Soybean salt-tolerant related gene GmRLP15, and coding protein and application thereof
By cloning and overexpressing the soybean salt tolerance-related gene GmRLP15 and its encoded protein, the problem of limited soybean growth in saline-alkali environments was solved, and the salt tolerance of soybeans was significantly improved. Salt-tolerant soybean varieties were then bred by constructing a recombinant expression system.
Patent Information
- Application Number
- CN202410711226.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-04
- Publication Date
- 2025-12-05
- Estimated Expiration
- 2044-06-04
AI Technical Summary
Current technologies have made slow progress in improving the salt tolerance of soybeans. Traditional breeding strategies are time-consuming and have limited effects, and the lack of in-depth understanding of molecular mechanisms has limited the growth of soybeans in saline-alkali environments.
The soybean salt tolerance-related gene GmRLP15 and its encoded protein were cloned and overexpressed. Recombinant expression vectors and recombinant bacteria were constructed and introduced into soybean plants. Enhanced promoters and translation control signals were used to ensure gene expression. Salt-tolerant soybean varieties were bred by combining stress screening technology.
Significantly improves the salt tolerance of soybeans, cultivates transgenic plants that can thrive under salt stress, enhances their growth performance in saline-alkali environments, and solves the salt tolerance problem that has not been effectively addressed in existing technologies.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of genetic engineering, and particularly relates to a salt-tolerant related gene GmRLP15 of soybean, and a coding protein and application thereof. BACKGROUND
[0002] Soybean is a major source of oil and protein, and is an important economic oil crop. With the intensification of global climate change, soil salinization is becoming increasingly serious, which has caused great threat to agricultural production. Therefore, it is of great significance to use modern biological means to improve the salt tolerance of soybean and screen soybean plants with excellent salt tolerance traits for solving the influence of salinized soil on soybean production.
[0003] Salt stress is usually caused by high concentrations of sodium ions and chloride ions in soil. Salt stress includes osmotic stress, ion stress and secondary stress. When the salt content in the external environment is high enough to significantly change the water potential, it will affect plants. This osmotic stress makes it difficult for plant root cells to absorb water, leading to high intracellular osmotic pressure, cell shrinkage, and water absorption that cannot meet the needs of plants. At the same time, under high salt environment, the ion channels and transport proteins on the cell membrane of plants are also inhibited, thereby limiting the entry of water and nutrients, which will inhibit the growth and development of soybean plants, resulting in reduced soybean yield.
[0004] The response of soybean to salt stress is a complex molecular regulation network, which is the result of the delicate interweaving and synergistic action of multiple physiological and biochemical reactions. Traditional breeding strategies are time-consuming and have limited effect in improving soybean salt tolerance. In order to accelerate the breeding of salt-tolerant new soybean varieties, we turn to molecular breeding technology. However, the current understanding of the molecular mechanism of soybean salt tolerance is still insufficient, and the related research progress is relatively slow. Therefore, we urgently need to further explore more new genes that have the potential to improve the salt tolerance of soybean. These studies have great significance for improving the salt tolerance of soybean and cultivating new varieties that can grow vigorously in saline-alkali environment. SUMMARY
[0005] To solve the above technical problems in the prior art, the purpose of the present application is to disclose a salt-tolerant related gene GmRLP15 of soybean, and a coding protein and application thereof.
[0006] The first purpose of the present application is to provide a salt-tolerant related gene GmRLP15, which is a DNA molecule as described in 1) or 2) or 3) below:
[0007] 1) a DNA molecule with a gene sequence as shown in SEQ ID NO. 1;
[0008] 2) a DNA molecule with a CDS sequence as shown in SEQ ID NO. 2;
[0009] 3) DNA molecules hybridizing with the DNA sequences defined in 1) or 2) under stringent conditions and encoding the proteins.
[0010] A second object of the present application is to provide the protein encoded by the aforementioned gene GmRLP15.
[0011] In particular, the protein provided by the present application is selected from any one of the following (a) or (b):
[0012] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3;
[0013] (b) a protein derived from the target sequence selected from SEQ ID NO. 2.
[0014] SEQ ID NO. 3 in the sequence listing consists of 872 amino acids.
[0015] A third object of the present application is to provide a recombinant expression vector, an expression cassette or a recombinant bacterium comprising the aforementioned gene GmRLP15.
[0016] Further, the recombinant expression vector or expression cassette is obtained by inserting the gene GmRLP15 into the recombination site of the vector pBA002 digested by XbaI; and the recombinant expression vector or expression cassette is transformed into an engineering bacterium to obtain the recombinant bacterium.
[0017] The recombinant expression vector containing any one of the aforementioned genes also falls within the protection scope of the present application.
[0018] The recombinant expression vector containing the gene can be constructed by using the existing plant expression vector.
[0019] The plant expression vector includes Agrobacterium binary vector and vector that can be used for plant microprojectile bombardment, etc. The plant expression vector can further comprise the 3' untranslated region of the exogenous gene, i.e. comprising the polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium crown gall inducing (Ti) plasmid gene (such as the Nos gene of the nopaline synthase), the 3' untranslated region of the plant gene (such as the soybean storage protein gene) all have similar functions.
[0020] When the gene construct is used to construct a recombinant plant expression vector, any one of the enhanced promoters or constitutive promoters, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize, can be added before the transcription initiation nucleotide, which can be used alone or in combination with other plant promoters; in addition, when the gene construct of the present application is used to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used, and these enhancer regions can be ATG start codon or adjacent regions start codon, but must be the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.
[0021] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can express enzymes or luminescent compounds that can produce color changes in plants (GUS genes, luciferase genes, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.) or anti-chemical reagent marker genes (such as herbicide-resistant genes) and the like. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.
[0022] The recombinant expression vector can be a recombinant plasmid obtained by inserting the gene GmRLP15 into the recombination site of the restriction enzyme XbaI single enzyme digestion vector pBA002. The pBA002 containing GmRLP15 is named pBA002-GmRLP15.
[0023] The expression cassette containing any one of the above-mentioned genes GmRLP15, the transgenic cell line and the recombinant bacteria all belong to the protection scope of the present application.
[0024] The fourth object of the present application is to provide primers for amplifying the aforementioned gene GmRLP15, and the primer pair for amplifying the full length or any fragment of the gene GmRLP15 also belongs to the protection scope of the present application. In a specific example, the primers are shown as SEQ ID NO. 4 and SEQ ID NO. 5.
[0025] The fifth object of the present application is to provide the aforementioned gene GmRLP15, the aforementioned protein, the aforementioned recombinant expression vector, the expression cassette or the recombinant bacteria, or the aforementioned primer, expression vector or recombinant bacteria for use in improving the salt tolerance of soybeans.
[0026] Further, the salt tolerance of soybeans is improved. The overexpression of the aforementioned gene GmRLP15 improves the salt tolerance of soybeans.
[0027] Preferably, the aforementioned recombinant expression vector, expression cassette or recombinant bacteria is introduced into soybean to overexpress the aforementioned gene GmRLP15.
[0028] A sixth object of the present application is to provide a method for improving salt tolerance of soybean by overexpressing the aforementioned gene GmRLP15 in soybean plants, thereby improving salt tolerance of soybean.
[0029] The aforementioned gene GmRLP15 can be overexpressed in soybean plants by introducing the aforementioned recombinant expression vector, expression cassette or recombinant bacteria into soybean to overexpress the aforementioned gene GmRLP15 in soybean plants.
[0030] The expression vector carrying the gene can be transformed into plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.
[0031] A seventh object of the present application is to provide a method for cultivating a salt-tolerant soybean variety by overexpressing the aforementioned gene GmRLP15 in soybean plants to obtain a salt-tolerant soybean variety. Preferably, the aforementioned gene GmRLP15 can be overexpressed in soybean plants by introducing the aforementioned recombinant expression vector, expression cassette or recombinant bacteria into soybean to overexpress the aforementioned gene GmRLP15 in soybean plants.
[0032] Advantages:
[0033] The present application first discovers and clones a new plant salt-tolerance related protein gene GmRLP15. The plant salt-tolerance related protein of the present application affects the salt tolerance of plants. The introduction of the coding gene of the protein into plants can improve the salt tolerance of plants, so that salt-tolerant transgenic plants can be cultivated. The protein and its coding gene can be applied to plant genetic improvement. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 The expression amount of GmRLP15 gene in the GmRLP15-Empty vector and GmRLP15-OE soybean combined hairy root plants.
[0035] Figure 2 The phenotype (Figure a) and plant root length comparison (Figure b) of the GmRLP15-Empty vector and GmRLP15-OE soybean combined hairy root plants under salt stress. DETAILED DESCRIPTION
[0036] The following examples facilitate a better understanding of the present application, but do not limit the present application. The experimental methods in the following examples are all conventional methods, unless otherwise specified. The experimental materials used in the following examples are all commercially available from conventional biochemical reagent stores, unless otherwise specified. The soybean variety Williams 82 used for transgenesis is a publicly available variety.
[0037] Example 1, cloning of soybean gene GmRLP15
[0038] The following primers were designed:
[0039] Primer 1: 5'-ATGCCAACCATTAATCCAGTTGG-3';
[0040] Primer 2: 5'-TCATCCATGCACCTTTGATATTTTATTAGC-3'.
[0041] PCR amplification was performed using primer 1 and primer 2 as primers and the root cDNA of Williams 82 seedlings as a template to obtain the target gene GmRLP15.
[0042] The PCR amplification reaction was performed in a Bio-rad T100 PCR instrument, and the reaction system (50 μL) was as follows: 2x Phanta Max Buffer 25 μL, dNTP Mix (10 mM) 1 μL, primer 1 (10 μM) 1.5 μL, primer 2 (10 μM) 1.5 μL, template cDNA (50 ng / uL) 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, ddH2O 18 μL; the program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 2 min, 35 cycles; 72 °C extension for 5 min; 15 °C storage.
[0043] After the PCR product was recovered and purified, it was ligated to pEASY-Blunt (Beijing Zoman Biotech Co., Ltd.), and E. coli DH5α competent cells (Tiangen CB101, Beijing Tiangen) were transformed. After positive clones were selected, sequencing was performed.
[0044] The sequencing results showed that the GmRLP15 gene fragment obtained by PCR reaction had the nucleotide sequence shown in SEQ ID NO. 2, and encoded a protein (SEQ ID NO. 3) consisting of 872 amino acid residues, and the whole genome sequence was shown in SEQ ID NO. 1.
[0045] Example 2, Obtaining and identifying hairy root combined plants overexpressing soybean GmRLP15 gene
[0046] I. Construction of GmRLP15 gene overexpression vector
[0047] The root genomic cDNA of Williams82 seedlings was used as a template, and primer 1 and primer 2 were used for PCR amplification to obtain a full-length CDS sequence fragment of GmRLP15 gene (SEQ ID NO. 2).
[0048] Primer 1: 5'-ATGCCAACCATTAATCCAGTTGG-3' (SEQ ID NO. 4);
[0049] Primer 2: 5'-TCATCCATGCACCTTTGATATTTTATTAGC-3' (SEQ ID NO. 5).
[0050] The amplification product was digested with XbaI and ligated to the pBA002 vector, and then transformed into E. coli DH5α. The positive plasmid was extracted and sequenced. The sequencing results showed that a recombinant expression vector containing the sequence shown in SEQ ID NO. 2 was obtained, which was named pBA002-GmRLP15.
[0051] II. Obtaining of recombinant Agrobacterium
[0052] The pBA002-GmRLP15 was transformed into Agrobacterium K599 strain by heat shock method to obtain a recombinant strain. The plasmid was extracted and identified by PCR and enzyme digestion. The recombinant strain correctly identified was named K5-pBA002-GmRLP15.
[0053] III. Obtaining of transgenic plants
[0054] The K5-pBA002-GmRLP15 strain was transformed into soybean variety Williams82, and the specific method was as follows:
[0055] (1) Select mature seeds with large, full, and no disease spots, and sow them in wet vermiculite. Cover the seed surface with another layer of vermiculite and place it in a 26°C incubation room for 3-4 days. When the soybean cotyledons are about to open but have not yet opened (light green in color, with lateral root growth), hairy root infection can be performed.
[0056] (2) When the seeds germinate for 2-3 days, inoculate the K5-pBA002-GmCNGC300 strain on LB plates containing antibiotics str and spec + at 28°C for 2 days. Pick single bacteria (one day before infection) and inoculate them in 5 mL of LB medium containing antibiotics str and spec +In liquid LB medium, 28℃, 200rpm overnight culture for 12h or so;
[0057] (3) Inoculate the bacteria liquid 1:100 into new LB medium, 28℃, 200rpm shaking culture until OD600=0.6-0.8, collect the bacteria;
[0058] (4) Resuspend the centrifuged bacteria with the prepared and sterilized equal volume of co-culture medium (CCM).
[0059] (5) Select the sprouted seeds without contamination and cotyledon intact without damage, cut off the base with a scalpel, reserve about 2cm hypocotyl, and soak the soybean hypocotyl in the above resuspended bacteria liquid for 1h, 150rpm, 28℃. Then transplant the infected explants in moist vermiculite, seal the film, and culture in a 26℃ culture room.
[0060] (6) After one week of culture, when white callus grows at the wound of soybean hypocotyl, culture it in 1 / 2 Hoagland nutrient solution, and continue to culture for one week, then soybean rootlets grow at the callus, and the rootlets are transgenic soybean plants.
[0061] IV. Identification of transgenic plants
[0062] 1. PCR molecular identification
[0063] Extract the DNA of the root of the soybean rootlet combination plant, use it as a template for PCR amplification, and the PCR primers are as follows:
[0064] Primer 1: 5'-ATGCCAACCATTAATCCAGTTGG-3';
[0065] Primer 2: 5'-TCATCCATGCACCTTTGATATTTTATTAGC-3'.
[0066] The PCR product is detected by 1% agarose gel electrophoresis, and the target band can be detected in positive plants, but not in negative plants.
[0067] 2. Detection of GmRLP15 gene expression
[0068] Take the root system sample of the GmRLP15 soybean rootlet combination plant, freeze the sample in liquid nitrogen, extract RNA by TRIzol method, take appropriate amount of RNA, and use the transcription kit to obtain cDNA as the template for fluorescence quantitative RT-PCR detection. Take appropriate amount of template cDNA, use soybean GmELF as the internal reference gene, and use Hieff TM qPCR Green Master Mix (No RoxPlux) kit, and the expression amount of GmRLP15 gene was detected in Bio-Rad fluorescence quantitative PCR instrument CFX96. The sequences of the GmELF gene quantitative detection primers (primer 3 and primer 4) and the GmRLP15 gene quantitative detection primers (primer 5 and primer 6) used are as follows:
[0069] Primer 3: 5'-GTTGAAAAGCCAGGGGACAC-3';
[0070] Primer 4: 5'-TCTTACCCCTTGAGCGTGG-3'.
[0071] Primer 5: 5'-ATGCCAACCATTAATCCAGT-3';
[0072] Primer 6: 5'-GATCCAGAAGTGCAGCCTTG-3'.
[0073] The relative expression amount was quantitatively calculated by 2 -△△CT The results are shown in Table 1. Figure 1 Figure 1 GmRLP15-OE).
[0074] Example 3, salt tolerance identification of GmRLP15 gene overexpression hairy root combination plants
[0075] The identified GmRLP15-OE hairy root combination plants were further cultured with 1 / 2 Hoagland nutrient solution to the 2-3rd leaf expansion, and the soybean hairy root combination plants transformed with the empty strain K599 were used as controls. Then, the soybean hairy root combination plants with relatively consistent growth were selected and transferred to 1 / 2 Hoagland nutrient solution and 1 / 2 Hoagland nutrient solution containing 120 mM NaCl, respectively, for further culture. After 7-10 days of further growth, the phenotypes of the soybean hairy root combination plants were observed by taking photos, and the related physiological indexes were determined.
[0076] Figure 2 The experimental results show that, compared with the soybean hairy root combination plants transformed with the empty strain, the GmRLP15-OE soybean hairy root combination plants exhibit lighter leaf chlorosis and wilting symptoms under salt stress, and the plant height and root length are significantly higher than those of the control group.
[0077] Therefore, according to these results, it can be determined that the GmRLP15 gene has an important positive regulatory effect on soybean salt tolerance, and overexpression of the protein coding gene can improve the salt tolerance of soybean.
Claims
1. The application of the gene GmRLP15 shown in SEQ ID NO. 1 or SEQ ID NO. 2, the protein shown in SEQ ID NO. 3, a recombinant expression vector containing the gene GmRLP15 shown in SEQ ID NO. 1 or SEQ ID NO. 2, or a recombinant bacterial strain in improving the salt tolerance of soybeans, characterized in that, Overexpression of the gene GmRLP15 shown in SEQ ID NO. 1 or SEQ ID NO. 2 improves the salt tolerance of soybeans.
2. A method for regulating the salt tolerance of soybeans, characterized in that, Overexpression of the gene GmRLP15 shown in SEQ ID NO. 1 or SEQ ID NO. 2 in soybean plants improves the salt tolerance of soybeans.
3. A method for breeding salt-tolerant soybean varieties, characterized in that, The method involves overexpressing the gene GmRLP15 shown in SEQ ID NO. 1 or SEQ ID NO. 2 in soybean plants to obtain salt-tolerant soybean varieties.