Application of AhSTF1 gene in regulating plant salt tolerance

Abstract

This invention belongs to the field of plant genetic engineering, specifically involving AhSTF1 The application of genes in regulating plant salt tolerance. AhSTF1 The base sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it expresses is shown in SEQ ID NO.2. Experimental results of this invention demonstrate... AhSTF1 The genes work synergistically to alleviate the damage caused by salt stress by increasing POD activity, reducing MDA content, and accumulating soluble proteins, ultimately resulting in higher germination rates, more developed root systems, and better growth. These results fully demonstrate... AhSTF1 Genes play a crucial and positive role in improving plant salt tolerance, providing important genetic resources and theoretical basis for peanut stress-resistance breeding, and have significant application value for cultivating new salt-tolerant crop varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically involving AhSTF1 Application of genes in regulating plant salt tolerance. Background Technology

[0002] Salt stress is one of the major abiotic stresses affecting plant growth and yield. Under high-salt conditions, Na+... + and Cl - Accumulation within plants disrupts ion homeostasis, leading to ion toxicity, osmotic stress, and oxidative stress, ultimately resulting in stunted growth, wilting, and even death. The harm of salt stress to plants can be divided into two stages: direct salt damage, such as water deficit and membrane system damage, and secondary salt damage, such as enzyme activity inhibition and impaired mineral element absorption, which in turn interfere with normal physiological metabolism, manifesting as decreased photosynthesis, growth inhibition, and premature aging.

[0003] Peanuts (A) rachis hypogaea Peanuts (L.) are important oilseed and cash crops, possessing advantages such as drought resistance, tolerance to poor soil, and nitrogen fixation for soil improvement. They exhibit strong adaptability in slightly saline-alkali soils, with a salt tolerance threshold of 0.35%–0.45%. However, when soil salinity exceeds 0.3%, peanut growth is significantly inhibited, leading to a decrease in yield.

[0004] Therefore, identifying genes that can improve plant salt tolerance and elucidating their mechanisms is of great importance for increasing peanut yield. Summary of the Invention

[0005] To solve the above problems, the present invention provides AhSTF1 Application of genes in regulating plant salt tolerance.

[0006] This invention is achieved through the following technical solution:

[0007] AhSTF1 The application of genes in regulating plant salt tolerance, the aforementioned AhSTF1 The base sequence of the gene is shown in SEQ ID NO.1.

[0008] Preferably, the AhSTF1 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2.

[0009] Preferably, the aforementioned [material] in plants is obtained through genetic engineering methods. AhSTF1 Gene overexpression to enhance salt tolerance in plants; the plants are tobacco or peanut.

[0010] Preferably, the genetic engineering method is: using the AhSTF1 Genes were constructed to create recombinant overexpression vectors, which were then transformed into plants to improve their salt tolerance.

[0011] Preferably, the recombinant overexpression vector is constructed by amplification using specific primer pairs. AhSTF1 The gene was recovered, and the PCR product was ligated into the pCAMBIA1307 vector to obtain a recombinant overexpression vector.

[0012] Preferably, the base sequences of the specific primer pair are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] Preferably, the method for transforming the plant is Agrobacterium-mediated transformation.

[0014] Preferably, the Agrobacterium is GV3101.

[0015] A method to improve the salt tolerance of plants, making the plants... AhSTF1 Gene overexpression, or enhancement of the aforementioned gene expression in plants AhSTF1 The activity of the gene-expressed protein, or the enhancement of the protein in the plant. AhSTF1 The amount of protein expressed by the gene is adjusted to improve the salt tolerance of the plant; the plant is tobacco or peanut.

[0016] A breeding method for salt-tolerant plants, enabling the plants to... AhSTF1 Gene overexpression yields salt-tolerant transgenic plants; the plants are tobacco or peanut.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides AhSTF1 Application of genes in regulating plant salt tolerance AhSTF1 The base sequence of the gene is shown in SEQ ID NO.1. AhSTF1 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2. This invention discovers a MYB class transcription factor gene. AhSTF1 It plays a key regulatory role in plant salt stress response. Gene overexpression technology is used to create cells carrying the target gene. AhSTF1 The recombinant overexpression vector was used to verify the gene expression using an Agrobacterium-mediated infection method. AhSTF1 It plays a key regulatory role in plant salt stress response. Experiments show that, through overexpression technology according to the present invention... AhSTF1 A pair of highly specific primers with restriction enzyme sites were designed based on the CDS sequence. Using cDNA reverse transcribed from peanut line FZ080 RNA as a template, the CDS sequence was specifically amplified. Then, using T4 ligase as a ligase, the linearized vector was ligated with the target fragment, allowing... AhSTF1Gene overexpression; transgenic plant materials were obtained using tissue culture and transient expression experiments. By simulating natural salt stress conditions, the transgenic materials were subjected to salt stress treatment. Experimental results showed that the peroxidase (POD) activity in the transgenic materials was effectively increased, while the malondialdehyde (MDA) content decreased. Therefore, it is evident that… AhSTF1 Salt tolerance of plants can be improved by activating antioxidant enzyme activity to eliminate excessive accumulation of reactive oxygen species and increasing the content of osmotic stress substances. The above experimental results indicate that overexpression... AhSTF1 It can significantly improve the plant's tolerance to salt stress, providing important genetic resources and theoretical basis for peanut stress-resistant breeding, and has important application value for breeding new salt-tolerant crop varieties. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 For the purposes of this invention AhSTF1 The gene structure diagram.

[0021] Figure 2 For the present invention AhSTF1 MOTIF plot of the amino acid sequence encoded by the gene.

[0022] Figure 3 This is a schematic diagram of the gene overexpression vector pCAMBIA1307 of the present invention.

[0023] Figure 4 For the overexpression of this invention AhSTF1 Agarose gel electrophoresis image showing positive detection of genetically modified tobacco DNA at the DNA level.

[0024] Figure 5 For the overexpression of this invention AhSTF1Germination status and germination rate of transgenic tobacco under salt stress; where A represents the seed germination rate of WT, 11#1, 11#7, 11#10 and 11#11 tobaccos after 5, 8, 11 and 14 days of growth on MS medium plates with a NaCl concentration of 0 mM; B represents the seed germination rate of WT, 11#1, 11#7, 11#10 and 11#11 tobaccos after 5, 8, 11 and 14 days of growth on MS medium plates with a NaCl concentration of 100 mM. Seed germination rates at 5 days and 14 days; C represents the seed germination rates of WT, 11#1, 11#7, 11#10 and 11#11 after 5 days, 8 days, 11 days and 14 days respectively on MS medium plates with a NaCl concentration of 150mM; D represents the seed germination rates of WT, 11#1, 11#7, 11#10 and 11#11 after 5 days, 8 days, 11 days and 14 days respectively on MS medium plates with a NaCl concentration of 200mM. E represents the growth of WT, 11#1, 11#7, 11#10, and 11#11 tobacco varieties on MS medium plates with 0 mM NaCl for 5 days; F represents the growth of WT, 11#1, 11#7, 11#10, and 11#11 tobacco varieties on MS medium plates with 100 mM NaCl for 5 days; G represents the growth of WT, 11#1, 11#7, 11#10, and 11#11 tobacco varieties on MS medium plates with 150 mM NaCl for 5 days; H represents the growth of WT, 11#1, 11#7, 11#10, and 11#11 tobacco varieties on MS medium plates with 200 mM NaCl for 5 days; I represents... The growth of tobacco varieties WT, 11#1, 11#7, 11#10, and 11#11 after 14 days on MS medium plates with 0 mM NaCl is shown; J represents the growth of tobacco varieties WT, 11#1, 11#7, 11#10, and 11#11 after 14 days on MS medium plates with 100 mM NaCl is shown; K represents the growth of tobacco varieties WT, 11#1, 11#7, 11#10, and 11#11 after 14 days on MS medium plates with 150 mM NaCl is shown; L represents the growth of tobacco varieties WT, 11#1, 11#7, 11#10, and 11#11 after 14 days on MS medium plates with 200 mM NaCl is shown.

[0025] Figure 6 For the overexpression of this invention AhSTF1Vertical culture of transgenic tobacco under salt stress; where A represents the whole plant fresh weight (mg) of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 0 mM; B represents the whole plant fresh weight (mg) of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 100 mM; C represents the whole plant fresh weight (mg) of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants on MS medium plates with a NaCl concentration of 150 mM. Fresh weight of the whole plant after 7 days of growth, mg; D represents the fresh weight of the whole plant of WT, 11#1, 11#7, 11#10 and 11#11 tobacco after 7 days of growth on MS medium plates with a NaCl concentration of 200 mM, mg; E represents the taproot length of the plant of WT, 11#1, 11#7, 11#10 and 11#11 tobacco after 7 days of growth on MS medium plates with a NaCl concentration of 0 mM, cm; F represents the fresh weight of the whole plant of WT, 11#1, 11#7, 11#10 and 11#11 tobacco after 7 days of growth on MS medium plates with a NaCl concentration of 100 mM, mg; The taproot length of the plants after 7 days of growth (cm); G represents the taproot length of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 150mM; H represents the taproot length of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 200mM; I represents the lateral root length of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 0mM. Length, cm; J represents the lateral root length of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 100 mM, cm; K represents the lateral root length of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 150 mM, cm; L represents the lateral root length of WT, 11#1, 11#7, 11#10, and 11#11 tobacco plants after 7 days of growth on MS medium plates with a NaCl concentration of 150 mM, cm. * indicates... p <0.05, significant; ** indicates p <0.01, highly significant; *** indicates p <0.001, extremely significant; **** indicates p <0.0001, extremely significant.

[0026] Figure 7 For the overexpression of this invention AhSTF1Physiological indicators of transgenic tobacco under salt stress; where A is the result of POD activity assay; B is the result of MDA content assay; and C is the result of SP content assay. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0029] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0030] This invention is based on data obtained from the PeanutBase database. AhSTF1 Gene sequence information was obtained, and specific primers containing Xbal and Hind III restriction sites were designed using Snap gene software. Using cDNA from peanut line FZ080 as a template, the gene sequence was obtained via PCR amplification. AhSTF1 The coding sequence was obtained. After purification, the amplified product was cloned into the intermediate vector QVB3 using T4 DNA ligase to construct the recombinant vector QVB3-. AhSTF1 After double enzyme digestion verification, the pCAMBIA1307 plant expression vector carrying the MYC tag was linearized using the same restriction endonuclease.

[0031] The recombinant plasmid was introduced into Agrobacterium GV3101 competent cells via electroporation. After transformation, the bacterial culture was incubated on ice for 30 minutes, then added to antibiotic-free LB liquid medium and cultured at 28°C with shaking at 200 rpm for 2.5 hours for recovery. Subsequently, the culture was plated on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and cultured at 28°C for 48 hours to screen for positive clones.

[0032] Using an Agrobacterium-mediated genetic transformation system, target genes are integrated into the plant genome via Vir protein complex-mediated T-DNA transfer. This technology not only provides an effective tool for gene function research but also enables the direct creation of transgenic materials with specific agronomic traits, providing technical support for crop genetic improvement.

[0033] Example 1

[0034] AhSTF1 Construction and genetic transformation of recombinant overexpression vectors for genes:

[0035] use AhSTF1 A pair of specific primers were designed based on the gene coding region-specific DNA sequence to amplify the base sequence of the target gene. The PCR reaction system is shown in Table 1, and the PCR amplification procedure is shown in Table 2. AhSTF1 Gene structure diagram as shown Figure 1 As shown.

[0036] AhSTF1The base sequence of the gene is shown in SEQ ID NO.1 as follows: ATGGGTAGAGCTCCATGCTGTGAGAAACTAGGGTTGAAGAAGGGGCCTTGGACCCCAGAAGAAGATCAAATCCTCATCAATTATATCAACACAAATGGTCACAATAATTGGCGTGCCCTTCCCAAACAAGCTGGGTTATTGAGGTGTGGAAAGAGTTGCAGATTAAGATGGATAAATTATTTGAGGCCAGATATCAAACGGGGCAACTTTACCAATGAAGAAGAAGAGACAATACTCAAGTTACATGAAATGTTGGGAAACAGATGGTCAGCAATTGCAGCAAGATTGCCGGGTCGCACAGATAACGAGATTAAAAATGTTTGGCACACCCACTTGAAGAAAAGGTTGCCCCCACAAGAAAACAACAACATTAAGAGCACAAAGAAGCCACGTCCAAAACAAAAGAAGAAGTTGGAAATTACAAAAAAGTCCAACAAAACCTCATCAAAACAAAAACAAGAAAAAGAAGAAGAGGAACCCATCATCAAAATTGAGGGAAGGACAATGATGTCTCCTAATACACAGTGTTCAAGCAGTAGTAATAATAATAATAATAATGATGGTGTGTCAATGAACAATTCAAGTGGTGGTGAATCGATTAATAATGATGGTGATGGTAATAAGGATGATAATTTGGCATTGGACGAGGAGTTTTGGTCTGAAGTTTTGTCATCGGATAATTCGTATGATGAGGATGCAAGAAAATTTGAGGACATTGAATTTGGTGACTTAGATTTGTTCCACTTTCCGTTGTTATCTTCATCAGCATCAACTGTTGCATGTGATGGCATGGATTCTTGGTATGATTTTTGCACAAAATCTTTGGAATTGCCACAATTGTGA。

[0037] AhSTF1The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2, and is: MGRAPCCEKLGLKKGPWTPEEDQILINYINTNGHNNWRALPKQAGLLRCGKSCRLRWINYLRPDIKRGNFTNEEEETILKLHEMLGNRWSAIAARLPGRTDNEIKNVWHTHLKKRLPPQENNNIKSTKKPRPKQKKKLEITKKSNKTSSKQKQEKEEEEPIIKIEGRTMMSPNTQCSSSSNNNNNNDGVSMNNSSGGESINNDGDGNKDDNLALDEEFWSEVLSSDNSYDEDARKFEDIEFGDLDLFHFPLLSSSASTVACDGMDSWYDFCTKSLELPQL. AhSTF1 The MOTIF plot of the amino acid sequence of the protein expressed by the gene is shown below. Figure 2 As shown.

[0038] Amplification AhSTF1 Gene-specific primer pairs are AhSTF1- Xbal-F and AhSTF1- Hind III-R.

[0039] in, AhSTF1- The base sequence of Xbal-F is shown in SEQ ID NO.3: GACTTGAACTCGGTATCTAGAATGGGTAGAGCTCCATGC; 5'-3'.

[0040] AhSTF1- The base sequence of Hind III-R is shown in SEQ ID NO.4: GTCGACGGTATCGATAAGCTTCAATTGTGGCAATTCCAAAGA; 5'-3'.

[0041] Table 1 PCR reaction system

[0042]

[0043] Table 2 PCR amplification program

[0044]

[0045] The amplified target gene was cloned into pCAMBIA1307, which was preserved by the Crop Genetics and Breeding Research Group of Jiangxi Agricultural University, to construct the recombinant overexpression vector pCAMBIA1307- AhSTF1 The specific construction method is as follows:

[0046] The amplified target gene fragment was constructed into the intermediate vector QVB3, kindly provided by the Northeast Institute of Geography, Chinese Academy of Sciences, to create the recombinant vector QVB3-. AhSTF1 Using Xbal and Hind III restriction enzymes, a 50 μL restriction enzyme digestion system was set up in a PCR instrument to linearize the vector. The reaction conditions were 37℃ for 1.5 h. The recombinant vector was then double-digested and the target fragment was recovered by gel electrophoresis. Using the same restriction enzyme digestion system, the pCAMBIA1307 plant binary expression vector with the MYC tag was simultaneously linearized by Xbal and Hind III restriction endonucleases, and the linearized vector fragment was recovered. The linearized vector fragment was ligated to the target fragment using T4 ligase from Takara China to obtain the target recombinant plasmid. AhSTF1 Recombinant overexpression vectors for genes. A schematic diagram of the pCAMBIA1307 vector is shown below. Figure 3 As shown in Table 3, the connection reaction system is connected.

[0047] Table 3 Connection Reaction System

[0048]

[0049] Reaction conditions: 4℃ for 12 hours.

[0050] The target recombinant plasmid was transformed into E. coli, and bacteria carrying the plasmid were screened. AhSTF1 Positive clones of recombinant overexpression vectors of genes:

[0051] (1) Take 100µL of DH5α competent cells from Beijing Qingke Biotechnology Co., Ltd. that have been thawed on ice, add the target recombinant plasmid, mix gently, and let stand on ice for 30min.

[0052] (2) Heat shock in a 42℃ water bath for 60s, then quickly transfer to an ice bath and let stand for 2 minutes. Do not shake the sample during the standing process on ice, otherwise the conversion efficiency will be reduced.

[0053] (3) Add 700µL of antibiotic-free sterile LB liquid medium to the centrifuge tube, mix well, and revive at 37°C and 200rpm for 60min.

[0054] (4) According to the experimental needs, take 50µL of the liquid culture medium that has been transformed into the target recombinant plasmid after step (3) and spread it evenly on the LB solid culture medium containing kanamycin resistance. Place the plate upside down in an incubator at 37°C and incubate overnight.

[0055] (5) Filter out those with AhSTF1 Positive clones of gene recombinant overexpression vectors.

[0056] will have AhSTF1The positive bacterial culture plasmid of the gene recombinant overexpression vector was introduced into Agrobacterium tumefaciens GV3101.

[0057] will have AhSTF1 The steps for introducing the positive bacterial culture plasmid of the recombinant overexpression vector into Agrobacterium tumefaciens GV3101 are as follows: Transformation was performed using Agrobacterium tumefaciens GV3101 competent cells from Beijing Qingke Biotechnology Co., Ltd. First, 1 μL of the positive bacterial culture plasmid containing the gene recombinant overexpression vector was introduced into Agrobacterium tumefaciens GV3101. AhSTF1 The positive plasmid DNA of the recombinant overexpression vector was mixed with 100 μL of Agrobacterium GV3101 competent cells, and the bacterial culture was thoroughly mixed. Afterward, the mixture was incubated on ice for 5 min, followed by liquid nitrogen treatment for 5 min, then incubated in a 37°C water bath for 5 min, and finally cooled on ice for 5 min. Following these steps, 700 μL of antibiotic-free LB medium was added to the mixture, and the mixture was then incubated on a shaker at 28°C for 3 h. Finally, the cultures were plated on solid culture media containing the appropriate antibiotics for screening.

[0058] Example 2

[0059] AhSTF1 Creation and identification of transgenic tobacco materials with overexpressed genes

[0060] The specific steps for obtaining transgenic tobacco materials through overexpression are as follows:

[0061] Genetic transformation experiments of recombinant overexpression vectors were conducted using the Agrobacterium-mediated leaf disc method.

[0062] (1) Tobacco pre-culture

[0063] Select healthy, green, and thick leaves from 5-leaf-stage Nicotiana benthamiana. Rinse them 6 times with sterile water, air dry them, disinfect them with 70% ethanol for 1 minute, sterilize them with 25% sodium hypochlorite for 3 minutes, rinse them 8 times with sterile water, gently place them on sterilized filter paper, and air dry them completely. Then, cut the leaves into 1cm pieces with a sterilized knife and place them on pre-cultured MS medium for 3 days.

[0064] (2) Co-cultivation

[0065] Add 200 μL of 100 μg / mL AS to 100 mL MS, and resuspend Agrobacterium in the liquid for 30 min. After the pre-culture is completed, put the tobacco leaves into the resuspended Agrobacterium liquid, shake gently for 10 min, and then gently place them on sterilized filter paper. After confirming that the water is completely absorbed, put them into co-culture medium and incubate in the dark for 3 days.

[0066] (3) Screening and cultivation

[0067] After co-culturing, remove the leaves, rinse them 6 times with sterile water, and then use sterile filter paper to absorb excess water. Then transfer the leaves to the screening medium and repeat the transfer every half month.

[0068] (4) Rooting culture

[0069] Observe the rooting of the leaves daily. When the bud grows to about 1 cm, remove it from the culture medium, cut off the callus tissue at the bottom, and transfer it to the rooting medium. After the bud has rooted, remove it with tweezers, rinse it 6 times with sterile water, and then transfer it to the soil for cultivation. Place the seedling in the dark for 3 days to allow it to fully adapt to the environment before placing it under normal light conditions.

[0070] DNA was extracted from transgenic tobacco plants using the CTAB method and identified by agarose gel electrophoresis. The identification results are as follows: Figure 4 As shown, tobaccos 11#1, 11#7, 11#10, and 11#11 were identified as positive tobaccos.

[0071] The steps to obtain transgenic material that transiently expresses peanut leaf expression are as follows:

[0072] (1) First absorb the contents AhSTF1 Two mL of Agrobacterium, containing the recombinant overexpression vector pCAMBIA1307-MYC and the control empty vector, were added to LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan. The medium was incubated at 28°C and 220 rpm for 20 h.

[0073] (2) Transfer the cultured bacterial solution to 30 mL of LB liquid medium, add 50 mg / L rif and 50 mg / L kan, and continue to culture at 28 °C and 220 rpm until OD. 600 The value reached 0.6.

[0074] (3) Centrifuge the Agrobacterium tumefaciens culture at 12,000 rpm for 10 min, adjust the pH to 6.0 with induction buffer containing 10 mM MgCl2, 10 mM MMEs, and 150 μM acetylsyl syringone, resuspend, and continue until OD is reached. 600 The value reached 0.6.

[0075] (4) Let the bacterial suspension stand at 25°C for 3 hours.

[0076] (5) Selection of peanut varieties: The leaves of peanut variety Changhua 06 from the Peanut Genetics and Breeding Laboratory of Jiangxi Agricultural University were selected for injection treatment. The injection point was located on the back of the peanut leaf.

[0077] (6) The injected leaves were cultured in the dark at 25°C for 3 days and then used for gene expression analysis. The experimental setup included 3 replicates, each of which included 3 to 4 leaves, and 3 biological replicates were performed. The obtained transiently transformed leaf materials were then subjected to salt stress treatment.

[0078] Example 3

[0079] The specific steps for phenotypic identification of transgenic tobacco under salt stress are as follows:

[0080] Table 4. Composition of salt screening culture medium

[0081]

[0082] Note: " / " indicates that this item is not present.

[0083] (1) 120 WT and 120 transgenic tobacco seeds collected in the same batch were sterilized and then sown separately in MS medium with different NaCl concentrations for salt selection, as shown in Table 4. The NaCl concentrations were 0 mM, 100 mM, 150 mM and 200 mM, respectively. During the germination period, the number of germinating tobacco seeds under different NaCl concentrations was counted daily, and the germination potential and germination rate were calculated. The results are shown in Table 4. Figure 5 Wild-type tobacco (WT) and transgenic tobacco varieties 11#1, 11#7, 11#10, and 11#11 were planted in MS solid medium with different salt concentrations, and the germination rate and germination potential of tobacco seeds were recorded after 5, 8, 11, and 14 days. The results showed that under salt stress, the germination rates of transgenic tobacco varieties 11#1, 11#7, 11#10, and 11#11 were significantly higher than those of the wild type (WT).

[0084] Gp germination rate (%) = (Number of germinated seeds on day 14 / Total number of seeds) × 100%.

[0085] Gv germination potential (%) = (number of germinated seeds on day 7 / total number of seeds) × 100%.

[0086] (2) Tobacco seeds from WT and transgenic plants were sown on MS medium for germination. After germination, seedlings with uniform growth were selected and transplanted to media containing different concentrations of NaCl (0 mM, 100 mM, 150 mM, and 200 mM). The seedlings were vertically cultured at 25°C using a 16-hour light-8-hour dark alternation pattern for seven days, after which the growth of leaves and roots was observed. The fresh weight of the whole plant and the fresh weight of the aboveground parts were weighed for each line. The length of the taproot was measured with a ruler, and changes in root length and other root conditions were analyzed. The results are as follows: Figure 6 As shown, under NaCl stress, especially under 100 mM NaCl stress, compared with wild-type WT, AhSTF1 The transgenic lines 11#1, 11#7, and 11#10 exhibited more significant taproot elongation and lateral root development. A well-developed root system helps plants absorb water and nutrients more effectively under stress conditions, which is a direct phenotypic characteristic of enhanced salt tolerance in transgenic plants.

[0087] Phenotypic observations revealed that when the NaCl concentration was 100 mM, the transgenic tobacco showed significantly greater root elongation and lateral root growth compared to the wild type. Further investigation is needed... AhSTF1 The salt tolerance mechanism of the gene was investigated, and a series of key physiological indicators were further measured under 100 mM NaCl stress. The results of relevant physiological indicators of the tobacco transgenic material under salt stress are as follows: Figure 7 As shown, CK represents the physiological indicators of tobacco measured at a concentration of 0 mM NaCl. The specific measurement steps are as follows:

[0088] (1) Determination of peroxidase, i.e., POD activity

[0089] Preparation of the reaction mixture: Add 0.076 mL of guaiacol stock solution and 0.112 mL of 30% H2O2 to 200 mL of 0.2 M pH 6.0 PBS. Take 50 μL of crude enzyme solution and add 3 mL of the reaction mixture. Measure the OD value at 470 nm, reading once every 1 minute, repeating three times. The change in OD per minute represents the enzyme activity. The formula for calculating POD enzyme activity is as follows:

[0090] POD enzyme activity (U·g⁻¹ FW) = .

[0091] △A470: Absorbance change during reaction time. t: Reaction time, min. Vt: Total volume of sample extract, mL. Vs: Sample volume taken during measurement, mL. FW: Fresh weight of sample, g. One enzyme activity unit (U) is defined as a decrease of 0.01 in A470 within 1 min.

[0092] The results are as follows Figure 7 As shown in Figure A, compared to WT, AhSTF1 The activity of POD in transgenic tobacco plants was increased. POD is a key enzyme for scavenging reactive oxygen species (ROS) in the body. Under salt stress, the POD activity of transgenic plants was significantly higher than that of wild-type plants, indicating that they have a stronger ability to scavenge ROS and can effectively reduce oxidative damage to cell membranes and functional molecules.

[0093] (2) Determination of malondialdehyde (MDA)

[0094] Weigh 0.5g of tobacco leaves, add 5mL of 5% TCA (total carbon dioxide) and grind into a homogenate. Make up to 10mL and centrifuge at 4000rpm for 10min using a low-temperature centrifuge. Take 2mL of the supernatant, mix thoroughly, add 2mL of 0.6% TBA (total carbon dioxide) and mix well. Boil in a water bath for 15min, then centrifuge at 12000rpm for 15min. Take 800μL of the supernatant and measure the absorbance at 450nm, 532nm, and 600nm. The MDA content can be calculated using the following formula:

[0095] MDA content (nmol·g-1 FW) = [6.45×(A532-A600)-0.56×A450]×Vt / Vs / FW.

[0096] Vt: Total volume of sample extract, mL. Vs: Sampling volume during measurement, mL. FW: Fresh weight of sample, g.

[0097] The results are as follows Figure 7 As shown in Figure B, the decrease in MDA content indicates... AhSTF1 MDA (membrane osmotic substances) participates in regulating the accumulation of osmotic substances in plants to adapt to salt stress. MDA is the final product of membrane lipid peroxidation, and its content is an indicator of the degree of cell membrane damage. The significant decrease in MDA content in transgenic plants directly proves... AhSTF1 The gene mitigated membrane system damage caused by salt stress, which corroborates the result of increased POD activity.

[0098] (3) Determination of soluble protein, i.e., SP content

[0099] Preparation of a standard curve: Weigh 25 mg of bovine serum albumin and prepare a standard stock solution with a concentration of 100 μg / mL. Dilute the protein standard solution with the stock solution at concentration gradients of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Take 0.5 g of plant tissue, add pre-cooled phosphate buffer (pH 7.8), homogenize, centrifuge at 12000 × g for 20 min at 4℃, and collect the supernatant as the enzyme solution. The protein calculation formula is obtained from the standard curve as follows:

[0100] SP content (mg·g⁻¹FW) = .

[0101] C: Amount of soluble protein in the sample test tube, μg. Vt: Total volume of sample extract, mL. Vs: Sampling volume during testing, mL. FW: Fresh weight of the sample, g.

[0102] The results are as follows Figure 7 As shown in C, compared to WT, AhSTF1The activity of SP (Soluble Protein) in transgenic tobacco plants is increased. Soluble proteins are important osmotic regulators. The accumulation of SP in transgenic plants helps maintain osmotic balance in cells, prevents water loss, and may also protect the activity of intracellular enzymes, thereby ensuring normal physiological function of cells under stress.

[0103] In summary, the experiments of this invention show that AhSTF1 The gene works synergistically to alleviate the damage caused by salt stress by increasing POD activity to activate the plant's antioxidant system, reducing MDA content to protect cell membrane integrity, and accumulating soluble proteins to enhance osmotic regulation. This ultimately manifests phenotypically as higher germination rates, more developed root systems, and better growth. These results fully demonstrate... AhSTF1 Genes play a crucial and positive role in improving plant salt tolerance, providing an important theoretical basis for their application in crop stress resistance breeding.

[0104] It should be noted that the numerical ranges mentioned in the claims of this invention should be interpreted as including the two endpoints of the range and every specific value between the endpoints. For the sake of brevity, this specification only lists some preferred embodiments as examples.

[0105] It should be understood that although this specification has described the technical solution of the present invention in detail through specific embodiments, those skilled in the art, based on their understanding of the core innovative points of the present invention, can make appropriate adjustments, modifications, or equivalent substitutions to the described embodiments. Therefore, the scope of protection of the claims of the present invention should cover all embodiments that conform to the essential technical features of the present invention, including but not limited to the preferred embodiments described in the specification, as well as various reasonable variations and equivalent embodiments that those skilled in the art can conceive of based on the basic technical concept of the present invention.

[0106] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. AhSTF1 Use of a gene in modulating salt tolerance in plants, characterized in that, The AhSTF1 The base sequence of the gene is shown in SEQ ID NO.1; the gene is generated in the plant through genetic engineering methods. AhSTF1 Gene overexpression to improve plant salt tolerance; The plant is tobacco or peanut.

2. Use according to claim 1, characterized in that, The AhSTF1 The amino acid sequence of the protein of the gene expression is shown in SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, The genetic engineering method is: using the AhSTF1 The gene constructs a recombinant overexpression vector, and the recombinant overexpression vector is transformed into plants to improve the salt tolerance of the plants.

4. Use according to claim 3, characterized in that, The method for constructing the recombinant overexpression vector is to amplify the gene using specific primers, recover the PCR product, and connect the PCR product with a pCAMBIA1307 vector to obtain the recombinant overexpression vector. AhSTF1 gene, recover the PCR product, and connect the PCR product with a pCAMBIA1307 vector to obtain the recombinant overexpression vector.

5. Use according to claim 4, characterized in that, The base sequence of the specific primer pair is shown in SEQ ID NO. 3 and SEQ ID NO.

4.

6. Use according to claim 3, characterized in that, The method for transforming the plant is Agrobacterium transformation.

7. Use according to claim 6, characterized in that, The Agrobacterium is GV3101.

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