Ssnhx1 and ssos1 double gene plant expression vector and its application in improving salt tolerance of alfalfa

Abstract

This invention relates to the field of plant genetic engineering technology, and more particularly to... SsNHX1 and SsSOS1 A dual-gene plant expression vector and its application in improving salt tolerance in alfalfa. The plant expression vector uses pCAMBIA3301 as its backbone and inserts genes derived from Suaeda salsa. SsNHX1 Genes and SsSOS1 It is constructed from genes. SsNHX1 Genes and SsSOS1 The nucleotide sequences of the gene are shown in SEQ ID NO:1 and SEQ ID NO:2. 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 advantages are: the synergistic effect of the two genes 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 SsSOS1 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 highly sensitive to salt stress, and severe soil salinization restricts its yield and the expansion of its planting area. Plant salt tolerance is a complex quantitative trait involving multiple physiological and molecular mechanisms. Among these, the maintenance of intracellular ion homeostasis, especially Na+, is crucial. + Regional isolation and external discharge are key salt tolerance strategies.

[0003] vacuolar membrane Na + / H + Antitransporter protein ( NHX1 By removing excess Na+ from the cytoplasm + Separation into the vacuoles, thereby reducing cytoplasmic Na+. + Concentration, to avoid toxicity. Na+ in the plasma membrane. + / H + Antitransporter protein ( SOS1 Then they are responsible for Na + It is expelled from the cell and participates in long-distance Na+ transport. + Transportation. Studies have shown that overexpression alone... NHX1 or SOS1 Genes can improve a plant's salt tolerance to some extent, but the effect is limited.

[0004] Suaeda salsa is a typical halophyte with extremely strong salt tolerance. SsNHX1 and SsSOS1 Genes are efficiently expressed under salt stress and synergistically regulate Na+. + Intracellular compartmentalization and efflux regulate intracellular Na+ + Homeostasis is a key genetic resource for the high salt tolerance of *Suaeda salsa*. Given its functional complementarity, how to utilize key salt-tolerant genes from halophytes is a crucial issue. SsNHX1 and SsSOS1 Genetic improvement of alfalfa, leveraging synergistic effects between genes to significantly enhance its salt tolerance, and cultivating new alfalfa germplasm that can adapt to high-salt environments, has become an important issue that urgently needs to be addressed in the field of alfalfa salt-tolerant breeding. Summary of the Invention

[0005] To solve the above problems, the present invention provides a... SsNHX1 and SsSOS1 Dual-gene plant expression vectors and their application in improving salt tolerance in alfalfa.

[0006] The primary objective of this invention is to provide SsNHX1 and SsSOS1 A dual-gene plant expression vector, into which a gene is inserted. SsNHX1 Genes and SsSOS1 Obtained through gene construction; The SsNHX1 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO:1; The SsSOS1 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO:2.

[0007] Preferably, the plant expression vector is pCAMBIA3301.

[0008] Preferably, the plant expression vector carries a kanamycin resistance gene as a prokaryotic selection marker.

[0009] The second objective of this invention is to provide SsNHX1 and SsSOS1 Application of dual-gene plant expression vectors in improving salt tolerance in alfalfa.

[0010] Preferably, the salt tolerance is 200~300mM NaCl stress.

[0011] 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 SsSOS1 Genes were stably co-expressed, and positive plants were obtained through screening.

[0012] Preferably, Agrobacterium-mediated genetic transformation is used, and the recipient organ of the alfalfa is the leaf of a sterile seedling.

[0013] The fourth objective of this invention is to provide an alfalfa, wherein the aforementioned SsNHX1 and SsSOS1 The dual-gene plant expression vector was introduced into alfalfa plants and cultured.

[0014] Compared with the prior art, the present invention can achieve the following beneficial effects: (1) Synergistic effect: compared with individual transformation SsNHX1 or SsSOS1 Compared to other genes, co-expression of these two genes can more effectively maintain ion homeostasis in transgenic alfalfa cells; SsSOS1 Mediated Na + External discharge and long-distance transportation SsNHX1 Mediated vacuolar Na + This compartmentalization, combined with other features, forms a dual Na+ network extending from the cytoplasm to the extracellular space and from the cytoplasm to the vacuoles. +Clearing the pathways significantly enhanced the plant's salt tolerance.

[0015] (2) Significantly improved salt tolerance: The transgenic alfalfa plants obtained under high salt stress (such as 200~300 mM NaCl) showed a more vigorous growth trend than non-transgenic wild-type plants and single-gene transgenic plants.

[0016] (3) Stable agronomic traits: Under normal conditions without salt stress, the main agronomic traits (growth rate, plant height, number of branches) of transgenic plants were not significantly different from those of wild type, indicating that the co-expression of exogenous genes did not have an adverse effect on the normal growth of plants.

[0017] (4) Broad application prospects: This invention provides efficient gene resources and technical approaches for cultivating new salt-tolerant alfalfa varieties, which is of great significance for developing and utilizing large areas of saline land, ensuring safe production of forage grass and improving the ecological environment. Attached Figure Description

[0018] Figure 1 It is provided according to the embodiments of the present invention. SsNHX1 and SsSOS1 A schematic diagram of the T-DNA region structure of the plant binary expression vector pCAMBIA3301-NHX1-T2A-SOS1.

[0019] 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.

[0020] Figure 3 The genetically modified alfalfa provided according to embodiments of the present invention SsSOS1 Electrophoresis diagram of gene PCR molecular identification; M: DNA molecular weight standard; +: positive control; -: negative control; 1-8: transgenic lines.

[0021] Figure 4 Under salt stress treatment (250 mM NaCl, 21 days) provided in the embodiments of the present invention, wild-type (WT) and co-expressed SsNHX1 / SsSOS1 Phenotypic comparison of two-gene alfalfa plants (T). Detailed Implementation

[0022] 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.

[0023] The purpose of this invention is to provide a method for co-expressing Suaeda salsa-derived... SsNHX1 and SsSOS1 A method to significantly improve the salt tolerance of alfalfa using genes; vacuolar membrane Na derived from Suaeda salsa + / H + reverse transporter gene SsNHX1 and plasma membrane Na + / H + reverse transporter gene SsSOS1 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.

[0024] 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.

[0025] Example 1 The specific methods for constructing plant expression vectors are as follows: cDNA was cloned from salt-stressed Suaeda salsa cDNA using RT-PCR. SsNHX1 Genes (as shown in sequence listing SEQ ID NO:1) and SsSOS1 The complete coding sequence of the gene (as shown in the sequence listing SEQ ID NO:2).

[0026] 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 SsSOS1 The complete coding region of a gene (including the stop codon) is fused into a single open reading frame, resulting in... SsNHX1-T2A- SsSOS1 Fragment fusion.

[0027] 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-SsSOS1.

[0028] In this carrier, SsNHX1 and SsSOS1 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.

[0029] 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.

[0030] The recombinant plasmid pCAMBIA3301-SsNHX1-T2A-SsSOS1 was finally obtained.

[0031] Example 2 The genetic transformation of alfalfa and the acquisition of transgenic plants are detailed below: Aseptic leaves of alfalfa cultivar 'Gongnong 5' were used as explants. The recombinant plasmid pCAMBIA3301-SsNHX1-T2A-SsSOS1 was introduced into the explants using Agrobacterium tumefaciens EHA105-mediated transformation. After infection, the explants were blotted dry with sterile filter paper to remove excess bacterial solution, and then inoculated onto a co-culture medium with the leaf underside down. The medium was incubated at 22℃ in a dark incubator for 3 days, maintaining a humidity of over 85% to prevent light and temperature fluctuations that could decrease Agrobacterium activity or cause browning of the explants. Following co-culture, callus induction, proliferation, and differentiation were performed, and resistant shoots were screened. The specific procedures were as follows: the explants were transferred to a sterile induction medium and incubated at 25℃ with a 16h / 8h light / dark cycle and a light intensity of 300 μmol·m⁻¹. -2 ·s -1 Cultured under the specified conditions for 10-14 days, pale yellow, loose callus tissue is induced. Vigorously growing callus tissue is selected and transferred to a proliferation medium, subcultured every 2 weeks for 3-4 weeks to obtain dense, light green resistant callus tissue (non-resistant callus tissue gradually yellows and dies). The proliferated resistant callus tissue is cut into 0.3-0.5 cm pieces and transferred to a shoot differentiation medium, and cultured under the above light and temperature conditions for another 2-3 weeks to induce the differentiation of green resistant shoots. The resistant shoots are then transferred to a rooting medium to induce rooting, resulting in complete regenerated plants.

[0032] Extract genomic DNA from regenerated plants and use targeted... SsNHX1 and SsSOS1 Gene-specific primers were used for PCR amplification. SsNHX1-F: 5'-ATGTTGTCACAGTTGAGCTCTT-3' SsNHX1-R: 5'-CTATGTTCTCTCTGTCATAT-3' SsSOS1-F: 5'-ATGGCAGCATCTCGAATTGA-3' SsSOS1-F: 5'-TCAAGGTGCTTGGCGGAAAGA-3'; The PCR reaction procedure is shown in Table 1 below: Table 1 PCR reaction procedure

[0033] Preliminary identification of positive transgenic plants, results are shown below. Figures 2-3 . Figure 2 and Figure 3 respectively SsNHX1 and SsSOS1 Gene-specific primers were used for PCR amplification, and positive transgenic plants could amplify genes that were similar to those in the target genome. SsNHX1 and SsSOS1 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 SsSOS1 Positive strains with specific bands.

[0034] Example 3 The salt tolerance identification experiment for transgenic alfalfa is as follows: Transgenic plants of the T1 generation co-expressing SsNHX1 / SsSOS1 gene, SsNHX1-transgenic plants, SsSOS1-transgenic plants, and wild-type (WT) alfalfa tissue culture seedlings that were identified as positive by PCR were transplanted into plastic pots containing equal amounts of nutrient soil (peat soil: vermiculite = 2:1, v / v) and kept in a greenhouse at 25℃ / 20℃ (day / night), a 16h / 8h light / dark cycle, and a light intensity of 300 μmol·m⁻¹. -2 ·s -1 Under suitable conditions, culture until the 4-5 leaf stage.

[0035] Salt stress treatment: Irrigate with 250mM NaCl solution every 3 days, with each irrigation amount being 80% of the pot's water holding capacity, for 21 days; at the same time, a control group was set up to be irrigated with an equal amount of clean water. Each treatment group was set up with 3 biological replicates, and each replicate contained 6 plants with the same growth.

[0036] Phenotypic observation and physiological index measurement: like Figure 3 As shown, after 21 days of salt stress, wild-type plants (WT) showed severe wilting, yellowing of leaves, and even death of the entire plant, while plants (T) that co-expressed the two genes grew normally, with leaves remaining bright green and only a few old leaves at the bottom showing slight yellowing. The overall plant height and biomass were significantly better than those of single-gene plants and wild-type plants, showing extremely strong salt tolerance.

[0037] Functional leaves of the treated plants were taken and their relative water content, chlorophyll content, and Na+ content were measured.+ / K + Parameters such as ratios showed that the relative water content and chlorophyll content of plants co-expressing the two genes were increased compared to the wild type, and Na... + / K + The ratio was significantly lower than that of wild-type and single-gene plants, further verifying the synergistic regulatory effect of dual-gene co-expression on ion homeostasis.

[0038] 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.

[0039] 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 SsSOS1 A dual-gene plant expression vector, characterized by: Inserted into plant expression vectors SsNHX1 Genes and SsSOS1 Obtained through gene construction; The SsNHX1 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO:1; The SsSOS1 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO:

2.

2. As described in claim 1 SsNHX1 and SsSOS1 A dual-gene plant expression vector, characterized by: The plant expression vector is pCAMBIA3301.

3. As described in claim 2 SsNHX1 and SsSOS1 A dual-gene plant expression vector, characterized by: The plant expression vector carries a kanamycin resistance gene as a prokaryotic selection marker.

4. The claim 1 SsNHX1 and SsSOS1 Application of dual-gene plant expression vectors in improving salt tolerance in alfalfa.

5. The method according to claim 4 SsNHX1 and SsSOS1 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.

6. 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 1-3 into alfalfa plants, so that... SsNHX1 Genes and SsSOS1 Genes were stably co-expressed, and positive plants were obtained through screening.

7. A method for improving the salt tolerance of alfalfa according to claim 6, characterized in that: The alfalfa was transformed using Agrobacterium-mediated genetic transformation, with the recipient organ being the leaf of a sterile seedling.

8. A type of alfalfa, characterized in that: The claim 1 SsNHX1 and SsSOS1 The dual-gene plant expression vector was introduced into alfalfa plants and cultured.

Images