Application of peanut exocrine peptide AhSIP1 in improving salt tolerance of plants

Abstract

The invention discloses application of peanut exocrine peptide AhSIP1 in improving salt tolerance of plants, and belongs to the technical field of molecular breeding. The nucleotide sequence of the peanut exocrine peptide AhSIP1 is as shown in SEQ ID NO: 1, and the sequence of amino acid coded by the peanut exocrine peptide AhSIP1 is as shown in SEQ ID NO: 2. Experiments prove that when the AhSIP1 is mature and applied in vitro, peanut seed germination under salt stress can be remarkably promoted, 35S: AhSIP1 transgenic apple callus grows faster (fresh weight is larger) under salt stress, and it is indicated that the peanut exocrine small peptide AhSIP1 positively regulates plant salt tolerance. Functional identification of the peanut exocrine small peptide AhSIP1 provides theoretical basis and technical guidance for seedling emergence, full seedling and high-yield cultivation of saline-alkali soil plants, and has important application value in salt-tolerant crop breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to the application of peanut exocrine peptide AhSIP1 in improving plant salt tolerance. Background Technology

[0002] Saline-alkali land is an important existing and potential agricultural resource, but its current rate of protective development and utilization is low. The rational development and utilization of saline-alkali land is crucial for increasing the reserve of arable land resources. The numerous defects of saline-alkali land, such as high salinity, water scarcity, and poor soil quality, severely restrict the planting of most crops, resulting in low farmer enthusiasm and the gradual abandonment of much saline-alkali land. Peanuts, a nitrogen-fixing crop with root nodules, are tolerant of poor soil and drought, and relatively salt-alkali conditions, making them one of the most suitable economic oil crops for planting in saline-alkali areas. The seed germination period is the most sensitive stage to salinity; "slow germination and low emergence rate" have long been bottlenecks restricting large-scale peanut production in saline-alkali land. How to "promote germination, ensure full seedling emergence, and cultivate strong seedlings" has become a key objective for agricultural researchers.

[0003] Improving peanut resistance through stress-resistance breeding is currently the main approach to increasing the germination rate of peanuts in saline-alkali land. Stress-resistance breeding mainly includes traditional conventional breeding, mutation breeding, and genetic transformation breeding. Traditional conventional hybridization breeding has a long cycle and low efficiency. Although mutation breeding significantly increases the mutation frequency, the frequency of beneficial mutations is low, the direction of mutation is difficult to control, and many chemical mutagens are highly toxic and have residual effects. Genetic transformation improves crop quality with targeted efficiency, but peanut transgenic technology is currently immature, with low success rates and long cycles.

[0004] In recent years, a large number of stress-resistant peptides have been screened and functionally identified, especially those that enhance plant salt and alkali tolerance. Therefore, identifying key peptides that regulate salt stress and promote seed germination during peanut seed germination is crucial. This research can provide a theoretical basis and technical support for improving the germination rate and production capacity of peanuts in saline-alkali land through efficient application of exogenous peptides in cultivation practices (such as seed coating technology). Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide the application of peanut exocrine peptide AhSIP1 in improving plant salt tolerance.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of peanut exocrine peptide AhSIP1 in improving plant salt tolerance, wherein the amino acid sequence encoded by the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:2.

[0008] Based on the above scheme, the nucleic acid sequence of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:1.

[0009] Based on the above approach, salt tolerance of plants can be improved by overexpressing peanut exocrine peptide AhSIP1 or by applying mature peptide fragments of peanut exocrine peptide AhSIP1 exogenously.

[0010] Based on the above scheme, the amino acid sequence of the mature peptide segment of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:5.

[0011] Based on the above scheme, the plant is either peanut or apple.

[0012] A method for improving plant salt tolerance involves overexpressing peanut exocrine peptide AhSIP1 in plants or applying a mature peptide fragment of peanut exocrine peptide AhSIP1 exogenously to improve plant salt tolerance; the amino acid sequence encoded by said peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:2.

[0013] Based on the above scheme, the nucleic acid sequence of the AhSIP1 gene was constructed into an expression vector, transformed into plants, and AhSIP1 was overexpressed in plants. The nucleic acid sequence of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:1.

[0014] Based on the above scheme, the amino acid sequence of the mature peptide segment of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:5.

[0015] Based on the above scheme, the mature peptide concentration of the peanut exocrine peptide AhSIP1 is 5-20 μM.

[0016] Application of peanut exocrine peptide AhSIP1 in the preparation of formulations to improve plant salt tolerance, wherein the amino acid sequence encoded by peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:2.

[0017] An agent for improving plant salt tolerance, the active ingredient being a mature peptide fragment of peanut exocrine peptide AhSIP1, the amino acid sequence of which is shown in SEQ ID NO:5.

[0018] A method to improve the germination rate of peanuts under salt stress involves exogenously applying 5-20 μM of the mature peptide fragment of the peanut exocrine peptide AhSIP1.

[0019] A method to improve the salt tolerance of apple callus involves constructing a plant expression vector from the nucleic acid sequence of the peanut exocrine peptide AhSIP1, transforming it into apple callus, and overexpressing it in the apple callus.

[0020] Advantages of the technical solution of this invention

[0021] This invention cloned the peanut exocrine peptide AhSIP1 gene from peanuts. The full-length CDS sequence of this gene is 462 bp, encoding a small protein of 143 amino acids. SignalP4.1 software prediction showed a signal peptide cleavage site between alanine and aspartic acid at the N-terminus of AhSIP1, suggesting that it may be an exocrine peptide.

[0022] A genetic transformation method using peanut hairy root systems was employed to obtain 35S:AhSIP1 transgenic peanut hairy roots. Liquid culture medium was collected, and proteins were extracted. Larger protein molecules were removed using molecular sieves. MALDI-TOF-TOF mass spectrometry identified the mature form of the exocrine peptide AhSIP1 as PYLPSKCYGTEATEFPSSNLFAAAGDGIWDNGAACGRQY (40-78 aa), containing 39 amino acids and approximately 4.3 kDa, a typical small exocrine peptide. Experimental results showed that in vitro application of mature AhSIP1 significantly promoted peanut seed germination under salt stress, and 35S:AhSIP1 transgenic apple callus grew faster (with greater fresh weight) under salt stress, indicating that the peanut exocrine peptide AhSIP1 regulates plant salt tolerance. This functional identification of the peanut exocrine peptide AhSIP1 provides a theoretical basis and technical guidance for seedling emergence, full emergence, and high-yield cultivation of plants in saline-alkali land, and has important application value in salt-tolerant crop breeding. Attached Figure Description

[0023] Figure 1 To identify the salt-induced gene AhSIP1 in peanut germination (where A is differentially expressed gene heatmap cluster analysis; B is the fold increase of AhSIP1 induced by salt by qRT-PCR; C is the expression level of AhSIP1 in different peanut varieties by qRT-PCR; and D is the prediction of AhSIP1 signal peptide and cleavage site by SignalP4.1 software).

[0024] Figure 2 Electrophoresis diagrams of AhSIP1 expression vectors were constructed (where A is the PCR amplification result of AhSIP1 gene; B is the PCR electrophoresis result of pMD19-T simple-AhSIP1 bacterial culture; C is the enzyme digestion electrophoresis identification of pCAMBIA1300-AhSIP1 and pBI121-AhSIP1 expression vectors).

[0025] Figure 3 Expression, purification, and identification of mature AhSIP1;

[0026] Figure 4Germination rates of peanut seeds treated with different concentrations of AhSIP1 peptide under different salt stress treatments (where A represents the germination rate of peanut seeds treated with different concentrations of AhSIP1 peptide under normal conditions; B represents the germination rate of peanut seeds treated with different concentrations of AhSIP1 peptide under 50 mM NaCl treatment; C represents the germination rate of peanut seeds treated with different concentrations of AhSIP1 peptide under 100 mM NaCl treatment; D represents the germination rate of peanut seeds treated with different concentrations of AhSIP1 peptide under 150 mM NaCl treatment; and E represents the germination rate of peanut seeds treated with different concentrations of AhSIP1 peptide under 200 mM NaCl treatment).

[0027] Figure 5 Growth status of apple callus transgenic with AhSIP1 gene under salt stress (where A represents the growth status of different apple callus in solid culture medium with or without NaCl; B represents the fresh weight of apple callus). Detailed Implementation

[0028] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0030] In the following embodiments:

[0031] The components of Agrobacterium rhizogenes YEP medium are: 10g tryptone, 10g yeast extract, 5g sodium chloride (NaCl), 1000mL distilled water, pH adjusted to 7.0, and 15g agar powder added for solid medium. Sterilize by autoclaving at 121℃ for 20min.

[0032] Composition of MS medium for induction and propagation of hairy roots and apple callus: 25 mL 20×MS macro (3.8 g KNO3, 3.3 g NH4NO3, 0.34 g KH2PO4, 100 mL distilled water), 5 mL 100×MS micro (0.223 g MnSO4·4H2O, 0.086 g ZnSO4·7H2O, 0.062 g H3BO3, 0.0083 g KI, 2.5 mg Na2MoO4·2H2O, 0.25 mg CuSO4·5H2O, 0.25 mg CoCl2·6H2O, 100 mL distilled water), 5 mL 100× iron salt (0.374 g EDTA-Na2, 0.278 g FeSO4·7H2O), 10 mL 100× VB (inositol 1 g, VB1 0.1 g, VB3 0.1 g). 0.01g, VB6 0.01g, add 100mL of water), 5mL 100×Ca 2+ 5mL 100×Mg 2+ Add 15g sucrose and 1000mL distilled water to adjust the pH to 5.8. For solid culture media, add 20g agar powder. Autoclave at 121℃ for 20min.

[0033] Tifrunner peanuts are bred and preserved by the Shandong Peanut Research Institute.

[0034] The pMD19-T simple cloning vector was purchased from Takara, 3271.

[0035] The Escherichia coli DH-5α strain was purchased from Solarbio, C1100.

[0036] The empty expression vectors pCAMBIA1300 and pBI121 were preserved by the Shandong Peanut Research Institute.

[0037] Agrobacterium rhizogenes R1601 was preserved by the Shandong Peanut Research Institute.

[0038] Agrobacterium rhizogenes LBA4404 was preserved by the Shandong Peanut Research Institute.

[0039] The callus tissue of the embryonic embryo of 'Wanglin' ​​apple (Malus × domestica 'Orin') was donated by Zheng Chengchao's research group from the College of Life Sciences, Shandong Agricultural University.

[0040] Acquisition of the AhSIP1 gene

[0041] Salt-tolerant peanut HYST seeds were germinated in the dark for 5 days in 0 and 150 mM NaCl petri dishes. RNA-seq analysis was performed to screen for salt-induced differentially expressed genes (fold change ≥ 2, P-value ≤ 0.05), with a focus on genes with large upregulation. Among them, the gene Araip.Z3YEK (AhSIP1) with unknown function showed the largest response amplitude. Figure 1 China A, Figure 1 (B). It was first named *Arachis hypogaea* Salt Induced Peptide 1 (AhSIP1). The expression of this gene was tested in 10 randomly selected peanut varieties (5 salt-tolerant and 5 salt-sensitive). It was found that its expression level was generally higher in salt-tolerant varieties than in salt-sensitive varieties. Figure 1 The presence of C in the middle suggests that the expression level of this gene is closely related to the salt tolerance of peanuts.

[0042] Those skilled in the art can determine the basic features of this invention, and various changes and modifications can be made to this invention without departing from its spirit and scope to achieve similar purposes. For example, vector construction can utilize the enzyme digestion and ligation method of this invention, or the gateway system construction method; processes such as gel recovery, plasmid extraction, RNA extraction, and cDNA reverse transcription can also be performed using kits from other companies or other methods; the screening method for transgenic peanut hairy root positive lines can also be changed, and qRT-PCR or RT-PCR can be used; apple callus can be selected from 'Wanglin' ​​apple callus, or other apple callus can be selected.

[0043] Example 1

[0044] Cloning of the AhSIP1 gene

[0045] Total RNA was extracted from Tifrunner (propagated and preserved by Shandong Peanut Research Institute) seedlings grown hydroponically for approximately 15 days. The resulting cDNA was reverse transcribed into a template. Using AhSIP1 gene-specific primers and novizan high-fidelity enzyme (2×Phanta MaxMaster Mix, p515), PCR amplification was performed to obtain the AhSIP1 gene CDS sequence, as shown in SEQ ID NO:1, and its encoded amino acid sequence, as shown in SEQ ID NO:2. The full-length AhSIP1 gene CDS sequence is 462 bp, encoding a small protein of 143 amino acids. SignalP4.1 software prediction showed a signal peptide cleavage site between alanine and aspartic acid at the N-terminus of AhSIP1. Figure 1 (D) is speculated to be an exocrine peptide.

[0046] SEQ ID NO:1(5'→3')

[0047] ATGTCATCATCATTAGCAACACTCATTCTATCATCGTTATTCATAGTTTCCTTCATAGTTCTCCACCTTTTCTTCCCCTGCAGAGCCGATGTTGGAACCGCTGCTAGTTACTCCCCCATATTTACCTTCGAAGTGTTATGGGACGGAAGCCACAGAGTTCCCATCGAGCAATCTGTTTTGCGGCGGCGGGAGATGGAATATGGGACAATGGGGCAGCTTGCGGTAGGCAG TATCTGGTGAGATGCATCAGTGCAGAGCAACCAAGGACCTGCATTCCCGACCAAAGCATTCAGATCAAGATCGTTGATTACGCTGCCACCGCTGTTTCGGCCGCCTCGGCTAGCGGCACTACCTTGGTGTTGTCCGACAAGGCATTCGGCAGTATTGCCAATACAACTGCCATCTTAATCAACATAGAATTTCAACAGTAATAATTAATTAATTAATTAATTAAACTCTGA

[0048] SEQ ID NO:2

[0049] MSSSLATLILSSLFIVSFIVLHLFFPCRADVGTAASYSPPYLPSKCYGTEATEFPSSNLFAAAGDGIWDNGAACGRQYLVRCISAEQPRTCIPDQSIQIKIVDYAATAVSAASASGTTLVLSDKAFGSIANTTAILINIEFQQ

[0050] The AhSIP1 gene-specific primer sequences are as follows:

[0051] upstream primer 5'- GGATCC ATGTCATCATCATTAGCAACACTC-3' (SEQ ID NO: 3);

[0052] Downstream primer 5'- GAGCTC TCAGAGTTTAATTAATTAATTAATTAATTATTACTG-3' (SEQ ID NO: 4).

[0053] The underlined areas represent enzyme cleavage sites. The upstream primer's cleavage site is BamHI, and the downstream primer's cleavage site is SacI.

[0054] The PCR reaction system consisted of: 12.5 μL of 2×Phanta Max Master Mix, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of cDNA template, and water to a final volume of 25 μL.

[0055] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 25 s, 50℃ annealing for 25 s, 72℃ extension for 20 s, 35 cycles; 72℃ final extension for 5 min.

[0056] Example 2

[0057] Construction of AhSIP1 gene cloning vector, expression vector, and recombinant bacteria

[0058] (1) Cloning vector

[0059] After the PCR reaction in Example 1 was completed, agarose gel electrophoresis was performed, and the target band was observed as shown in the figure. Figure 2 As shown in Figure A, the gel was then cut and the gel was recovered according to the steps of the Tiangen Ordinary Agarose Gel DNA Recovery Kit (DP209).

[0060] After gel recovery of PCR products and overnight ligation with pMD19-T simple (Takara, 3271) vector, the cells were transformed into Escherichia coli DH-5α strain (Solepro, C1100) by heat shock and grown overnight on LB plates containing ampicillin (50 μg / mL).

[0061] Select a single white colony for colony PCR ( Figure 2 (B) Select positive colonies and transfer them to 5 mL shake tubes for further culture.

[0062] Plasmid DNA was extracted using the Tiangen Plasmid Mini-Prep Kit (DP106); sequencing was performed by Sangon Biotech Co., Ltd., and the correctly sequenced cloning vector pMD19-T simple-AhSIP1 was successfully constructed.

[0063] (2) Expression carrier

[0064] The pMD19-T simple-AhSIP1 cloning vector and the empty expression vectors pCAMBIA1300 and pBI121 (containing 35S) were digested with BamHI and SacI (preserved by Shandong Peanut Research Institute). The DNA was ligated using T4 DNA ligase (Thermo, EL0014) for gel extraction and recovery. The mixture was then transformed into DH-5α strain using the heat shock method and grown overnight on LB agar plates containing kanamycin (50 μg / mL). Positive colonies were selected by colony PCR, and plasmids were extracted and digested for identification. Figure 2As shown in Figure C, two overexpression vectors of 35S:AhSIP1 (pCAMBIA1300-AhSIP1 and pBI121-AhSIP1) were constructed.

[0065] (3) Obtaining recombinant bacteria

[0066] 2.0 μL of pCAMBIA1300-AhSIP1 plasmid was transformed into Agrobacterium rhizogenes R1601 (preserved by Shandong Peanut Research Institute) using the freeze-thaw method. Positive colonies were selected by colony PCR and amplified to obtain recombinant bacteria AhSIP1-R1601.

[0067] 2.0 μL of pBI121-AhSIP1 plasmid was transformed into Agrobacterium rhizogenes LBA4404 (preserved by Shandong Peanut Research Institute) using the freeze-thaw method. Positive colonies were selected by colony PCR and amplified to obtain recombinant bacteria AhSIP1-LBA4404.

[0068] Example 3

[0069] Preparation of AhSIP1 gene-transgenic peanut hairy roots

[0070] The method for inducing AhSIP1 gene-transformed peanut hairy roots was performed according to the authorized invention patent (Application of peanut hairy root lines in improving peanut's tolerance to low nitrogen and high salt ZL202010312520.1). The specific steps are as follows:

[0071] (1) When the Luhua 11 peanut has grown to the stage of 3-6 compound leaves, select 2-4 compound leaves from the bottom up as experimental materials. Wash with sterile water, soak in 4% sodium hypochlorite solution for 2 minutes, wash gently with sterile water 3-4 times, soak in sterile water for 10 minutes, and dry for later use;

[0072] (2) Agrobacterium AhSIP1-R1601 stored at -80℃ was inoculated into 100 mL of YEP liquid medium containing kanamycin (100 μg / mL) and hygromycin (200 μg / mL) for activation, and cultured at 28℃ with shaking at 180 rpm for 18 h. The next day, 100 μL of the bacterial culture was inoculated into 100 mL of fresh YEP liquid medium and cultured at 28℃ with shaking. This activation was repeated 2-3 times until the OD600 value reached 1.5 for later use. Agrobacterium pCAMBIA1300-R1601 carrying the empty vector was used as a negative control, and the procedure was the same as above.

[0073] (3) Cut the peanut leaves disinfected in step (1) into small pieces of about 1 cm, make 2-3 scratches on the leaves, and immerse them in the AhSIP1-R1601 and pCAMBIA1300-R1601 bacterial solutions prepared in step (2) for 5 min each. During this time, gently shake to ensure that the leaf surface is fully covered with bacterial solution. Remove the leaves, blot dry with sterile filter paper, and spread them evenly on the surface of MS solid medium, about 8-12 peanut leaves per plate. After 24 h, transfer the leaves to MS solid medium supplemented with 100 mg / L cefotaxime sodium (to inhibit the growth of Agrobacterium rhizogenes). After 24 h, transfer them to MS solid medium again. Repeat the transfer between the two mediums until Agrobacterium no longer grows. Then culture them on MS solid medium containing kanamycin to induce differentiation and produce transgenic AhSIP1 peanut hairy roots and control hairy roots.

[0074] Example 4

[0075] Preparation of mature AhSIP1 peptide

[0076] Peanut hairy roots longer than 2 cm transgenic with the AhSIP1 gene, prepared according to the method in Example 3, were transferred to MS solid medium and subcultured for 2 weeks. RNA was extracted from a portion of the hairy roots and reverse transcribed into cDNA. The reverse-transcribed cDNA was diluted 25-fold and used as a template for RT-PCR. RT-PCR amplification was performed using primers for AhSIP1 and the internal reference gene AhActin11, respectively. Hairy roots induced by Agrobacterium tumefaciens transgenic with the empty vector pCAMBIA1300-R1601 were used as a control. The AhSIP1 expression level in the positive transgenic peanut hairy root lines was significantly higher than that in the control. The transgenic line with a high AhSIP1 expression level (35S:AhSIP1) was selected for the preparation of the mature AhSIP1 peptide.

[0077] The specific steps are as follows:

[0078] The selected positive AhSIP1 gene-transformed peanut hairy root branches were transferred to MS liquid medium and subcultured at 23-26℃ and 160 rpm for 4 weeks. The liquid medium was collected, and proteins were extracted using a plant protein extraction kit (Solarbio BC3720). Protein electrophoresis was performed, and after gel excision, large molecular weight contaminants were removed using molecular sieves. MALDI-TOF-TOF mass spectrometry identified the mature AhSIP1 peptide as a polypeptide containing 39 amino acids with a molecular weight of approximately 4.3 kDa. Figure 3 The specific sequence is: PYLPSKCYGTEATEFPSSNLFAAAGDGIWDNGAACGRQY (SEQ ID NO:5).

[0079] The mature form of AhSIP1 was synthesized by Sangon Biotech Co., Ltd. using the mature form chemical synthesis method for future use.

[0080] Example 5

[0081] Effects of exogenous application of AhSIP1 mature peptide on peanut germination rate under salt stress

[0082] The synthesized AhSIP1 mature peptide powder was dissolved in DMSO to prepare a 1mM stock solution, which was then diluted with ddH2O to prepare four different concentrations of AhSIP1 mature peptide: 5μM, 10μM, 15μM, and 20μM.

[0083] 150mm petri dishes were selected and lined with two layers of filter paper. Fifteen Tifrunner peanut seeds were evenly placed in each dish. In the control group, 5mL of ddH2O with or without different concentrations of mature AhSIP1 peptide was added to the filter paper using a pipette. In the salt stress treatment group, 5mL of 50mM, 100mM, 150mM, and 200mM NaCl aqueous solutions with or without different concentrations of mature AhSIP1 peptide were added to the filter paper. The seeds were placed in a shaded climate chamber for germination. Germination rate was recorded every 12 hours from the date of sowing. The ddH2O or NaCl aqueous solution with or without mature peptide was replaced every 2 days until 10 days after sowing, and the germination rate was calculated (germination was defined as a white tip appearing 3mm). All experiments were performed in triplicate.

[0084] Germination rate (%) = (Number of germinated seeds / Total number of peanut seeds tested) × 100%.

[0085] The results are as follows Figure 4 As shown. Under normal conditions, Tifrunner peanut seeds begin to germinate on day 1, with rapid germination rates from day 2.5 to 3.5. External application of different concentrations of AhSIP1 mature peptide had no significant effect on the germination rate of peanut seeds. Figure 4 (A) Different concentrations of salt stress significantly inhibited peanut seed germination, with the germination rate decreasing as the salt concentration increased. Under 50 mM NaCl treatment, peanut seeds grew slowly in the first 2 days, and the germination rate increased from the 4th to the 6th day. However, exogenous application of AhSIP1 accelerated peanut seed germination, shortened the germination cycle, and increased the germination rate. Although all treatments eventually reached 100% germination rate, the 15 μM AhSIP1 treatment only required 7.5 days to reach 100% germination rate, while the control required 9 days. Figure 4 (B); Applying AhSIP1 externally under 100mM NaCl and 50mM NaCl yielded similar results: peanut seeds germinated earlier and the germination rate accelerated, but the seed germination rate was 100% for both the control and the standard. Figure 4(C). Under 150 mM NaCl treatment, the exogenous application of 5 μM mature peptide had no significant effect on the germination rate and germination rate of peanut seeds. With increasing AhSIP1 mature peptide concentration, the peanut germination rate continuously increased, but gradually reached saturation. The effects of 20 μM (germination rate 90.1%) and 10 μM (germination rate 88.8%) mature peptides were basically the same, both significantly higher than the control (germination rate 78.1%), but lower than the 15 μM mature peptide treatment (germination rate 95.4%). 15 μM is the optimal concentration for exogenous application of AhSIP1 mature peptide, which can significantly improve the germination rate of peanuts under salt stress (150 mM NaCl). Figure 4 (D). 200 mM NaCl showed the highest inhibitory effect on seed germination rate. Peanut seeds grew slowly in the first 3.5 days, and the germination rate increased rapidly from day 4.5 to 6.5, but the final germination rate was only 64.7%. External application of AhSIP1 mature peptide increased the germination rate of peanut seeds in all cases. Among them, 15 μM AhSIP1 mature peptide had the most significant effect on the germination rate of peanut seeds, increasing it by 23.50% compared to the control. The other three concentrations of mature peptide had basically the same effect on the germination rate of peanut seeds. Figure 4 (E). The above results indicate that exogenous application of AhSIP1 mature peptide can improve the germination rate and germination rate of peanut seeds. Among all concentrations, the exogenous application of 15 μM AhSIP1 mature peptide has the most significant effect on the germination rate of peanut seeds, and the exogenous application of AhSIP1 mature peptide at 150 mM NaCl has the most obvious effect on the germination rate of peanut seeds.

[0086] Example 6

[0087] Obtaining AhSIP1 gene-transferred apple callus

[0088] 1. Agrobacterium AhSIP1-LBA4404 stored at -80℃ was inoculated into 100 mL of YEP liquid medium containing kanamycin (100 μg / mL) and hygromycin (200 μg / mL) for activation, and cultured at 28℃ with shaking at 200 rpm for 18 h;

[0089] 2. Secondary activation of Agrobacterium: Take 200 μL of activated bacterial culture and add it to 20 mL of YEP (containing kanamycin and hygromycin). Shake until OD600 is about 0.5. Add 100 μmol / L acetylsuccinone (AS) in the last 1-2 hours of shaking. Incubate at 28℃ and 200 rpm.

[0090] 3. Collect the bacterial cells at 5000 rpm for 5 minutes, then discard the supernatant;

[0091] 4. After resuspending in sterile water, collect the bacterial cells again, rotate at 5000 rpm for 5 minutes, and discard the supernatant;

[0092] 5. Add the infection solution (MS + 6-BA 0.4 mg / L + 2,4-D 1.5 mg / L + sucrose 30 g / L, pH 5.2) to 100 μmol / LAS, resuspend the bacterial cells in a small amount of infection solution, and add the resuspended bacterial cells to 20 mL of infection solution, controlling OD600 between 0.2 and 0.3;

[0093] 6. Callus tissue from immature embryos of 'Wanglin' ​​apple (Malus × domestica 'Orin') was cultured in the dark at 24°C on subculture medium (MS + 6-BA 0.4 mg / L + 2,4-D 1.5 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH 5.9), and transferred to fresh subculture medium every 10-14 days for further subculture.

[0094] 7. Break up the upper callus and add it to the infection solution containing Agrobacterium tumefaciens AhSIP1-LBA4404. Shake at room temperature for 15-20 minutes.

[0095] 8. Filter the callus tissue through two layers of gauze, and slightly blot the bacterial solution with filter paper (do not dry it too much). Transfer the callus to co-culture medium (MS + 6-BA 0.4 mg / L + 2,4-D 1.5 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH 5.2), so that the callus is in a clump and fully in contact with the medium. Incubate in the dark at 22°C for 1.5-3 days.

[0096] 9. Spread the callus onto a selection plate (subculture medium + 500 mg / L termethin + 50 mg / L kanamycin resistance), spread it into a thin layer, and incubate in the dark for selection;

[0097] 10. Transfer the newly grown individual callus spheres to a medium with the same resistance, culture for 14 days, and repeat three times;

[0098] 11. Detection of transgenic callus tissue: DNA and RNA were extracted and screened to obtain 35S:AhSIP1 transgenic callus.

[0099] Example 7

[0100] Application of AhSIP1 gene in improving salt tolerance of apple callus

[0101] The apple callus transgenic with the AhSIP1 gene screened out in Example 6 was cultured for 14 days in subculture medium (containing 500 mg / L termethin + 50 mg / L kanamycin resistance) with or without 100 mM and 200 mM NaCl, and the fresh weight of the callus was measured. The callus transgenic with the empty vector pBI121-LBA4404 Agrobacterium was used as a negative control. The preparation method was the same as in Example 6.

[0102] Observe the growth of apple callus on different culture media, such as Figure 5 As shown, the growth rate of different apple calluses on normal culture medium was basically the same, with no difference in fresh weight. Salt treatment significantly inhibited the growth and development of apple calluses. 35S:AhSIP1 overexpressing transgenic calluses grew faster and had greater fresh weight than the control. Specifically, at 100 mM NaCl, the fresh weight of 35S:AhSIP1 transgenic calluses increased by 23.0% compared to the control, and at 200 mM NaCl, it increased by 32.6%. These results indicate that peanut AhSIP1 positively regulates the salt tolerance of apple calluses.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. The application of peanut exocrine peptide AhSIP1 in improving plant salt tolerance, characterized in that, The amino acid sequence encoded by the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:

2.

2. The application of the peanut exocrine peptide AhSIP1 according to claim 1 in improving plant salt tolerance, characterized in that, The nucleic acid sequence of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:

1.

3. The application of the peanut exocrine peptide AhSIP1 according to claim 1 or 2 in improving plant salt tolerance, characterized in that, Salt tolerance in plants can be improved by overexpressing the peanut exocrine peptide AhSIP1 or by exogenously applying the mature peptide fragment of the peanut exocrine peptide AhSIP1.

4. The application of the peanut exocrine peptide AhSIP1 according to claim 3 in improving plant salt tolerance, characterized in that, The amino acid sequence of the mature peptide segment of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:

5.

5. A method for improving the salt tolerance of plants, characterized in that, Salt tolerance of plants is improved by overexpressing peanut exocrine peptide AhSIP1 or by exogenously applying mature peptide fragments of peanut exocrine peptide AhSIP1; the amino acid sequence encoded by said peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:

2.

6. The method for improving plant salt tolerance according to claim 5, characterized in that, The nucleic acid sequence of the AhSIP1 gene was constructed into an expression vector and transformed into plants to overexpress AhSIP1 in plants. The nucleic acid sequence of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:

1.

7. The method for improving plant salt tolerance according to claim 5, characterized in that, The amino acid sequence of the mature peptide segment of the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:

5.

8. The method for improving plant salt tolerance according to claim 7, characterized in that, The mature peptide of the peanut exocrine peptide AhSIP1 is applied at a concentration of 5-20 μM.

9. The application of peanut exocrine peptide AhSIP1 in the preparation of formulations to improve plant salt tolerance, characterized in that, The amino acid sequence encoded by the peanut exocrine peptide AhSIP1 is shown in SEQ ID NO:

2.

10. A preparation for improving the salt tolerance of plants, characterized in that, The active ingredient is the mature peptide segment of peanut exocrine peptide AhSIP1, whose amino acid sequence is shown in SEQ ID NO:5.

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