Application of SaHKT1;8 gene in plant adaptation to salt stress

Abstract

This invention belongs to the field of SaHKT1;8 gene application technology, and particularly relates to the application of SaHKT1;8 gene in plant adaptation to salt stress, specifically the application of the Spartina alterniflora SaHKT1;8 gene in improving plant salt tolerance. The plant is rice, wheat, corn, cotton, or other crops that require salt tolerance improvement. The nucleotide sequence of the coding region of the Spartina alterniflora SaHKT1;8 gene is shown in SEQ ID NO:6.

Description

Technical Field

[0001] This invention belongs to the field of SaHKT1;8 gene application technology, and particularly relates to the application of SaHKT1;8 gene in plant adaptation to salt stress. Background Technology

[0002] Soil salinization is one of the most serious challenges facing sustainable agricultural development globally. With accelerated industrialization and inadequate irrigation, the area of ​​salinized soil continues to expand, leading to inhibited crop growth and significantly reduced yields, severely restricting the improvement of agricultural production efficiency. Traditional methods for improving crop salt tolerance mainly rely on hybridization breeding, but these methods suffer from problems such as long cycles, complex genetic backgrounds, and limited effectiveness in improving salt tolerance traits, making it difficult to meet the urgent needs of modern agriculture for salt-tolerant crop varieties. The development of molecular biotechnology has provided a new pathway for crop salt tolerance breeding, among which the HKT gene family plays a crucial role in regulating plant salinity. Key genes for balance play a central role in plant adaptation to salt stress and have become important targets for salt tolerance genetic engineering improvement.

[0003] Spartina alterniflora, a typical halophyte, has long thrived in the high-salt intertidal environment and has evolved a strong salt stress adaptation mechanism. Its genome contains abundant salt-tolerant functional genes. Currently, research on the Spartina alterniflora HKT gene family is still in its early stages, especially regarding the specific salt-tolerant function, expression regulation characteristics, and application value of the SaHKT1;8 gene in crop genetic improvement, which have not yet been systematically explored and verified. The limitations of existing salt-tolerant gene resources make the screening and utilization of novel, highly efficient salt-tolerant genes from halophytes a key direction for overcoming the bottlenecks in current crop salt tolerance improvement and enriching the salt-tolerant gene pool. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned technical problems by providing the application of the SaHKT1;8 gene in plant adaptation to salt stress.

[0005] In view of this, the present invention provides the application of the SaHKT1;8 gene in plant adaptation to salt stress, specifically the application of the Spartina alterniflora SaHKT1;8 gene in improving plant salt tolerance.

[0006] The plants mentioned are rice, wheat, corn, cotton, or other crops that require salt tolerance improvement.

[0007] The coding region nucleotide sequence of the Spartina alterniflora SaHKT1;8 gene is shown in SEQ ID NO:6.

[0008] The beneficial effects of this invention are: From the perspective of salt tolerance, the SaHKT1;8 gene in Spartina alterniflora exhibits a sensitive response to salt stress, with its expression significantly upregulated under treatment with 100mM and 400mM NaCl. Applying this gene to crop genetic improvement can effectively enhance plant salt tolerance. Taking rice as an example, transgenic plants subjected to 100mM–150mM NaCl stress for 5 days during the seedling stage, followed by a 5-day recovery period in a salt-free nutrient solution, showed significantly higher survival rates than wild-type plants. Furthermore, leaf yellowing and wilting were significantly reduced, even surpassing the OsHKT2;4 overexpression positive control line, providing crucial gene support for the normal growth of crops in saline-alkali soils.

[0009] Meanwhile, this gene also possesses additional technical value in improving crop grain traits. Under conventional field conditions, transgenic rice lines stably expressing SaHKT1;8 generally exhibit increased grain length and a more elongated morphology in the main panicle at maturity. This overcomes the limitations of traditional salt-tolerant genes, which only focus on stress resistance and do not significantly improve yield-related traits. While enhancing crop salt tolerance, it provides a new pathway for subsequent optimization of crop yield traits through genetic engineering, achieving a synergistic improvement in salt tolerance and yield. Attached Figure Description

[0010] Figure 1 This is a graph showing the relative expression levels of the SaHKT1;8 gene under different NaCl concentrations.

[0011] Figure 2 The construction map of the plant expression vector HRG-UBI-GFP::SaHKT1;8.

[0012] Figure 3 This is a diagram showing the positive PCR test results for genetically modified rice.

[0013] Figure 4 This is a phenotypic comparison and survival rate statistics of transgenic lines and controls under salt stress.

[0014] Figure 5 This is a comparison of the grain morphology of the transgenic line and Zhonghua 11 under field conditions.

[0015] Figure 6 This is a schematic diagram illustrating the survival rate after salt stress. Detailed Implementation

[0016] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0017] It should be noted that all directional and positional terms used in this invention, such as "up," "down," "left," "right," "front," "back," "vertical," "horizontal," "inner," "outer," "top," "lower," "lateral," "longitudinal," and "center," are only used to explain the relative positional relationships and connections between components in a specific state (as shown in the accompanying drawings). They are merely for the convenience of describing the invention and do not require the invention to be constructed and operated in a specific orientation; therefore, they should not be construed as limitations on the invention. Furthermore, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0018] In the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0019] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0020] The main reagents and materials used in this embodiment include: SteadyPure Universal Genomic DNA Extraction Kit, SteadyPure Plant RNA Extraction Kit, SYBR Green Pro Taq HS Premixed qPCR Kit II, and Evo M-MLV Reverse Transcription Premixed Kit (purchased from Aike Rui Biotechnology Co., Ltd.); Universal DNA Purification and Recovery Kit and Plasmid Mini-Extraction Kit (purchased from Tiangen Biotech Co., Ltd.); 2×Phanta Max Master Mix (Dye Plus) High-Fidelity DNA Polymerase Mixture (purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.); Ampicillin, kanamycin, Escherichia coli competent cells, Agrobacterium tumefaciens competent cells, NBT staining solution, and DAB staining solution (purchased from Beijing Coollab Technology Co., Ltd.); Anhydrous ethanol, chloroform, sodium chloride, glacial acetic acid, EDTA, and other analytical grade reagents (purchased from Aladdin Reagent Co., Ltd.); and plant nutrient solution (purchased from Beijing Xiangshan Co., Ltd.).

[0021] The materials used in the experiment included: rice inbred line Zhonghua 11; Escherichia coli strain DH5α; Agrobacterium tumefaciens strain GV3101; and the plant expression vector was the SaHKT1;8 gene, which was modified based on the pCAMBIA1301 backbone and contained the maize Ubiquitin promoter-driven expression, with GFP marker and hygromycin resistance selection marker, abbreviated as HRG-UBI-GFP.

[0022] Example 1: Expression analysis of SaHKT1;8 response to salt stress in Spartina alterniflora; Healthy Spartina alterniflora plants with uniform growth were selected and treated for 48 hours in nutrient solutions containing different concentrations of NaCl (0 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM). After treatment, leaf samples were collected, flash-frozen in liquid nitrogen, and used for total RNA extraction.

[0023] RNA extraction was performed using the SteadyPure Plant RNA Extraction Kit, and reverse transcription was performed using the Evo M-MLV premixed kit. Using the SaHKT1;8 gene as the target gene and the Actin gene as an internal control, real-time quantitative PCR analysis was performed using the SYBR Green Pro Taq HS premixed qPCR kit II. The primer sequences used for real-time quantitative PCR are as follows: SEQ ID NO: 1: 5'-ACCTTCTTCTTCGGTGCTTTG-3'; SEQ ID NO: 2: 5'-TGATCCATCGTAGAGTTTGCAT-3'; Experimental results showed that the expression of the SaHKT1;8 gene was induced under different salt concentrations, especially significantly upregulated under 100mM and 400mM NaCl conditions, reflecting its sensitive response to salt stress.

[0024] This expression characteristic indicates that SaHKT1;8 can serve as a key functional element for regulating plant salt tolerance, and is applicable to the molecular design and genetic improvement of salt-tolerant crops (see...). Figure 1 ).

[0025] Example 2: Cloning and expression vector construction of the SaHKT1;8 gene; This embodiment provides a method for cloning the full-length coding sequence of the high-affinity potassium ion transporter gene SaHKT1;8 from the halophyte Spartina alterniflora and constructing a plant expression vector, providing technical support for subsequent functional studies and genetic improvement of crop salt tolerance.

[0026] Fresh leaves of Spartina alterniflora were collected, and total RNA was extracted using the SteadyPure Plant RNA Extraction Kit. First-strand cDNA was synthesized using the EvoM-MLV Reverse Transcription Premix Kit.

[0027] Specific primer pairs were designed based on the coding region of the SaHKT1;8 gene for amplification. The primer sequences used are as follows: SEQ ID NO: 3: 5'-ATGCATCCAGCCAGTTCAATTC-3'; SEQ ID NO: 4: 5'-TCATGGAAGTGGTAGTTGTGTA-3'.

[0028] The total volume of the PCR reaction system was 50 μL, including: 25 μL of 2× Phanta Max Master Mix (Dye Plus), 2 μL each of primers (10 μM), and 1 μL of cDNA template. Bring the volume to 50 μL. The PCR amplification conditions are as follows: 95°C pre-denaturation for 3 min, 30 cycles (95°C 15 s, 56°C 15 s, 72°C 1 min), 72°C extension for 5 min.

[0029] The amplified products were detected by 1% agarose gel electrophoresis. The target band was excised and recovered, purified, and ligated into the pEASY-Blunt zero-background cloning vector, which was then transformed into E. coli DH5α competent cells. Positive clones were selected, identified by bacterial PCR, and plasmids were extracted and sequenced for verification.

[0030] Sequencing results showed that the cloned SaHKT1;8 CDS sequence was completely identical to the reference sequence, with no base mutations or deletions. The obtained nucleotide sequence of the SaHKT1;8 coding region is shown in SEQ ID NO:6.

[0031] The SaHKT1;8 coding sequence was treated with restriction endonucleases and then directionally cloned into the plant expression vector HRG-UBI-GFP. This vector, with pCAMBIA1301 as its backbone, contains a maize Ubiquitin (UBI) promoter, a green fluorescent protein (GFP) reporter gene, and a hygromycin resistance selection marker, enabling efficient expression of the target gene in plants (see [link to documentation]). Figure 2 ).

[0032] After PCR validation, the recombinant expression vector was transformed into E. coli DH5α for amplification, and the plasmid was extracted and sequenced again for confirmation. Positive plasmids were then transformed into Agrobacterium GV3101 competent cells, amplified, and cryopreserved for subsequent plant genetic transformation experiments.

[0033] Example 3: Plant transformation and positive strain identification of SaHKT1;8 expression vector; This embodiment provides a method for genetic transformation of rice using the constructed SaHKT1;8 plant expression vector, and obtains positive plants by screening through molecular detection, providing a material basis for subsequent functional verification.

[0034] The transformation material was the rice variety "Zhonghua 11 (ZH11)" (Oryza sativa L.). Hulled and surface-sterilized seeds were inoculated onto callus induction medium, and callus formation was induced under suitable conditions.

[0035] Normal callus tissue was transferred to co-culture medium and co-cultured at 25°C in the dark for 2–3 days.

[0036] After co-culture, the callus tissue was transferred to a selection medium containing hygromycin for resistance selection. The resistant callus tissue obtained by selection was transferred to a shoot differentiation medium to induce shoot formation, and then transferred to a rooting medium to induce root development, thus obtaining complete transgenic plants.

[0037] The complete plant was transferred to a potting substrate for acclimatization. Once it reached the three-leaf stage, leaves were collected for molecular testing.

[0038] Genomic DNA was extracted from plants at the three-leaf stage, and SaHKT1;8 specific primer pairs were designed for PCR amplification to detect the integration of the target gene. The primer sequences are as follows: SEQ ID NO:8: 5'-TGGCAAACAAGAGTATATTCACA-3'; SEQ ID NO:9: 5'-GAGTACCCCACATTGCCGAA-3'.

[0039] The PCR products were detected by 1% agarose gel electrophoresis, and some plants obtained specific amplification bands consistent with SaHKT1;8. Sequencing confirmed that the amplified sequence was accurate, indicating that the target gene has been stably integrated into the rice genome.

[0040] Molecular detection and growth phenotypic analysis were performed on the obtained T1, T2, and T3 generation positive transgenic plants to screen out several stable expression lines. The PCR detection results of representative T3 generation plants are shown below. Figure 3 As shown.

[0041] Example 4: Salt tolerance assessment of SaHKT1;8 transgenic plants during seedling stage This embodiment evaluates the function of SaHKT1;8 in enhancing salt tolerance in rice seedlings through a seedling salt stress experiment. Experimental materials included: rice Zhonghua 11 (ZH11, WT) as a negative control, rice OsHKT2;4 overexpression lines as a positive control, and the stable SaHKT1;8 transgenic line SaHKT1;8-8 as the experimental group.

[0042] All materials were hydroponically cultured in rice nutrient solution until the three-leaf stage (approximately 21 days), followed by salt stress treatment. NaCl concentration gradients were set at 0 mM (control), 100 mM, and 150 mM, with three biological replicates for each treatment group, each replicate consisting of 24 plants.

[0043] Salt treatment solutions were based on nutrient solution, with 5.844 g / L and 8.775 g / L NaCl added, respectively, and the stress treatment lasted for 5 days. Morphological changes such as yellowing and wilting of plant leaves were recorded during the treatment period.

[0044] After treatment, the plants were transferred to salt-free nutrient solution for recovery culture for 5 days. The number of surviving plants in each group was counted, and the survival rate was calculated as an evaluation index of salt tolerance.

[0045] The results showed that under 100 mM and 150 mM NaCl treatments, the SaHKT1;8-8 transgenic plants exhibited stronger salt tolerance, with significantly lower levels of leaf yellowing and wilting compared to ZH11. Under 150 mM treatment, the survival rate of SaHKT1;8-8 was significantly higher than that of the wild type and superior to the positive control OsHKT2;4 line (see...). Figure 4 ).

[0046] The above results indicate that overexpression of SaHKT1;8 can effectively enhance the salt tolerance of rice seedlings and has good application potential under high salt stress conditions.

[0047] Case Study 5: Grain Trait Analysis of SaHKT1;8 Transgenic Rice To preliminarily assess the effects of the SaHKT1;8 gene on rice grain development and yield traits under conventional field conditions, several transgenic lines stably expressing SaHKT1;8 were planted in experimental fields in Hainan Province.

[0048] Using non-transgenic rice Zhonghua 11 (ZH11, WT) as a control, grain samples from the main panicle of each transgenic line were collected at maturity, photographed, and recorded (see [link to relevant documentation]). Figure 5 ).

[0049] The results showed that the grains of the SaHKT1;8 overexpression lines were generally longer than those of the control lines, with a more elongated morphology and a clear trend of increased grain length.

[0050] The above results suggest that SaHKT1;8 may be involved in the regulation of grain development and has potential application value in improving rice yield traits.

[0051] The above embodiments are preferred embodiments of the present invention, intended to illustrate the principles and effects of the invention, and do not constitute a limitation on the scope of protection. All equivalent modifications, substitutions, or improvements made within the spirit and scope of the present invention should be covered within the scope of protection of the present invention, as specified in the claims.

[0052] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. SaHKT1;8 The application of genes in improving salt tolerance and increasing grain length in rice is characterized by: By overexpressing Spartina alterniflora SaHKT1;8 Genes enhance rice salt tolerance and increase grain length. The Spartina alterniflora SaHKT1;8 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO:

6.

2. A method for cultivating transgenic plants with salt tolerance and increased grain length, characterized in that: Includes the following steps: Cloning the Spartina alterniflora as described in claim 1 SaHKT1;8 Gene; The gene was inserted into a plant expression vector to construct a recombinant expression vector; The recombinant expression vector was transformed into plant cells or tissues; The transformed plant cells or tissues are regenerated into complete plants, and positive transgenic plants are obtained by molecular detection screening. The plant in question is rice.

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