt damage symptoms such as leaf burn appeared. On day 15 of salt treatment, the degree of salt damage to the leaves was assessed: 1 = healthy green leaves, no salt damage observed; 2 = mild salt damage, slight yellowing of true leaves; 3 = moderate salt damage, yellowing of trifoliate leaves; 4 = severe salt damage, more than 75% of leaves turned yellow; 5 = death, complete wilting of the plant. Levels 1 and 2 were salt-tolerant, while levels 3-5 were salt-sensitive. For the control group, the pots were simply watered.
[0040] 1.3 Plasmid Construction
[0041] Yeast two-hybrid (Y2H) assay was used to detect... GtCOBL9 and GmAKT1 ( Ion inward rectifier channel ） , GmCAX1 The in vitro interactions of (calcium / proton antiporters involved in sodium ion isolation) were investigated using homologous recombination. GtCOBL9 The CDS (SEQ ID NO.1) was linked to the carrier pXGY17 containing the Cub domain to obtain GtCOBL9-pXGY17, and simultaneously, the InFusion method was used to... GmAKT1 and GmCAX1 The three molecules were fused with the nub-containing vector pXGY18 to obtain GmAKT1-pXGY18 and GmCAX1-pXGY18, respectively. The in vivo interactions of these three molecules were verified using a luciferase-luciferase (LUC) complementation assay. GtCOBL9 GtCOBL9-nLUC was obtained by fusing with the pCAMBIA1300-nLUC vector. GmAKT1 and GmCAX1 were respectively linked with the pCAMBIA1300-cLUC vector to obtain GmAKT1-cLUC and GmCAX1-cLUC.
[0042] 1.4 Yeast Two-Hybrid Experiment
[0043] Yeast two-hybrid (Y2H) experiments were performed using a splitting ubiquitin membrane system. Bait vectors GtCOBL9-pXGY17, GmAKT1-pXGY18, and GmCAX1-pXGY18 were co-transformed into yeast strain NMY51 and grown on SD / -Leu-Trp solid medium at 28°C for three days. After successful transformation, several clones were picked and suspended in sterile ddH2O. After serial dilutions, the samples were spotted onto SD / -Leu-Trp, SD / -His-Leu-Trp, and SD / -Ade-His-Leu-Trp media and incubated at 28°C for 48 hours, and colony growth was observed. The empty vector GtCOBL9-pXGY17 was used as a negative control.
[0044] 1.5 Luciferase (LUC) Complementation Assay
[0045] examine GtCOBL9 and GmAKT1 and GmCAX1 To investigate the interaction between GtCOBL9-nLUC, GmAKT1-cLUC, and GmCAX1-cLUC, a fission-LUC complementation experiment was conducted. GtCOBL9-nLUC, GmAKT1-cLUC, and GmCAX1-cLUC were transfected into Agrobacterium GV3101 to obtain GV3101 strains carrying these GtCOBL9-nLUC, GmAKT1-cLUC, and GmCAX1-cLUC. These GV3101 strains were cultured at 28°C for 48 hours and then resuspended in injection buffer (10 mM MgCl2, 10 mM MES, and 100 μM acetylsyleugenol, pH 5.6). Equal volumes of the corresponding cLUC and nLUC constructs were then mixed (final concentration OD600 = 0.5 / each) and infiltrated into leaves of *Nicotiana benthamiana*. The infiltrated leaves were covered with plastic caps to maintain high humidity. Two days after immersion, the LUC substrate D-luciferin (Promega) was applied to leaves co-expressing different constructs, and LUC activity was detected using a CCD camera equipped with AllDoc_x software (Tanon 4800Multi).
[0046] 1.6 Real-time quantitative PCR
[0047] Identification GtCOBL9 Overexpression and detection of gene expression changes after NaCl stress treatment were performed by germinating seeds in vermiculite under greenhouse conditions (26°C, 16 hours light / 8 hours dark, 60% humidity). At 5 days, seedlings were transferred to a hydroponic system containing a 1 / 2 strength Hoagland solution. For 10-day-old seedlings... GtCOBL9-OE and Zhongji 602 seedlings were treated with 150 mM / L NaCl (added to 1 / 2 strength Hoagland nutrient solution) for 3 h, 6 h, 12 h, 24 h, and 48 h, respectively. Seedlings grown in nutrient solution without NaCl served as a 0 h control. All required samples were collected using liquid nitrogen at specific time intervals, and total RNA was extracted using the Vazyme RNA Extraction Kit (RC411-01) and converted to cDNA using the Takara (RR037A) kit. All RT-qPCR was performed using a ChamQ Universal SYBR qPCR MasterMix. The internal control gene Actin11 was used as a control.
[0048] 1.7 Stress Index Measurement
[0049] ROS accumulation was tested using GtCOBL9-OE and Zhongji 602 materials as described above. ROS accumulation in leaves was detected using seedlings grown in 1 / 2 strength Hoagland solution for 12 days as a control. For treatment, the growth solution was supplemented with 150 mM / L NaCl for 24 hours. Then, the detached leaves were vacuum-sealed for 20 minutes and stained overnight at room temperature with NBT (BCIP, AR0043) and DAB (R24611) working solutions. Finally, the staining was decolorized with 75% ethanol, and images were taken under a stereomicroscope (MSHOT). Furthermore, to test enzyme activity, proline, MDA, and cellulose content, treatment and sample collection were performed as described above. To test CAT, POD, proline, and MDA in leaves, and cellulose in roots and leaves, 150 mM NaCl was applied for 24 hours, and samples were collected. Physiological parameters were measured by Qingdao Future Testing Technology Co., Ltd. The following kits from Suzhou Greens Biotechnology Co., Ltd. were used: (CAT; G0105W48), (POD; G0107W48), (Proline; G0111W48), and (MDA; G0109W48). In addition, cellulose content was determined by Nanjing Hanguang Testing Technology Co., Ltd. using the anthraquinone sulfuric acid colorimetric method. For the measurement of Na... + and K + The throughput was measured by treating the sample with 100 mM NaCl for 24 hours and then performing NMT measurements.
[0050] 1.8 Statistical Analysis
[0051] Statistical analysis was performed using IBM SPSS Statistics software version 25 (IBM). For comparisons between two sample groups, the following methods were applied: Student's t - Tests. The results of qRT-PCR and physiological parameters were analyzed using analysis of variance (ANOVA).
[0052] 2. Results
[0053] 2.1 Overexpression GtCOBL9 Creation and Salt Tolerance Verification of Transgenic Soybean Plants
[0054] Results of salt stress treatment on transgenic plants as follows Picture 1 As shown, GtCOBL9 The survival rate of the overexpression lines was significantly increased, which indicates that... GtCOBL9 Overexpression plays a key role in conferring salt tolerance.
[0055] 2.2 GtCOBL9 Participating in ion steady state
[0056] Under NaCl stress, compared to the wild type, GtCOBL9 -OE seedlings exhibited characteristic flux patterns in their roots, including enhanced Na+. + Ion efflux and increased K + Ion influx leads to higher K + / Na + Ratio. Furthermore... GtCOBL9 and GmAKT1 and GmCAX1 Physical interactions provide the molecular basis for regulating the activity of these ion channels and promoting efficient ion homeostasis, such as... Picture 2 As shown.
[0057] 2.3 GtCOBL9 Protect soybeans from oxidative stress
[0058] GtCOBL9 Soybeans are able to cope with salt-induced excess reactive oxygen species by activating their antioxidant defense system. Under stress conditions, this system encodes core antioxidant enzymes (such as catalase). GmCAT ) and peroxidase ( GmPOD The expression level of CAT gene was significantly increased in GtCOBL9-OE plants. Correspondingly, the in vitro enzyme activity of CAT was significantly higher in transgenic plants. Furthermore, these plants accumulated more of the compatible solute proline, which functions as an osmotic protectant and free radical scavenger. In addition, the GtCOBL9-OE lines exhibited lower MDA accumulation (a key indicator of membrane lipid peroxidation), representing... GtCOBL9 Its role in membrane stability.
[0059] like Picture 3 As shown.
[0060] 2.4 GtCOBL9 Regulation of cellulose biosynthesis
[0061] GtCOBL9It also promotes structural tolerance by enhancing the cell wall. The GtCOBL9 protein shares conserved sequence domains with the COBRA family of known regulators of cellulose biosynthesis and cell wall structure. Picture 4 As shown, the increase in cellulose content in soybean roots and the upregulation of cellulose synthesis (CESA) genes (GmCESA1 is Glyma.04G067900 and GmCESA2 is Glyma.08G117500) in GtCOBL9-OE plants reveal their importance in cell wall modification under salt stress.
[0062] As illustrated in the above embodiments, a protein called GtCOBL9, which promotes salt tolerance in soybeans, along with its encoding gene and applications, is disclosed. This invention enables the stable genetic transformation of the soybean gene GtCOBL9 (SEQ ID NO.1) into cultivated soybeans, thereby conferring salt tolerance. Furthermore, GtCOBL9 endows soybeans with excellent and stable stress resistance by coordinating ion homeostasis, mitigating oxidative stress, and enhancing cell wall structure. This invention also provides transgenic soybean plants expressing the soybean gene GtCOBL9, which exhibit higher salt tolerance than the wild type, providing a basis for constructing salt-tolerant soybean plants and having significant commercial implications for increasing soybean yield.
[0063] 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 gene GtCOBL9, characterized in that, The soybean gene GtCOBL9 is shown in SEQ ID NO.
1.
2. The protein encoded by the soybean gene GtCOBL9 according to claim 1, characterized in that, As shown in SEQ ID NO.
2.
3. The use of the soybean gene GtCOBL9 of claim 1 or the protein of claim 2 in the preparation of products that improve plant salt tolerance, stress resistance, or cultivate salt-tolerant transgenic plants, wherein the products comprise any one of the following: (1) Recombinant expression vector; (2) Recombinant bacteria; The method for constructing the recombinant expression vector is as follows: the soybean gene GtCOBL9 described in claim 1 is linked into the pBC12 vector; The method for constructing the recombinant bacteria is as follows: the soybean gene GtCOBL9 described in claim 1 or the recombinant expression vector described in (1) is transferred into Agrobacterium EHA105.
4. The application of the soybean gene GtCOBL9 as described in claim 1 in improving plant salt tolerance, stress resistance, or cultivating salt-tolerant transgenic plants, characterized in that... The steps include: constructing a soybean gene containing the one shown in SEQ ID NO.
1. GtCOBL9 The recombinant expression vector was transformed into Agrobacterium and cultured to obtain recombinant bacteria carrying the recombinant expression vector. The recombinant bacteria carrying the recombinant expression vector were then used to infect plant plants. The empty vector used to construct the recombinant expression vector was pBC12; The Agrobacterium was EHA105.
5. An expression cassette for improving plant salt tolerance, stress resistance, or cultivating salt-tolerant transgenic plants, characterized in that, Includes any one of the following: (1) Recombinant expression vector; (2) Recombinant bacteria; The method for constructing the recombinant expression vector is as follows: the soybean gene GtCOBL9 described in claim 1 is linked into the pBC12 vector; The method for constructing the recombinant bacteria is as follows: the soybean gene GtCOBL9 described in claim 1 or the recombinant expression vector described in (1) is transferred into Agrobacterium EHA105.
6. The primer pair for amplifying the soybean gene GtCOBL9 shown in SEQ ID NO.1, characterized in that, As shown in SEQ ID NO.3~4.
7. A primer pair for detecting the expression level of the soybean gene GtCOBL9 shown in SEQ ID NO.1, characterized in that, As shown in SEQ ID NO.7~8.
8. The application of the primer pair according to claim 7 in screening soybean varieties with strong salt tolerance and stress resistance.
9. A method for improving the salt tolerance and stress resistance of plants, characterized in that, Includes the following steps: Overexpress the soybean gene GtCOBL9 shown in SEQ ID NO.1 in plants; or construct a recombinant expression vector containing the soybean gene GtCOBL9 shown in SEQ ID NO.1, transform the recombinant expression vector into Agrobacterium, culture it to obtain recombinant bacteria carrying the recombinant expression vector, and use the recombinant bacteria carrying the recombinant expression vector to infect plant plants. The Agrobacterium was EHA105.
10. A method for cultivating a salt-tolerant transgenic plant, characterized in that, Includes the following steps: A recombinant expression vector containing the soybean gene GtCOBL9 shown in SEQ ID NO.1 was constructed. The recombinant expression vector was transformed into Agrobacterium and cultured to obtain recombinant bacteria carrying the recombinant expression vector. The recombinant bacteria carrying the recombinant expression vector were used to infect plant plants. The Agrobacterium was EHA105.