Application of plant stress resistance gene GsZIP7 in salt stress

Abstract

Plant stress resistance genes GsZIP7 In the field of plant genetic engineering technology, this invention relates to the application of salt stress. To identify genes that regulate salt stress, this invention provides a plant stress-resistance gene for regulating plant salt stress tolerance. GsZIP7 The plant stress resistance gene GsZIP7 The nucleotide sequence is shown in SEQ ID NO.1, and a recombinant vector and recombinant bacteria containing the above gene were constructed to obtain transgenic plants; the plant stress resistance gene GsZIP7 Positive regulation of a plant's ability to tolerate salt stress can lay the foundation for research into breeding salt-tolerant transgenic plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of a plant stress resistance gene in salt stress. Background Technology

[0002] Currently, the widespread distribution of saline-alkali soils has severely damaged the crop growing environment. It is estimated that as much as 831 × 10⁻⁶ tons of saline-alkali soils exist on land. 6 hm 2 The land is saline-alkali land, and its area is constantly increasing. Soil salinization seriously affects agricultural production and development, and is a major problem facing agricultural development worldwide. Therefore, understanding the damage caused by salt-alkali stress to plants, the plant response to salt-alkali stress, and the stress tolerance mechanisms of salt-alkali tolerant plants will lay a solid theoretical research foundation for enhancing the salt-alkali tolerance of crops and developing and utilizing saline-alkali land.

[0003] Salt stress primarily damages plants through ion stress, osmotic stress, and oxidative stress. Alkali stress, in addition to salt stress, also introduces high pH stress, resulting in more severe and complex damage. Therefore, saline-alkali stress is a major problem restricting agricultural production. Developing and utilizing these vast saline-alkali land resources is of significant strategic and practical importance for ensuring sustainable and efficient agricultural development and food security, and can create enormous economic, social, and ecological benefits.

[0004] With the rapid development of cutting-edge disciplines such as modern molecular biology, bioinformatics, genetic engineering, genomics, and proteomics, efficient and scientific technical means have been provided for discovering functionally significant genes and transgenic molecular breeding. Utilizing increasingly mature genetic engineering techniques to cultivate new crop varieties with superior traits and good stress tolerance has become one of the important means of modern crop improvement. Transgenic crop technology has become a priority development strategy and a key technological high ground to be seized globally. Therefore, those skilled in the art are eager to discover genes that regulate salt stress and control the salt stress resistance of plants. Summary of the Invention

[0005] This invention aims to discover genes that regulate salt stress, thereby providing plant stress resistance genes for regulating plant salt stress tolerance. GsZIP7 Application in salt stress.

[0006] One of the objectives of this invention is to provide a plant stress resistance gene. GsZIP7 The plant stress resistance gene GsZIP7 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] A second objective of this invention is to provide a recombinant vector containing the aforementioned plant stress resistance gene. GsZIP7 .

[0008] In a preferred embodiment of the present invention, the recombinant vector further comprises a 3' untranslated region of the exogenous gene, wherein the 3' untranslated region is a DNA fragment containing a polyadenylate signal and participating in mRNA processing or gene expression.

[0009] In a preferred embodiment of the present invention, the recombinant vector further comprises a translational enhancer or a transcriptional enhancer, wherein the enhancer is an ATG start codon or an adjacent region start codon that is the same as the reading frame of the coding sequence.

[0010] A third objective of this invention is to provide a recombinant bacterium containing the aforementioned recombinant vector.

[0011] The fourth objective of this invention is to provide a breeding method for regulating the salt stress resistance of plants, the specific steps of which are as follows:

[0012] S1: Using primers to target plant stress resistance genes GsZIP7 Cloning is performed to obtain gene clone sequences;

[0013] S2: Ligate the gene clone sequence obtained in S1 with the vector to obtain a recombinant vector;

[0014] S3: The recombinant vector obtained in S2 is introduced into microbial bacteria to obtain recombinant bacteria;

[0015] S4: The recombinant bacteria obtained in S3 are introduced into recipient plant cells to obtain transgenic plants.

[0016] In a preferred embodiment of the present invention, the upstream primer Primer-KS sequence in S1 is shown in SEQ ID NO.2, and the downstream primer Primer-KAS sequence is shown in SEQ ID NO.3.

[0017] In a preferred embodiment of the present invention, the vector in S2 is a plasmid, bacteriophage, or viral vector, and the microorganism in S3 is yeast, bacteria, algae, or fungi.

[0018] In a preferred embodiment of the present invention, the method for introducing the recombinant bacteria into the recipient plant cells in S4 is microinjection, electroporation, or Agrobacterium-mediated transformation; the recipient plant is a monocotyledonous plant or a dicotyledonous plant; the dicotyledonous plant is a legume, a cruciferous plant, or a styrax plant; the legume is soybean, Lobelia chinensis, alfalfa, or water hyacinth; the cruciferous plant is Arabidopsis thaliana or rapeseed; the styrax plant is sunflower; and the Arabidopsis thaliana is Colombian ecotype Arabidopsis thaliana col-0.

[0019] The fifth objective of this invention is to provide plants carrying the aforementioned stress resistance genes. GsZIP7The application of recombinant vectors or recombinant bacteria in plant salt stress, and the plant stress resistance genes mentioned above. GsZIP7 Positively regulates the ability of plants to tolerate salt stress.

[0020] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a plant stress resistance gene. GsZIP7 Application in salt stress, the plant stress resistance gene GsZIP7 The nucleotide sequence is shown in SEQ ID NO.1.

[0021] Through tissue localization analysis, plant stress resistance genes were discovered. GsZIP7 The expression level in soybean young stems was significantly higher than in other tissues; by analyzing... GsZIP7 Analysis of gene expression patterns under salt stress revealed that under this abiotic stress, salt... GsZIP The seven genes showed varying degrees of response in soybean leaves and roots, and GsZIP The relative expression levels of gene 7 changed with stress treatment time, indicating that... GsZIP7 Genes play a role in the soybean response to salt stress.

[0022] Relevant experiments have shown that overexpression GsZIP7 Transgenic Arabidopsis thaliana plants exhibit higher tolerance to salt stress, and salt stress-related... Marker Significantly increased gene expression levels indicate that the plant has genes for stress resistance. GsZIP7 By upregulating the expression of the antioxidant defense system, the plant's ROS scavenging ability is effectively enhanced, thereby maintaining cell homeostasis under salt stress and reducing the adverse effects of stress on plant growth. GsZIP7 The gene enhances the plant's superoxide anion scavenging efficiency under salt stress by strengthening ROS scavenging ability; GsZIP7 Genes participate in the early adaptation process of plants to salt stress by regulating the expression of these salt stress response marker genes; further illustrating, GsZIP7 Genes can lay the foundation for research into developing salt-tolerant transgenic plants. Attached Figure Description

[0023] Figure 1 Different tissues of wild soybean in Example 2 GsZIP7 Gene expression level statistics;

[0024] Figure 2 After salt stress treatment in Example 2 GsZIP7 Statistical graph of relative gene expression levels in soybean leaves and roots; A represents soybean leaf tissue; B represents soybean root tissue; * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0025] Figure 3 For the transfer in Example 3 GsZIP7 Image of DNA-level molecular identification in Arabidopsis thaliana plants;

[0026] Figure 4 For the transfer in Example 3 GsZIP7 Image showing molecular identification of RNA at the gene level in Arabidopsis thaliana plants;

[0027] Figure 5 For the transfer in Example 3 GsZIP7 Statistical graph of gene expression levels by qRT-PCR in Arabidopsis thaliana plants;

[0028] Figure 6 For the transfer in Example 3 GsZIP7 Phenotypic diagram of Arabidopsis thaliana plants under salt stress; A represents the transformation. GsZIP7 Phenotypic diagram of Arabidopsis thaliana seedlings; B represents transgenic plants. GsZIP7 Statistical graph of root length of Arabidopsis thaliana plants; C represents transformation GsZIP7 Statistical chart of fresh weight of Arabidopsis thaliana plants;

[0029] Figure 7 For example, wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana under salt stress in Example 3 GsZIP7 Graphs showing the determination of physiological indicators in Arabidopsis thaliana; A is a statistical graph of chlorophyll content; B is a statistical graph of MDA content; C is a statistical graph of SOD enzyme activity; D is a statistical graph of POD enzyme activity; E is a statistical graph of CAT enzyme activity.

[0030] Figure 8 For example, wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana under salt stress in Example 3 GsZIP7 NBT staining image of Arabidopsis thaliana;

[0031] Figure 9 Salt stress related to Example 3 Marker Gene expression analysis diagram; A is COR15A Gene expression level statistics; B represents... RD29A Gene expression level statistics; C represents H + -APase Gene expression level statistics; D represents NADP-ME Gene expression level statistics; E represents... KIEI Gene expression level statistics; F represents COR47 Gene expression level statistics chart. Detailed Implementation

[0032] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0034] The wild soybean G07256 in the following examples is disclosed in the literature "DuanMu Huizi,Wang Yang,Bai Xi,Cheng Shufei,Deyholos Michael K,Wong Gane Ka-Shu... Zhu Yanming.(2015).Wildsoybean roots depend on specific transcription factors and oxidation-reduction related genesin response to alkaline stress..Functional integrativegenomics(6),651-60.", and can be obtained from Harbin Normal University.

[0035] The DH5α competent cells, Agrobacterium GV3101 competent cells, and Agrobacterium K599 competent cells used in the following examples were all purchased from TransGen Biotech Ltd.

[0036] Example 1: Plant stress resistance genes GsZIP7 Screening and cloning

[0037] 1. GsZIP7 Gene screening

[0038] In the early stages, the laboratory collected 325 wild soybean accessions from saline-alkali land in Northeast China. Through screening under salt stress, a wild soybean line (G07256) that could tolerate 50 mM NaHCO3 (pH=9.02) stress was obtained. Transcriptome sequencing was used to identify differentially expressed genes in wild soybean (G07256) under 50 mM NaHCO3 stress. A salt stress regulatory network of wild soybean was constructed, and salt stress response genes were screened from it. GsZIP7 .

[0039] 2. Treatment of plant materials

[0040] Select plump wild soybeans (G07256), sterilize them in a 6% sodium hypochlorite solution for 10 minutes, rinse them 3-4 times with sterile water, and place them on moist filter paper. Incubate them in the dark at 25℃ for 4 days to promote germination. When the sprouts grow to 1-2 cm, transfer them to pots containing Hogrange culture medium, fix them with space cotton to immerse the sprouts in the culture medium, and place them in a greenhouse for cultivation. The greenhouse growth conditions are 25℃ / 20℃ (day / night) and a photoperiod of 16 hours of light / 8 hours of darkness. When the seedlings reach 3 weeks of age, take their leaves and place them in EP tubes for storage at -80℃.

[0041] 3. Extraction of total RNA and acquisition of cDNA

[0042] RNA was extracted from the leaves of 3-week-old soybean G07256 seedlings obtained above using the Plant Total RNA Isolation Kit (purchased from Chengdu Fuji Biotechnology Co., Ltd.); cDNA was obtained by reverse transcription using the total RNA obtained above as a template.

[0043] Using the cDNA obtained above as a template, PCR amplification was performed using Primer-KS (nucleotide sequence shown in SEQ ID NO.2) and Primer-KAS (nucleotide sequence shown in SEQ ID NO.3) as primers to obtain PCR amplification products. The PCR amplification system (25 μL) consisted of: 1 μL cDNA, 1 μL Primer-F, 1 μL Primer-R, 12.5 μL Prime Star Mix, and 9.5 μL ddH2O. The PCR amplification conditions were: 94℃ for 10 s, 58℃ for 5 s, 72℃ for 1 min and 30 s, for 35 cycles; 72℃ for 5 min; and the reaction was terminated at 4℃.

[0044] The PCR amplification products obtained above were detected by 1% agarose gel electrophoresis, yielding an amplification band with a molecular weight of approximately 1.1 Kb. The PCR amplification products were recovered using an agarose gel recovery kit (purchased from Shanghai Soba Biotechnology Co., Ltd.). The product was then ligated into the pCAMBIA-1300 vector to obtain the recombinant vector, which was named pCAMBIA1300- GsZIP7 and transform it into E. coli DH5α The competent cells were then sent for sequencing.

[0045] Sequencing results showed that the nucleotide sequence of the PCR amplification product, which was 1086 bp in size, was as shown in SEQ ID NO. 1, and it was named... GsZIP7 Gene.

[0046] Example 2: Plant stress resistance genes GsZIP7 Expression characteristics analysis

[0047] 1. Different tissues of soybean GsZIP7 Relative expression level of genes

[0048] (1) Treatment of plant materials

[0049] Wild soybean G07256 seeds were sterilized in a 6% sodium hypochlorite solution for 10 min and germinated in a petri dish with distilled water for 4 days. The seedlings were fixed in the holes of a foam board and cultured in a black plastic container containing 1 / 2 Hoagland nutrient solution under greenhouse conditions of 25℃ / 20℃ (day / night) and a photoperiod of 16 hours of light / 8 hours of darkness. Seedlings with good growth were selected and transplanted into sterilized soil. When the seedlings reached 3 months of age, different tissues of wild soybean (including young leaves, young roots, young stems, roots, stems, leaves, and pods) were quickly collected and stored at -80℃.

[0050] (2) Extraction of total RNA and acquisition of cDNA

[0051] Total RNA was extracted from different tissues of the above-mentioned wild soybean (including young leaves, young roots, young stems, roots, stems, leaves, and pods) using the Plant Total RNA Isolation Kit (purchased from Chengdu Fuji Biotechnology Co., Ltd.); cDNA was obtained by reverse transcription using the obtained total RNA as a template.

[0052] (3) GsZIP7 Detection of gene expression levels

[0053] Using the cDNA obtained above as a template, and Primer-qS (nucleotide sequence shown in SEQ ID NO.4) and Primer-qAS (nucleotide sequence shown in SEQ ID NO.5) as primers, Real-time PCR was performed to... GsZIP7 Gene expression levels were detected; the conditions for the Real-time PCR reaction were: 94℃ 10 min → [94℃ 30 s → 60℃ 30 s → 60℃ 30 s] × 40 → 72℃ 10 min → 94℃ 2 min.

[0054] Real-time PCR uses the comparative CT method (ΔΔCT) to calculate gene expression levels. GmGAPDH For internal reference gene (internal reference gene primers include) GsGAPDH S and GsGAPDH AS, the GsGAPDH The S nucleotide sequence is shown in SEQ ID NO.6. GsGAPDH The AS nucleotide sequence is shown in SEQ ID NO.7, with untreated samples as controls. Target gene expression differences were expressed as the fold increase in expression of the treated sample relative to the untreated sample at each time point. Each sample included three biological replicates and three technical replicates. Data were taken as the average of the three biological replicates; if one value had a large deviation, the average of the two data points was used. The raw data, after standardization, were analyzed for significant differences using a T-test. The relative expression level was calculated as follows: 2 - ΔΔCT = 2 - (ΔCT treatment - ΔCT control) = 2 - [(CT-treated target gene - CT-treated internal reference gene) - (CT control target gene - CT control internal reference gene)].

[0055] like Figure 1 As shown, the expression level in soybean young leaves is used as a relative reference. GsZIP7 The gene expression level was highest in young stems and mature roots, and relatively high in young roots and stems, while expression was lower in mature leaves and pods. This indicates that... GsZIP7 The gene expression levels differed significantly in different soybean tissues, indicating that... GsZIP7 Gene expression in wild soybean is specific.

[0056] 2. Soybean roots and leaves under different salt stress treatments for different durations GsZIP7 Gene expression pattern analysis

[0057] (1) Treatment of plant materials

[0058] Wild soybean G07256 seeds were sterilized in a 6% sodium hypochlorite solution for 10 min and germinated in petri dishes with distilled water for 4 days. The growing seedlings were fixed on the holes of a foam board and cultured in black plastic containers containing 1 / 2 Hoagland nutrient solution under greenhouse growing conditions of 25℃ / 20℃ (day / night) and a photoperiod of 16 hours of light / 8 hours of darkness. When the soybean seedlings reached 18 days of age, they were subjected to salt stress treatment under 150 mM NaCl. Three soybean seedlings were selected at each time point of 0 h, 1 h, 3 h, 6 h and 12 h. Leaves and 3 cm of root tips were cut from the soybean seedlings after different treatment times as tissue samples for testing. The samples were quickly frozen in liquid nitrogen and then stored at -80℃.

[0059] (2) Extraction of total RNA and acquisition of cDNA

[0060] Total RNA was extracted from soybean seedling leaves and root tip tissues after different treatment times using the Plant Total RNA Isolation Kit (purchased from Chengdu Fujie Biotechnology Co., Ltd.); cDNA was obtained by reverse transcription using the obtained total RNA as a template.

[0061] (3) GsZIP7 Detection of gene expression levels

[0062] Using the cDNA obtained above as a template, and Primer-qS (nucleotide sequence shown in SEQ ID NO.4) and Primer-qAS (nucleotide sequence shown in SEQ ID NO.5) as primers, Real-time PCR was performed to... GsZIP7 Gene expression levels were detected; the conditions for the Real-time PCR reaction were: 94℃ 10 min → [94℃ 30 s → 60℃ 30 s → 60℃ 30 s] × 40 → 72℃ 10 min → 94℃ 2 min.

[0063] Real-time PCR uses the comparative CT method (ΔΔCT) to calculate gene expression levels. GmGAPDH For internal reference gene (internal reference gene primers include) GsGAPDH S and GsGAPDH AS, the GsGAPDH The S nucleotide sequence is shown in SEQ ID NO.6. GsGAPDHThe AS nucleotide sequence is shown in SEQ ID NO.7, with untreated samples as controls. Target gene expression differences were expressed as the fold increase in expression of the treated sample relative to the untreated sample at each time point. Each sample included three biological replicates and three technical replicates. Data were taken as the average of the three biological replicates; if one value had a large deviation, the average of the two data points was used. The raw data, after standardization, were analyzed for significant differences using a T-test. The relative expression level was calculated as follows: 2 - ΔΔCT = 2 - (ΔCT treatment - ΔCT control) = 2 - [(CT-treated target gene - CT-treated internal reference gene) - (CT control target gene - CT control internal reference gene)].

[0064] like Figure 2 As shown, soybean tissue treated with salt stress for 0 h... GsZIP7 The relative expression level of the gene was compared with that of the control; under salt stress induced by 150 mmol / L NaCl, the expression level in soybean leaves was [data missing]. GsZIP7 The relative expression levels of genes showed a fluctuating trend of first increasing, then decreasing, then increasing again and decreasing again; among them, the relative expression levels of soybean leaves under salt stress for 6 h were... GsZIP7 The relative expression level of the gene reaches its peak in soybean roots. GsZIP7 The relative expression levels of the gene showed an increasing trend during 1-3 h of salt stress treatment, a rapid decreasing trend during 6 h of salt stress treatment, and a slight increasing trend during 12 h of salt stress treatment; among them, the expression levels of the gene in soybean roots after 6 h of salt stress treatment were... GsZIP7 The relative expression level of the gene reaches its peak.

[0065] Example 3: Plant stress resistance genes GsZIP7 Application in plant salt stress

[0066] 1. Preparation of transgenic plants

[0067] The recombinant vector pCAMBIA 1300- obtained in Example 1 was subjected to a freeze-thaw method. GsZIP7 Transformed into Agrobacterium tumefaciens GV3101, positive transformants were obtained by PCR identification (the positive transformants contain the nucleotide sequence shown in SEQ ID NO.1). GsZIP7 Genes were obtained to obtain recombinant Agrobacterium, which was then used to infect wild-type Arabidopsis thaliana (Columbia ecotype col-0) via the Floral-dip method.

[0068] Infected Arabidopsis thaliana were cultured to obtain T0 generation transgenic strains. GsZIP7 Arabidopsis seeds; T0 substitution GsZIP7 After surface disinfection, Arabidopsis thaliana seeds are sown in a solution containing 25 mg / L kanamycin ( Kanamycin SulfateT1 generation transfectants were obtained by screening on 1 / 2 MS medium (Biotopped, K6020). GsZIP7 Arabidopsis seedlings; this process is repeated until T3 generation is obtained. GsZIP7 Arabidopsis homozygous lines.

[0069] 2. Turn GsZIP7 Identification of positive Arabidopsis thaliana seedlings

[0070] (1) DNA-level molecular identification

[0071] To confirm pCAMBIA 1300- GsZIP7 To investigate the integration of recombinant plasmids into Arabidopsis thaliana, genomic DNA was extracted from rosette leaves of wild-type and transgenic Arabidopsis thaliana and amplified by PCR using specific primers.

[0072] Specifically: Extract the T3 generation obtained above. GsZIP7 The genomic DNA of Arabidopsis thaliana seedlings was used as a template for PCR identification. Primer-KS (nucleotide sequence shown in SEQ ID NO.2) and Primer-KAS (nucleotide sequence shown in SEQ ID NO.3) were used as primers for PCR amplification to obtain PCR amplification products.

[0073] The PCR amplification system (25 μL) consisted of: 1 μL cDNA, 1 μL Primer-F, 1 μL Primer-R, 12.5 μL PrimeStar Mix, and 9.5 μL ddH2O. PCR amplification conditions were: 94℃ for 10 s, 58℃ for 5 s, 72℃ for 1 min and 30 s, for 35 cycles; 72℃ for 5 min; and the reaction was terminated at 4℃.

[0074] The PCR amplification products obtained above were subjected to 1% agarose gel electrophoresis, and the results are as follows: Figure 3 As shown, the target band was not detected in the PCR amplification product of wild-type Arabidopsis plants, while the PCR amplification product of transgenic plants showed a clear band at the expected position, indicating that the exogenous gene has been successfully transferred into Arabidopsis.

[0075] (2) Identification of transcription level

[0076] Regarding the above transfer GsZIP7 The homozygous Arabidopsis lines #1 and #3 were further identified at the transcriptional level. The specific steps were as follows: The T3 generation transgenic lines were analyzed using the Plant Total RNA Isolation Kit (purchased from Chengdu Fuji Biotechnology Co., Ltd.). GsZIP7 Total RNA was extracted from Arabidopsis homozygous lines #1 and #3, and cDNA was obtained by reverse transcription.

[0077] Using the cDNA obtained above as a template, and Primer-qS (nucleotide sequence shown in SEQ ID NO.4) and Primer-qAS (nucleotide sequence shown in SEQ ID NO.5) as primers, ... Actin This is an internal reference gene, and the primers for the internal reference gene are... [[ID= S (nucleotide sequence shown in SEQ ID NO. 8) and ​ The AS primer pair (nucleotide sequence shown in SEQ ID NO.9) was used for Real-time PCR. ​ Gene expression levels were detected; the Real-time PCR reaction conditions were as follows: 94℃ 10 min → [94℃ 30 s → 60℃ 30 s → 60℃ 30 s] × 40 → 72℃ 10 min → 94℃ 2 min.

[0078] Real-time PCR was performed using the comparative CT method (ΔΔCT) to calculate gene expression levels. GmGAPDH was used as the internal reference gene (the primers for the internal reference gene included GsGAPDH S and GsGAPDH AS, the nucleotide sequence of GsGAPDH S is shown in SEQ ID NO.6, and the nucleotide sequence of GsGAPDH AS is shown in SEQ ID NO.7), and untreated samples were used as controls. Differences in target gene expression were expressed as the fold increase in the expression of the treated sample relative to the untreated sample at each time point. Each sample included three biological replicates and three technical replicates. The data were the average of the three biological replicates; if one value showed a large deviation, the average of the two data points was used. The raw data, after standardization, were analyzed for significant differences using a T-test. The relative expression level was calculated as follows: 2 - ΔΔCT = 2 - (ΔCT treatment - ΔCT control) = 2 - [(CT-treated target gene - CT-treated internal reference gene) - (CT control target gene - CT control internal reference gene)].

[0079] The PCR amplification products obtained above were subjected to 1% agarose gel electrophoresis, and the results are as follows: ​ As shown, PCR of wild-type Arabidopsis plants produced no amplification products, while T3 generation transgenic plants produced no amplification products. ​ Arabidopsis homozygous lines #1 and T3 generation ​ The target band could be amplified in the homozygous Arabidopsis line #3, indicating the presence of a foreign gene. ​ The gene has been successfully integrated into the Arabidopsis genome.

[0080] Real-time PCR test results as follows ​ As shown, ​The gene was efficiently transcribed and expressed in transgenic Arabidopsis thaliana; among them, the selected T3 generation transgenic gene... ​ Arabidopsis homozygous lines #1 and T3 transgenic ​ Arabidopsis homozygous line #3 ​ Gene expression levels were significantly higher in the T3 generation than in the wild type, indicating that the obtained T3 generation transgenic genes... ​ The homozygous Arabidopsis lines #1 and #3 can be used for the next step of phenotypic analysis.

[0081] 3. Turn ​ Phenotypic analysis of Arabidopsis thaliana plants under salt stress

[0082] (1) Turn ​ Phenotyping and root length of Arabidopsis thaliana seedlings under salt treatment

[0083] For wild-type Arabidopsis thaliana WT and the T3 generation obtained above ​ Seeds from homozygous Arabidopsis lines #1 and #3 were disinfected and cleaned, vernalized for 3 days, and then sown on 1 / 2 MS medium for cultivation. After 5 days of growth, 12 seedlings with excellent growth and uniform vigor were selected from each line and transferred to 1 / 2 MS solid medium containing NaCl concentrations of 0 mmol / L, 75 mmol / L, 100 mmol / L, and 125 mmol / L, respectively. These seedlings were then cultured vertically under light for 7 days. Phenotypic results after stress were photographed and recorded, and root length and fresh weight were measured. All experiments were repeated 3 times technically and 3 times biologically, with 12 seedlings from each line per experiment.

[0084] The results are as follows ​ As shown, under normal culture conditions, Arabidopsis seedlings from all genetic lines exhibited similar growth patterns. When different concentrations of NaCl were added to the culture medium for salt stress treatment, root length growth in all lines was significantly affected, showing concentration-dependent growth restriction; however, under salt stress conditions, the T3 generation of transgenic... ​ The root lengths of the homozygous Arabidopsis lines #1 and #3 were significantly longer than those of the wild type (WT), and their overall growth was significantly better than that of the WT plants. Under salt stress treatments of 75 mmol / L, 100 mmol / L, and 125 mmol / L, the T3 generation of transgenic plants... ​ There was no significant difference in root length and fresh weight between the homozygous Arabidopsis lines #1 and #3, but both were significantly higher than the root length and fresh weight of the WT line.

[0085] It is evident that under salt stress conditions, the conversion... ​ The taproot length and fresh weight of Arabidopsis seedlings from different lines showed a high degree of consistency and were significantly better than those of wild-type (WT) plants; indicating that... ​ Genes positively regulate the plant's response mechanism to salt stress, thereby enhancing the plant's salt stress tolerance.

[0086] 4. Determination of physiological indicators in transgenic Arabidopsis thaliana under salt stress

[0087] (1) Treatment of plant materials

[0088] Wild-type Arabidopsis thaliana (WT) and T3 generation plants of normal growth at 28 days old and with uniform growth were transferred. ​ The homozygous Arabidopsis lines #1 and #3 were divided into a control group and an experimental group that was treated with 200 mM NaCl salt stress for 72 hours.

[0089] Accurately weigh leaf tissue from the same leaf position of each group of Arabidopsis plants (sample size 0.1g ± 0.005g) and quickly transfer it into pre-cooled 1.5 mL centrifuge tubes; all experimental samples were immediately immersed in liquid nitrogen for flash freezing after collection, and then stored separately in an ultra-low temperature freezer at -80℃ for later use.

[0090] (2) Measurement of physiological indicators

[0091] Leaf tissues from the same leaf position of each group of Arabidopsis plants were analyzed for chlorophyll content, malondialdehyde (MDA) content (a lipid peroxidation product), superoxide dismutase (SOD) activity, peroxidase (POD) activity, and catalase (CAT) activity. The experimental design employed seven biological replicates, with sampling time strictly controlled within 10:00 ± 0.5 h to avoid the influence of circadian rhythms on physiological indicators. All physiological indicators were analyzed and measured according to the methods previously summarized by our laboratory.

[0092] The results are as follows ​ As shown, under normal growth conditions, wild-type (WT) and T3 generation transgenic... ​ No significant differences were observed in antioxidant-related physiological parameters between homozygous Arabidopsis lines #1 and #3.

[0093] After salt stress treatment, wild-type (WT) and T3 generation transgenic ​ The chlorophyll content of homozygous Arabidopsis lines #1 and #3 both showed a decreasing trend, but the T3 generation... ​ The homozygous Arabidopsis lines #1 and #3 showed significantly lower chlorophyll degradation than the wild type, indicating less damage to their photosynthetic system. (T3 generation) ​ The malondialdehyde (MDA) accumulation in homozygous Arabidopsis lines #1 and #3 was significantly lower than that in the wild type, reflecting a lower degree of cell membrane lipid peroxidation. Further analysis of antioxidant enzyme activity revealed that the T3 generation of transgenic lines... ​ The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the homozygous Arabidopsis lines #1 and #3 were significantly higher than those in the wild type. This enhanced enzyme activity suggests that the activity of these enzymes helps to scavenge reactive oxygen species (ROS) in cells and reduce oxidative damage.

[0094] Therefore, ​ By upregulating the expression of the antioxidant defense system, the gene effectively enhances the plant's ROS scavenging ability, thereby maintaining cell homeostasis under salt stress and reducing the adverse effects of stress on plant growth.

[0095] 5. NBT staining analysis of transgenic Arabidopsis under salt stress

[0096] (1) Treatment of plant materials

[0097] Wild-type Arabidopsis thaliana (WT) and T3 generation transgenic plants that have grown to the two-leaf stage and exhibit uniform growth will be used. ​ The homozygous Arabidopsis lines #1 and #3 were divided into a control group and an experimental group that was treated with 75 mM, 100 mM, and 125 mM NaCl salt stress for 12 hours.

[0098] (2) NBT staining and destaining

[0099] NBT staining was performed on Arabidopsis seedlings in each group that had undergone stress treatment. The specific procedure was as follows: the seedlings were completely immersed in 1 mg / mL NBT staining solution and stained in a dark environment for 6 hours; after staining, decolorization was performed using a decolorizing solution (volume ratio of anhydrous ethanol: glacial acetic acid: glycerol = 3:1:1) in a 100℃ water bath until the leaves showed a clear staining effect; leaves of similar size and completely decolorized were selected and photographed for record.

[0100] The results are as follows ​ As shown, under unstressed conditions, wild-type Arabidopsis thaliana (WT) and T3 generation transgenic Arabidopsis thaliana... ​ Leaves from homozygous Arabidopsis lines #1 and #3 showed light staining with no significant difference; under 75 mmol / L NaCl salt stress, WT leaves stained deeper to light blue, while T3 transgenic leaves stained more deeply. ​ The leaves of homozygous Arabidopsis lines #1 and #3 showed lighter staining and smaller staining areas; under 100 mmol / L NaCl salt stress, the WT and T3 generations of transgenic... ​ The leaves of homozygous Arabidopsis lines #1 and #3 stained a deeper blue, but compared to WT, the T3 generation showed a more pronounced blue color. ​ The homozygous Arabidopsis lines #1 and #3 still showed relatively light staining and large unstained areas; under 125 mmol / L NaCl salt stress, the WT and T3 generations of transgenic lines... ​ The leaf staining differences between the homozygous Arabidopsis lines #1 and #3 were more significant.

[0101] Therefore, ​ The gene enhances the plant's ability to scavenge superoxide anions under salt stress by strengthening its ROS scavenging capacity.

[0102] 6. Salt stress-related effects of transgenic Arabidopsis thaliana ​ Gene expression level measurement

[0103] (1) Treatment of plant materials

[0104] Transgenic wild-type Arabidopsis thaliana WT and T3 generations ​ After disinfection and cleaning, the seeds of Arabidopsis homozygous lines #1 and #3 were vernalized for 3 days, evenly spotted in 1 / 2 MS medium, and horizontally cultured for 2 weeks before being subjected to stress treatment.

[0105] Treatment groups: CK (1 / 2 MS medium), experimental group (200 mM NaCl);

[0106] Sampling time: 0 h, 3 h and 6 h.

[0107] Accurately weigh leaf tissue from the same leaf position of each group of Arabidopsis plants (sample size 0.1g ± 0.005g) and quickly transfer it into pre-cooled 1.5 mL centrifuge tubes; all experimental samples were immediately immersed in liquid nitrogen for flash freezing after collection, and then stored separately in an ultra-low temperature freezer at -80℃ for later use.

[0108] (2) Extraction of total RNA and acquisition of cDNA

[0109] Total RNA was extracted from leaf tissues at the same leaf position of each group of Arabidopsis plants using the Plant Total RNA Isolation Kit, and cDNA was obtained by reverse transcription.

[0110] Using the cDNA obtained above as a template, salt stress-related... ​ The primers for the gene (as shown in Table 1) are used to... ​ This is an internal reference gene, and the primers for the internal reference gene are... ​ S (nucleotide sequence shown in SEQ ID NO. 8) and ​ The AS primer pair (nucleotide sequence shown in SEQ ID NO.9) was used to detect salt stress-related factors via real-time PCR. ​ The expression levels of the genes were detected separately; the Real-time PCR reaction conditions were as follows: 94℃ 10 min → [94℃ 30 s → 60℃ 30 s → 60℃ 30 s] × 40 → 72℃ 10 min → 94℃ 2 min.

[0111] Table 1

[0112]

[0113] The results are as follows ​As shown, compared with the CK control group (treatment for 0 hours), T3 generation... ​ Salt stress-related in Arabidopsis homozygous lines #1 and #3 ​ Gene( ​ + ​ The relative expression levels of all genes showed significant upregulation.

[0114] Specifically, ​ Gene transfer in T3 generation ​ The expression levels in the homozygous Arabidopsis lines #1 and #3 showed a continuous upward trend and reached a highly significant level after 6 hours of treatment. ​ and ​ Gene transfer in T3 generation ​ The expression patterns in the homozygous Arabidopsis lines #1 and #3 showed a trend of first increasing and then decreasing, and both reached their peak after 3 hours of salt stress treatment, exhibiting highly significant differences.

[0115] Therefore, ​ Genes participate in the early adaptation process of plants to salt stress by regulating the expression of these salt stress response marker genes.

[0116] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A breeding method for regulating the salt stress resistance of plants, characterized in that, The specific steps of the breeding method are as follows: S1: Using primers to target plant stress resistance genes GsZIP7 Cloning is performed to obtain gene clone sequences; S2: Ligate the gene clone sequence obtained in S1 with the vector to obtain a recombinant vector; S3: The recombinant vector obtained in S2 is introduced into microbial bacteria to obtain recombinant bacteria; S4: The recombinant bacteria obtained in S3 are introduced into recipient plant cells to obtain transgenic plants; The plant stress resistance gene described in S1 GsZIP7 The nucleotide sequence is shown in SEQ ID NO.1; The recipient plant mentioned in S4 is Arabidopsis thaliana, specifically the Colombian ecotype Arabidopsis thaliana col-0.

2. The breeding method according to claim 1, characterized in that, The upstream primer Primer-KS sequence in S1 is shown in SEQ ID NO.2, and the downstream primer Primer-KAS sequence is shown in SEQ ID NO.

3.

3. Plant stress resistance genes GsZIP7 Its application in plant salt stress is characterized by... The application refers to the overexpression of plant stress resistance genes. GsZIP7 Improve the ability of plants to tolerate salt stress; The plant is Arabidopsis thaliana, and the Arabidopsis thaliana is the Colombian ecotype Arabidopsis thaliana col-0; The plant stress resistance gene GsZIP7 The nucleotide sequence is shown in SEQ ID NO.1.

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