SsNHX1 and SsSOS2 dual-gene plant expression vectors and their application in improving salt tolerance in alfalfa

Abstract

This invention relates to the field of plant genetic engineering technology, and more particularly to... SsNHX1 and SsSOS2 Dual-gene plant expression vector and its application in improving salt tolerance in alfalfa. The plant expression vector uses pCAMBIA1300 as its backbone and inserts genes derived from Suaeda salsa. SsNHX1 Genes and SsSOS2 It is constructed from genes. SsNHX1 Genes and SsSOS2 The CDS coding sequence of the gene is shown in SEQ ID NO:1 and SEQ ID NO:2 of the sequence listing. Using Agrobacterium tumefaciens EHA105-mediated transformation, the vector was introduced into the leaves of sterile alfalfa seedlings. Positive transgenic plants were obtained through co-culture, callus induction differentiation, and PCR identification. The advantage lies in the synergistic effect of the two genes, which effectively enhances the salt tolerance of alfalfa, enabling it to grow normally under 200–300 mM NaCl stress, providing a new solution for forage cultivation in saline soils.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more particularly to... SsNHX1 and SsSOS2 Dual-gene plant expression vectors and their application in improving salt tolerance in alfalfa. Background Technology

[0002] Alfalfa is one of the world's most important legume forage crops, but it is quite sensitive to salt stress, and severe soil salinization restricts its yield and the expansion of its planting area. my country has a large area of ​​saline-alkali land resources, and cultivating new alfalfa varieties with strong salt tolerance is of great significance for developing and utilizing marginal land and ensuring the security of forage supply.

[0003] Plant salt tolerance mechanisms involve a complex signal regulatory network, among which the SOS signaling pathway is one of the core regulatory mechanisms in plant responses to salt stress. When the external salt concentration is high, the calcium signal induced by salt stress is sensed by the calcium-binding protein SOS3. SOS3 binds to and activates the protein kinase SOS2; the activated SOS2 further phosphorylates Na+ in the plasma membrane. + / H + The reverse transporter SOS1 enhances its activity, expels sodium ions from cells, and thus confers salt tolerance in plants. Studies have shown that SOS2 plays a central regulatory role in this pathway, not only regulating the activity of SOS1 but also participating in the regulation of other ion transport systems and the expression of downstream salt-tolerant response genes.

[0004] Except Na + In addition to efflux mechanisms, excess Na+ in the cytoplasm is eliminated. + Separation into vacuoles is another important strategy for plant salt tolerance. (Valvular membrane Na...) + / H + The antitransporter NHX utilizes the vacuolar membrane H + -ATPase and H + - The proton-driven force produced by pyrophosphatase will transfer Na+ + Transporting it to the vacuoles for storage reduces cytoplasmic sodium levels. + Concentration can also be determined by Na + It acts as an osmotic regulator to maintain cell turgor pressure.

[0005] Suaeda salsa ( Suaeda salsa Suaeda salsa is a typical halophyte, capable of growing normally in high-salt environments. Previous studies have shown that clones from Suaeda salsa... SsNHX1 Gene expression in yeast and tobacco significantly improved the salt tolerance of transformants, and overexpression in alfalfa... SsNHX1This allows transgenic plants to tolerate up to 400 mM NaCl, while the control group gradually dies under 200 mM conditions. However, while introducing a single salt-tolerance gene can improve the salt tolerance of transgenic plants, the effect is often limited and cannot meet the actual needs of production in severely saline-alkali land.

[0006] Currently, no evidence has been found of using Na+ in vacuolar membranes. + / H + Na+ transport protein-mediated reverse transport + This research focuses on the synergistic integration of the segmentation mechanism and the salt stress signaling regulation mechanism mediated by the core kinase of the SOS signaling pathway to systematically improve the salt tolerance of alfalfa. Current salt tolerance genetic improvement technologies still suffer from problems such as single salt tolerance pathways, insufficient regulatory efficiency, and limited improvement in salt tolerance. There is an urgent need to develop more efficient and systematic molecular strategies for improving salt tolerance in alfalfa. Summary of the Invention

[0007] To solve the above problems, the present invention provides a... SsNHX1 and SsSOS2 Dual-gene plant expression vectors and their application in improving salt tolerance in alfalfa.

[0008] The primary objective of this invention is to provide SsNHX1 and SsSOS2 A dual-gene plant expression vector, into which a gene is inserted. SsNHX1 Genes and SsSOS2 Obtained through gene construction; The SsNHX1 The CDS coding sequence of the gene is shown in the sequence listing SEQ ID NO:1; The SsSOS2 The CDS coding sequence of the gene is shown in the sequence listing SEQ ID NO:2.

[0009] Preferably, the plant expression vector is pCAMBIA3301.

[0010] Preferably, the plant expression vector contains a selective marker gene.

[0011] Preferably, the selective marker gene is the hygromycin resistance gene. HptII Glufosinate resistance gene Bar or kanamycin resistance gene NptII .

[0012] Preferably, it includes at least two independent expression boxes, each driving... SsNHX1 Genes and SsSOS2 Gene transcription; or polycistronic molecules containing a 2A peptide sequence, to achieve SsNHX1 Genes and SsSOS2 Coordinated expression of genes.

[0013] The second objective of this invention is to provide SsNHX1 and SsSOS2 Application of dual-gene plant expression vectors in improving salt tolerance in alfalfa.

[0014] Preferably, the salt tolerance is 200~300mM NaCl stress.

[0015] The third objective of this invention is to provide a method for improving the salt tolerance of alfalfa, wherein the method involves introducing the aforementioned dual-gene plant expression vector into alfalfa plants, thereby enabling... SsNHX1 Genes and SsSOS2 Genes were stably co-expressed, and positive plants were obtained through screening.

[0016] Preferably, Agrobacterium-mediated genetic transformation is used, and the recipient organ of the alfalfa is the leaf of a sterile seedling.

[0017] The fourth objective of this invention is to provide an alfalfa, wherein the aforementioned SsNHX1 and SsSOS2 The dual-gene plant expression vector was introduced into alfalfa plants and cultured.

[0018] Compared with the prior art, the present invention can achieve the following beneficial effects: (1) Put Na + Vacuole partitioning mechanism ( SsNHX1 ) and the core regulatory mechanism of the SOS signaling pathway ( SsSOS2 By combining these methods, a two-level salt tolerance system of "segregation-signal regulation" was constructed in alfalfa. SsNHX1 Responsible for transporting Na+ into the cell + Efficient transport to vacuolar storage, utilizing Na + It acts as an osmotic regulator to maintain cell turgor pressure; SsSOS2 As a protein kinase, it activates endogenous SOS1-mediated Na+. + It can be effluxed and may also regulate other downstream salt-tolerant target proteins through phosphorylation, synergistically enhancing the overall salt tolerance of plants; the two work together to produce a significant synergistic effect.

[0019] (2) Significantly optimized ion steady state: co-expression SsNHX1 and SsSOS2 Transgenic alfalfa can more effectively maintain lower Na levels in the cytoplasm under salt stress. + Concentration and higher K + Concentration, Na + / K + The ratio was significantly lower than that of wild-type and single-gene transformed lines. Studies have shown that SOS2 not only activates SOS1 to promote Na+ absorption, but also... + External discharge, and also participate in the regulation of K +Absorption channels (such as AKT1) work together to maintain Na + / K + Steady state.

[0020] (3) Significantly increased salt tolerance threshold: through co-expression SsSOS2 Further enhancing the regulatory capacity of the SOS signaling pathway, the salt tolerance threshold of transgenic alfalfa is further increased, maintaining better growth performance under severely saline-alkali soil planting conditions.

[0021] (4) Excellent agronomic traits: Under normal growth conditions, it expresses a total of SsNHX1 and SsSOS2 The genetically modified alfalfa showed no significant differences from the wild type in terms of growth vigor and morphological characteristics, and exhibited no adverse agronomic traits, demonstrating good potential for production and application.

[0022] (5) Outstanding application value: The salt-tolerant alfalfa material cultivated by this invention can be directly used for planting in mild to moderate saline-alkali land, or used as a breeding parent for conventional hybridization breeding. It is of great significance for expanding the planting area of ​​alfalfa, improving the productivity of saline-alkali land and improving the ecological environment. Attached Figure Description

[0023] Figure 1 It is provided according to the embodiments of the present invention. SsNHX1 and SsSOS2 Plant binary expression vectors for genes pCAMBIA3301 - SsNHX1 - SsSOS2 A schematic diagram of the T-DNA region structure.

[0024] Figure 2 The genetically modified alfalfa provided according to embodiments of the present invention SsNHX1 Electrophoresis diagram of gene PCR molecular identification; M: DNA molecular weight standard; +: positive control; -: negative control; 1-8: transgenic lines.

[0025] Figure 3 The genetically modified alfalfa provided according to embodiments of the present invention SsSOS2 Electrophoresis diagram of gene PCR molecular identification; M: DNA molecular weight standard; +: positive control; -: negative control; 1-8: transgenic lines.

[0026] Figure 4 Under salt stress treatment (300 mM NaCl, 21 days) provided in the embodiments of the present invention, wild-type (WT) and co-expressed SsNHX1 / SsSOS2 Phenotypic comparison of two-gene alfalfa plants (T). Detailed Implementation

[0027] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0028] The purpose of this invention is to provide a method for co-expressing Suaeda salsa-derived... SsNHX1 and SsSOS2 Genes, methods to significantly improve the salt tolerance of alfalfa; vacuolar membrane Na+ derived from Suaeda salsa + / H + reverse transporter gene SsNHX1 and plasma membrane Na + / H + reverse transporter gene SsSOS2 The two genes were co-introduced into the alfalfa genome, enabling stable co-expression of the two genes in alfalfa. The co-introduction of these two functionally complementary genes into alfalfa produces a significant synergistic effect, far exceeding the effect of expressing either gene alone, thereby breeding a new salt-tolerant alfalfa variety that can still grow normally in high-salt environments.

[0029] SsNHX1 The CDS coding sequence of the gene is shown in the sequence listing SEQ ID NO:1; SsSOS2 The CDS coding sequence of the gene is shown in the sequence listing SEQ ID NO:2.

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0031] Example 1 The methods for gene cloning and construction of plant expression vectors are as follows: Plant materials and stress treatment: Select vigorous Suaeda salsa plants, treat them with 200 mM NaCl for 24 hours, collect leaf and root tissues, flash freeze them with liquid nitrogen and store them at -80℃ for later use. Total RNA extraction and cDNA synthesis: Total RNA was extracted from Suaeda salsa leaves and root tissues using the Trizol method and analyzed by agarose gel electrophoresis; the first strand of cDNA was synthesized using a reverse transcription kit with total RNA as a template and stored at -20℃ for later use. Gene cloning: Based on reported Suaeda salsa... SsNHX1 Gene sequence and SsSOS2 Gene sequence-specific primers: SsNHX1-F: 5'-ATGTTGTCACAGTTGAGCTCTT-3' SsNHX1-R: 5'-CTATGTTCTCTCTGTCATAT-3' SsSOS2-F: 5'-ATGGCCTTGTTTAACCTTACAATG-3' SsSOS2-R: 5'-TTATCTGCCATCCACATTAGTGAA-3'; Using Suaeda salsa cDNA as a template, PCR amplification was performed. The PCR reaction conditions were as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 sec, 58℃ annealing for 5 sec, 72℃ extension for 20 sec, 31 cycles; 72℃ extension for 10 min. After the PCR products were recovered by agarose gel electrophoresis, they were ligated into the pMD18-T vector and transformed into Escherichia coli DH5α. The target plasmids T-SsNHX1 and T-SsSOS2 were obtained by colony PCR and sequencing verification.

[0032] The specific method for constructing the expression vector is as follows: Using overlap extension PCR technology, SsNHX1 The coding region of the gene (with the stop codon removed), the coding sequence of the T2A peptide, and SsSOS2 The complete coding region of a gene (including the stop codon) is fused into a single open reading frame, resulting in... SsNHX1 - T2A - SsSOS2 Fragment fusion.

[0033] The fusion fragment was cloned into a single multiple cloning site of the pCAMBIA3301 vector and placed between the CaMV35S promoter and the NOS terminator to construct the polycistronic expression vector pCAMBIA3301-SsNHX1-T2A-SsSOS2.

[0034] In this carrier, SsNHX1 and SsSOS2 Transcription into a single mRNA is driven by the same promoter, and the two proteins are expressed independently during translation via ribosome jumping mediated by the T2A peptide.

[0035] After the ligation product was transformed into E. coli DH5α competent cells, resistance selection was performed on LB solid medium containing kanamycin. Plasmids were extracted from resistant single colonies and subjected to enzyme digestion to verify the correct size and orientation of the inserted fragment. DNA sequencing confirmed that the inserted sequence was consistent with SEQ ID NO:1 and SEQ ID NO:2, without mutations, deletions, or frameshifts.

[0036] The recombinant plasmid pCAMBIA3301-SsNHX1-T2A-SsSOS2 was finally obtained.

[0037] Example 2 The genetic transformation of alfalfa is detailed below: Explant preparation: Select plump "Gongnong No. 5" alfalfa seeds, disinfect them with 75% ethanol for 30 seconds, disinfect them with 0.1% mercuric chloride for 8 minutes, rinse them with sterile water 5 times, and then inoculate them on MS basic medium for germination. Take the cotyledons of sterile seedlings that have grown for 7-10 days as transformation explants. Preparation of Agrobacterium infection solution: A single colony of Agrobacterium EHA105 containing recombinant plasmid was inoculated into YEB liquid medium containing the corresponding antibiotic and cultured at 28°C with shaking until the OD600 was approximately 0.6-0.8; the bacterial suspension was centrifuged at 4°C and 5000 rpm for 10 minutes to collect the bacterial cells, resuspended in MS liquid medium (containing 100 μM acetylsyl syringone), and the OD600 was adjusted to 0.5 for later use; Infection and co-culture: Immerse the explants in Agrobacterium bacterial suspension for 15-20 minutes, gently shaking during the process; remove the explants, blot off excess bacterial suspension with sterile filter paper, inoculate onto co-culture medium, and incubate in the dark at 25°C for 3 days; Screening and differentiation: After co-culture, the explants were transferred to the screening induction medium for callus induction, and subcultured every 2 weeks; the well-grown and dense resistant callus was transferred to the differentiation medium to induce adventitious shoot differentiation, and subcultured every 3 weeks. Rooting and transplanting: When the resistant buds grow to 3-4cm, cut them off and transfer them to a rooting medium to induce rooting; after the root system has developed well, open the culture bottle cap and harden the seedlings for 3-5 days. Carefully take out the seedlings, wash the culture medium off the roots, and transplant them into nutrient soil for greenhouse cultivation, eventually obtaining alfalfa plants.

[0038] Example 3 DNA extraction and PCR detection: Approximately 100 mg of tender leaves from successfully transplanted regenerated plants were collected to extract genomic DNA. Using the genomic DNA as a template, PCR amplification was performed using specific primers for SsNHX1 and SsSOS2, respectively. Positive transgenic plants showed amplification of DNA similar to that of SsNHX1 and SsSOS2. SsNHX1 and SsSOS2 The specific bands in the wild-type plants (negative control) were of the same expected size, while no specific bands were observed in the wild-type plants. This indicates that lanes 1-8 were simultaneously amplified. SsNHX1 and SsSOS2 Positive strains with specific bands.

[0039] RNA extraction and expression analysis: Total RNA was extracted from PCR-positive lines, reverse transcribed into cDNA, and then analyzed by qRT-PCR to detect its expression. SsNHX1 and SsSOS2 Gene transcription level. Two lines with high expression levels were selected for subsequent functional analysis.

[0040] Southern hybridization verification: 20 μg of genomic DNA was extracted from the positive lines and analyzed using restriction endonucleases. Eco After complete digestion with RI and separation by 0.8% agarose gel electrophoresis, the samples were transferred to a nylon membrane and hybridized with digoxigenin-labeled SsNHX1 and SsSOS2 probes to confirm the integration of the foreign gene and its copy number.

[0041] Example 4 The salt tolerance assessment of genetically modified organisms is as follows: Co-expression lines with consistent growth and non-transgenic wild-type (WT) alfalfa were selected for propagation by cuttings to obtain experimental materials with consistent genetic backgrounds. Seedlings of each line were transplanted into pots containing a 1:1 mixture of vermiculite and nutrient soil, and after 4 weeks of routine management, they were subjected to salt treatment.

[0042] Salt stress treatment: The treatment group was irrigated with 1 / 2 Hoagland nutrient solution containing 300 mM NaCl, while the control group was irrigated with normal nutrient solution. Irrigation was carried out every 3 days to ensure a relatively stable salt concentration in the substrate. Various indicators were measured after 21 days of treatment. Each line was replicated 3 times, with 5 plants in each replicate.

[0043] Phenotypic observation: such as Figure 3 As shown, after 21 days of salt stress treatment, wild-type plants showed severe wilting and even death, while plants co-expressing the two genes grew normally, with leaves remaining green and only the lower old leaves showing slight yellowing, demonstrating extremely strong salt tolerance.

[0044] This invention uses Suaeda salsa. SsNHX1 Genes and SsSOS2 Gene co-expression in alfalfa successfully created a new transgenic alfalfa germplasm with significantly enhanced salt tolerance. This strategy utilizes Na+... + The vacuolar compartmentalization mechanism combines with the core regulatory mechanism of the SOS signaling pathway to synergistically enhance Na+ + vacuole partitioning and SOS signaling pathway-mediated Na + Efflux regulation optimizes ion homeostasis and physiological metabolism, with significantly better results than single gene transformation. It provides a highly efficient new approach for the genetic improvement of salt tolerance in alfalfa and has important application value for the development and utilization of saline-alkali land and the development of the forage industry.

[0045] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0046] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. SsNHX1 and SsSOS2 A dual-gene plant expression vector, characterized by: Inserted into plant expression vectors SsNHX1 Genes and SsSOS2 Obtained through gene construction; The SsNHX1 The CDS coding sequence of the gene is shown in the sequence listing SEQ ID NO:1; The SsSOS2 The CDS coding sequence of the gene is shown in the sequence listing SEQ ID NO:

2.

2. As described in claim 1 SsNHX1 and SsSOS2 A dual-gene plant expression vector, characterized by: The plant expression vector is pCAMBIA3301.

3. As described in claim 2 SsNHX1 and SsSOS2 A dual-gene plant expression vector, characterized by: The plant expression vector contains a selective marker gene.

4. The method according to claim 3 SsNHX1 and SsSOS2 A dual-gene plant expression vector, characterized by: The selective marker gene is a hygromycin resistance gene. HptII Glufosinate resistance gene Bar or kanamycin resistance gene NptII .

5. The method according to claim 1 SsNHX1 and SsSOS2 A dual-gene plant expression vector, characterized by: It contains at least two independent expression boxes, each driving its own expression box. SsNHX1 Genes and SsSOS2 Gene transcription; or polycistronic molecules containing a 2A peptide sequence, to achieve SsNHX1 Genes and SsSOS2 Coordinated expression of genes.

6. The claim 1 SsNHX1 and SsSOS2 Application of dual-gene plant expression vectors in improving salt tolerance in alfalfa.

7. The method according to claim 6 SsNHX1 and SsSOS2 The application of dual-gene plant expression vectors in improving salt tolerance in alfalfa is characterized by: The salt tolerance is described as being able to withstand 200~300mM NaCl stress.

8. A method for improving the salt tolerance of alfalfa, characterized in that: The method involves introducing the dual-gene plant expression vector according to any one of claims 1-5 into alfalfa plants, so that... SsNHX1 Genes and SsSOS2 Genes were stably co-expressed, and positive plants were obtained through screening.

9. A method for improving the salt tolerance of alfalfa according to claim 8, characterized in that: The alfalfa was transformed using Agrobacterium-mediated genetic transformation, with the recipient organ being the leaf of a sterile seedling.

10. A type of alfalfa, characterized in that: The claim 1 SsNHX1 and SsSOS2 The dual-gene plant expression vector was introduced into alfalfa plants and cultured.

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