Peptide signal molecule GhEG45 and application of gene engineering product of peptide signal molecule GhEG45 in plant growth regulation and disease resistance

Abstract

The invention belongs to the technical field of biology, and discloses a peptide signal molecule GhEG45 and application of a gene engineering product of the peptide signal molecule GhEG45 in plant growth regulation and disease resistance. The gene coding sequence of the peptide signal molecule GhEG45 is as shown in SEQ ID NO. 1, and the amino acid sequence of the peptide signal molecule GhEG45 is as shown in SEQ ID NO. 2. The peptide signal molecule GhEG45 can prevent and treat cotton verticillium wilt and regulate plant growth, can also be used for cultivating new varieties of transgenic plants resisting V.dahliae infection, improves the disease resistance and stress resistance of the plants, plays a role in regulating plant growth, increases both production and income, and improves crop quality.

Description

Technical Field

[0001] The present invention generally relates to the field of biotechnology, and in particular to the application of a peptide signal molecule GhEG45 and a genetically engineered product thereof in regulating plant growth and disease resistance. Background Art

[0002] Cotton Verticillium wilt, as an important disease threatening cotton production, has become a serious obstacle to cotton production, causing huge economic losses to cotton farmers every year. Cotton Verticillium wilt has a high incidence rate, causing a 20%-30% loss of cotton yield in mild cases, and can cause the death of the entire plant in severe cases. Verticillium wilt is called "cotton cancer" and is difficult to prevent and control. Verticillium dahliae is a soil-borne pathogenic fungus that causes systemic diseases by infecting the vascular tissues of upland cotton, seriously threatening the sustainable production of cotton. V. dahliae microsclerotia survive in the soil for a long time. After the germinated hyphae invade the host root system, the cotton leaves turn yellow, wilt, and the plants age prematurely. Traditional prevention and control measures are difficult to effectively remove pathogens in the soil. V. dahliae can secrete a variety of effector proteins, infect cotton and enter plant tissues, and invade epidermal cells at the roots or wounds, expand longitudinally along the vascular system, and spread to the aboveground part. During the process of V. dahliae infecting cotton, whether cotton can recognize the pathogen and quickly activate an effective immune response is a key factor in its formation of disease resistance. If identification fails, the pathogen's effector factors can easily break through the defense line, successfully colonize, and induce systemic diseases. Therefore, analyzing the molecular mechanisms of cotton-pathogen interactions and discovering high-resistance genes have become the key to sustainable prevention and control of cotton Verticillium wilt.

[0003] In recent years, with the changes in soil management and cotton planting methods, the scope of cotton Verticillium wilt has continued to expand, and innovative measures for the prevention and control of Verticillium wilt in cotton production are urgently needed. Since traditional disease-resistant breeding and chemical control methods have limited effectiveness, analyzing the molecular mechanism of genes related to the regulation of cotton disease-resistant trait expression, especially the use of genetic engineering to create new disease-resistant cotton varieties, has become a major issue that needs to be solved. In the plant pathogen recognition-response system, pattern recognition receptors (PRRs) on the cell membrane surface and intracellular resistance proteins (NLRs) jointly perceive pathogen invasion and activate downstream immune responses, and this process is closely related to signal linkage mechanisms such as cell ion homeostasis, reactive oxygen species (ROS) accumulation, and cell wall reinforcement. Among them, the cell's response to Na + , K + The effective perception and regulation of plasma has been proven to play a pivotal regulatory role in plant stress resistance and immunity.

[0004] Plant Natriuretic Peptides (PNPs) are a class of peptide signaling molecules with small molecular weight and systemic mobility. They were originally named because of their functional similarity to mammalian atrial natriuretic peptides (ANPs). PNPs are a class of natural peptide signaling substances widely found in higher plants. They have the ability to regulate cellular water and salt balance, activate immune signals and promote cell proliferation. PNPs can induce stomatal movement, regulate membrane potential and plasma membrane H + -ATPase activity, thereby maintaining cellular K + / Na + The ion gradient and osmotic pressure homeostasis show significant stress resistance effects under stress conditions such as drought and high salt.

[0005] However, the current research on the role of peptide signaling molecule GhEG45 in regulating Na + With K + Systematic research on homeostasis, regulation of plant growth and disease resistance is still a blank, especially the lack of practical technical means and commercial models for applying it to important economic crops such as cotton (tomato, cucumber, strawberry). Summary of the Invention

[0006] To address the aforementioned problems in the prior art, the present invention provides a peptide signaling molecule, GhEG45, and its genetically engineered products for use in regulating plant growth and disease resistance. The peptide signaling molecule, GhEG45, can prevent and control Verticillium wilt in cotton and regulate plant growth. It can also be used to cultivate new transgenic plant varieties resistant to V. dahliae infection, improve plant disease and stress resistance, and regulate plant growth, while also increasing yields and improving crop quality.

[0007] According to a first aspect of the present invention, a peptide signal molecule GhEG45 is provided. The gene coding sequence of the peptide signal molecule GhEG45 is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0008] Specifically, the preparation method of the peptide signal molecule GhEG45 is:

[0009] Specific primers were designed based on the GhEG45 gene sequence cloned from upland cotton. After PCR amplification, the gene was ligated into the pET-22b(+) expression vector with NdeI and SalI restriction sites to construct the recombinant plasmid pET-22b-GhEG45. This plasmid was transformed into Escherichia coli DH5α for amplification, and after verification by restriction enzyme digestion and sequencing, it was further transformed into the BL21 expression strain.

[0010] Under IPTG induction conditions, the GhEG45 protein was induced to express; the cells were resuspended in PBS and then ultrasonically disrupted, and the resulting lysate was purified by chromatography to obtain the recombinant GhEG45 protein containing the His tag; after the tag was removed by enzymatic cleavage, the target protein was treated with high temperature to inactivate the enzyme and freeze-dried to prepare a powder;

[0011] The specific primer sequences are:

[0012] eGhEG45-F:5'-GGAATTCCATATGGATCAGG GGAAGGCTGTTTT-3'

[0013] eGhEG45-R:5'-ACGCGTCGACAACCTCATTAAATTCAACTTGG-3'.

[0014] According to a second aspect of the present invention, a genetically engineered product is provided. The genetically engineered product is recombined from the GhEG45 gene, and the cDNA sequence of the GhEG45 gene is shown in SEQ ID NO.1.

[0015] In specific cases, the genetic engineering products include recombinant vectors and recombinant strains.

[0016] According to the third aspect of the present invention, there is provided use of the peptide signal molecule GhEG45 according to the first aspect of the present invention or the genetically engineered product according to the second aspect of the present invention in regulating plant growth and disease resistance.

[0017] In a specific case, the plant is a dicotyledonous plant. Preferably, the plant is cotton, tomato, cucumber, or strawberry.

[0018] Specifically, the disease resistance refers to cotton Verticillium wilt caused by Verticillium dahliae.

[0019] GhEG45 can regulate Na + / K + Homeostasis maintains intracellular homeostasis and effectively regulates ion channel disturbances during pathogen infection. Exogenous application or overexpression of GhEG45 accelerates plant root development, increases growth rate, and enhances salt and disease resistance, demonstrating its potential as a plant growth regulator and immune inducer. More importantly, GhEG45 is a biopeptide molecule composed of natural amino acids that is rapidly metabolized after use. It is non-toxic, residue-free, environmentally friendly, and harmless to humans and animals, making it suitable for promotion in green agricultural production systems.

[0020] The present invention is based on the above functional characteristics of GhEG45 and proposes for the first time its role in regulating Na + With K +Innovative application paths in ion homeostasis, coordinated plant growth and disease control provide new ideas and methods for achieving green, efficient and sustainable plant disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 GhEG45 amino acid sequence, functional domains, and gene structure annotation. (A) Nucleotide and amino acid sequences of the GhEG45 protein. Red letters indicate the signal peptide, and the green portion indicates the DPBB domain. (B) Schematic diagram of the DPBB domain and the location of the signal peptide. (C) Gene structure diagram of GhEG45. Yellow indicates the CDS region, blue indicates upstream and downstream regulatory regions, and black lines indicate introns.

[0022] Figure 2 : Temporal expression changes of the GhEG45 gene after V. dahliae infection in cotton;

[0023] Data are mean ± SE (n = 3). Asterisks indicate significant differences compared with 0 h (*P < 0.05, **P < 0.01, Student's t test).

[0024] Figure 3 :Identification of silencing efficiency of GhEG45 gene-silenced cotton plants;

[0025] (A) TRV:CLA1, a positive control, demonstrates leaf albino phenotype. (B) Phenotypic observations of TRV:00 and TRV:GhEG45. (C) RT-qPCR validation of GhEG45 gene silencing efficiency.

[0026] Figure 4 :Susceptibility of GhEG45 gene-silenced cotton plants to V. dahliae;

[0027] (A) Phenotypic comparison of plants 14 days after inoculation with V. dahliae. (B) Disease index. (C) Longitudinal section of a vascular stem segment of a TRV:GhEG45 plant. (D) Pathogen recovery assay. (E) Fungal biomass accumulation. (F) Trypan blue staining. Error bars represent the standard deviation of three biological replicates. Two-way ANOVA was used (ns, not significant, *P < 0.05 and **P < 0.01).

[0028] Figure 5 : GhEG45 expression level in transgenic Arabidopsis thaliana.

[0029] Figure 6 : Overexpression of GhEG45 enhances resistance of Arabidopsis thaliana to V. dahliae.

[0030] (A) Comparison of disease phenotypes between the GhEG45-overexpressing strain and the WT strain 15 days after inoculation. (B) Disease index at three stages (12, 15, and 18 dpi). (C) Fungal biomass accumulation. Error bars represent the standard deviation of three biological replicates. One-way ANOVA was used (ns, not significant, *P < 0.05 and **P < 0.01).

[0031] Figure 7 :GhEG45 regulates Na + , K + Ion content;

[0032] (A–C) Na in GhEG45 gene-silenced cotton plants + , K + Content and K + / Na + Ratio determination results. (D–F) Na in GhEG45 transgenic Arabidopsis + , K + Content and K + / Na + Error bars represent the standard deviation of three biological replicates, and statistical significance was assessed using the Student's t test (*P < 0.05 and **P < 0.01).

[0033] Figure 8 :Exogenous application of peptide signal molecule GhEG45 enhances cotton resistance to V. dahliae;

[0034] The left picture shows the water-treated control group (H2O), and the right picture shows the GhEG45-treated group.

[0035] Figure 9 :Exogenous spraying of peptide signal molecule GhEG45 improves the growth of upland cotton seedlings;

[0036] The left picture shows the water-treated control group (H2O), and the right picture shows the GhEG45-treated group. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention are described below by way of specific examples. The examples described in this field are provided to help understand the present invention and should not be considered as specific limitations of the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0039] The terms "includes" and "including" and any variations thereof herein are intended to cover a non-exclusive inclusion.

[0040] Example 1: V. dahliae infection induces relative expression of GhEG45 in cotton

[0041] First, we screened the genes induced by V. dahliae through the transcriptome library of V. dahliae-infected upland cotton, and found the gene encoding the GhEG45 peptide signaling molecule with significantly upregulated expression. + With K + GhEG45, a peptide signaling molecule that regulates plant growth and disease resistance, is involved in homeostasis.

[0042] To study the expression pattern of GhEG45 under V. dahliae infection, an experiment was conducted on upland cotton seedlings at the two-leaf, one-heart stage. 7 spores / mL of V. dahliae spore suspension was used for pathogen infection. The treatment time was set at 0h, 1h, 3h, 6h, 12h and 24h. Cotton root tissue was quickly collected for expression analysis. All samples were immediately frozen with liquid nitrogen after collection and stored in a -80℃ freezer. Total RNA was extracted from V. dahliae-infected upland cotton using the EASYspin Plant RNA Rapid Extraction Kit according to the instructions. Total RNA was extracted and analyzed by 1.2% agarose gel electrophoresis and NanoDrop 2000 (A 260 / A 280 =1.8–2.0, A 260 / A 230 >2.0) to check RNA quality. 1 μg RNA was used for reverse transcription to synthesize first-strand cDNA, and RT-qPCR was used to detect GhEG45 expression. cDNA was synthesized using oligo(dT) primers and amplified using SYBR Green. -ΔΔCt The relative expression level was calculated by the method.

[0043] The GhEG45 quantitative primers in the above real-time fluorescence quantitative PCR reaction system are:

[0044] qGhEG45-F: 5`-GATCAGGGGAAGGCTGTTTTC-3`

[0045] qGhEG45-R:5`-CTTGCAAGGATGTGGTGCTTC-3`

[0046] The above-mentioned real-time fluorescence quantitative PCR reaction system is:

[0047]

[0048] The expression pattern of V. dahliae was analyzed using RT-qPCR. Figure 2 The results showed that GhEG45 showed significant induction upon V. dahliae infection, with its expression level increasing 3.4-fold within 24 hours after pathogen inoculation, indicating that GhEG45 is involved in the plant's early response to cotton Verticillium wilt.

[0049] Example 2: Susceptibility of GhEG45 gene-silenced cotton plants to Verticillium wilt

[0050] In order to study the function of GhEG45 in the disease resistance process of cotton and its sensitivity to cotton Verticillium wilt, a TRV:GhEG45 vector was constructed to conduct virus-induced gene silencing (VIGS) experiments. Based on the tobacco rattle virus (TRV) vector system, primers targeting specific sites of the GhEG45 gene were designed, and a 300bp fragment was amplified from the cotton seedling cDNA and cloned into the TRV2 vector. The recombinant plasmids TRV2:GhEG45 (target gene), TRV2:00 (empty vector control) and TRV2:GhCLA1 (positive control) were transformed into Agrobacterium GV3101. The Agrobacterium culture liquid (OD 600 =1.0) in a 1:1 ratio and injected into the cotyledons of 10-day-old cotton seedlings. After 24 hours of incubation in the dark, the plants were transferred to a 25°C / 20°C day / night cycle with a 16-hour light cycle. After 14 days, the albinism phenotype of GhCLA1-silenced lines was observed to verify VIGS efficiency. After 21 days, plant RNA was extracted and RT-qPCR was used to determine the silencing efficiency of GhEG45. Effectively silenced lines were selected for subsequent pathogen inoculation experiments.

[0051] The primers targeting the specific sites of the GhEG45 gene are:

[0052] TRV-GhEG45-F: 5`-CGACGACAAGACCGTGACCATGGTGGCGGGAGTTAGTGA-3`

[0053] TRV-GhEG45-R: 5`-GAGGAGAAGAGCCGTCATTAGATTATACCGGCGTCAGG-3`

[0054] The highly pathogenic V. dahliae strain V991 was selected and cultured on PDA medium for 4-6 days, then transferred to Czapek liquid medium (200 rpm, 25°C) for 7 days. The spore suspension concentration was adjusted to 1×10 7spores / mL. Cotton plants were inoculated with the pathogen via the root dip method and replanted in soil. The disease phenotype of GhEG45-silenced plants was observed between 7 and 28 days after infection. Disease severity was assessed using a 0-4 scale, and the disease index (DI) was calculated. Fungal biomass was quantified by RT-qPCR using the specific primers ITS1-F / STVe1-R to amplify the V. dahliae ITS region. GhUBQ7 was used as an internal reference gene in each cotton plant.

[0055] Primers for the above-mentioned V. dahliae biomass determination:

[0056] ITS1-F:5`-TGAGTTCGAGGCTGGTATCT-3`

[0057] STVe1-R:5`-CACTTGGTGGTGTCCATCTT-3`

[0058] The quantitative primers for the above-mentioned GhUBQ7 as an internal reference gene are:

[0059] qGhUBQ7-F:5`-AGGCATTCCACCTGACCAAC-3`

[0060] qGhUBQ7-R:5`-CAGCGAGCTTGACCTTCTTC-3`

[0061] To elucidate the regulatory role of the GhEG45 gene in pathogen colonization during disease resistance, cotton stem segments infected with V. dahliae were observed and subjected to pathogen recovery experiments. Cell death staining was performed using trypan blue stain. The leaves to be examined were placed in a 50 mL glass bottle, and the stain was added to completely cover the sample. Vacuum the bottle for 15 minutes to facilitate penetration of the stain into the tissue. The bottle was then placed in a 100°C boiling water bath for 5-8 minutes and continued staining at room temperature for 6-8 hours. After staining, the sample was rinsed several times with distilled water to remove excess stain, and then decolorized with chloral hydrate solution until the tissue was transparent and clear. The staining results were observed under a microscope and photographed.

[0062] To study the function of GhEG45 in cotton disease resistance, a TRV:GhEG45 vector was constructed to conduct virus-induced gene silencing (VIGS) experiments. Figure 3 ), RT-qPCR results showed that the expression level of GhEG45 in TRV:GhEG45 plants was significantly reduced, indicating that the gene was effectively silenced ( Figure 3 ), verifying the effectiveness of the VIGS system in this experiment.

[0063] After inoculation with V. dahliae, TRV:GhEG45 plants showed more severe disease phenotypes compared with the control group (TRV:00), such as leaf curling, wilting, yellowing and necrosis ( Figure 4 A). 14 days after V. dahliae infection, the disease index of TRV:GhEG45 plants increased to 38.85, which was significantly higher than that of the control group ( Figure 4 B) (P < 0.01), indicating that silencing GhEG45 increased the susceptibility of cotton plants to V. dahliae. To clarify the regulatory role of the GhEG45 gene in pathogen colonization during disease resistance, cotton stem segments infected with V. dahliae were observed and pathogen recovery experiments were conducted. Compared with the empty vector control (TRV:00), the vascular tissues of the TRV:GhEG45 plant stem segments showed more severe browning, indicating a wider range of pathogen infection ( Figure 4 C). In the PDA plate recovery experiment, the TRV:GhEG45 stem segments produced more fungal colonies and denser mycelial growth ( Figure 4 D). Trypan blue staining showed that a large number of dark blue necrotic plaques appeared in the leaves of GhEG45 target gene silenced plants, with increased cell death and severe tissue damage ( Figure 4 E). RT-qPCR analysis showed that the fungal-specific DNA content in the stem segments of GhEG45 gene-silenced cotton plants was 4.5 times higher than that in the control group ( Figure 4 F).

[0064] Example 3: Disease resistance of GhEG45-transgenic Arabidopsis plants to V. dahliae

[0065] To construct Arabidopsis thaliana overexpressing GhEG45, PCR amplification was performed using cotton cDNA as a template and specific primers; the specific primers are as follows:

[0066] ZW-GhEG45-F:5`-AAACTGCAGATGGGTGGAAGATATACT-3`

[0067] ZW-GhEG45-R:5`-CGCGTCGACCTCATTAAATTCAACTTGG-3`

[0068] The obtained PCR product was ligated with the pCAMBIA1300-GFP cloning vector to obtain the ligation product 35S:GhEG45-GFP. The ligation product 35S:GhEG45-GFP was transformed into Escherichia coli DH5α and spread on LB solid medium containing kanamycin. A single colony was picked and shaken to extract the plasmid. After plasmid PCR, the plasmid was sent for sequencing. The sequencing results are shown as follows: Figure 1The sequence of A is correct. The recombinant plasmid (35S:GhEG45-GFP) was transformed into Arabidopsis thaliana via the inflorescence infiltration method mediated by Agrobacterium GV3101. Seeds of transgenic plants expressing the GhEG45 gene were screened on a selective medium containing hygromycin until a T3 generation homozygous transgenic line expressing GhEG45 was obtained. Disease resistance testing was performed to evaluate the response of transgenic plants to infection with V. dahliae.

[0069] The highly pathogenic V. dahliae strain V991 was selected and cultured on PDA medium for 4–6 days, then transferred to Czapek liquid medium (200 rpm, 25°C) for 7 days. The spore suspension concentration was adjusted to 1×10 7 spores / mL. Arabidopsis plants were inoculated with the pathogen via the root dip method and replanted in soil. The disease phenotypes of GhEG45-silenced and transgenic Arabidopsis plants were observed between 7 and 28 days after infection. Disease severity was assessed using a 0-4 scale, and the disease index (DI) was calculated. Fungal biomass was quantified by RT-qPCR using the specific primers ITS1-F / STVe1-R to amplify the V. dahliae ITS region. AtEF1α was used as an internal reference gene in Arabidopsis plants.

[0070] The above-mentioned specific primers ITS1-F / STVe1-R:

[0071] ITS1-F:5`-TGAGTTCGAGGCTGGTATCT-3`

[0072] STVe1-R:5`-CACTTGGTGGTGTCCATCTT-3`

[0073] The above-mentioned internal reference genes are:

[0074] qAtEF1α-F:5`-CCTGGATTGCCACACC-3`

[0075] qAtEF1α-R:5`-AGTCTGCCTCATGTCC-3`

[0076] GhEG45 overexpressing Arabidopsis was constructed, and the expression levels of the gene in each transgenic Arabidopsis strain of GhEG45 were detected by RT-qPCR. The results showed that the expression levels of strains 1, 4, and 12 were relatively high ( Figure 5 ), genetically stable T3 generation plants were selected for disease resistance identification, and the response of transgenic plants to V. dahliae infection was evaluated.

[0077] After inoculation of V. dahliae, transgenic plants overexpressing GhEG45 showed significantly enhanced disease resistance. Compared with the wild type, Arabidopsis plants overexpressing OE showed milder symptoms at 12, 15, and 18 dpi, with leaves maintaining good morphology, while WT plants showed severe yellowing, wilting, and necrosis ( Figure 6 A). The disease index statistics showed that the disease index of OE1, OE4 and OE12 at each time point was lower than that of WT ( Figure 6 B), indicating that the expression of GhEG45 effectively delays the progression of the disease. The relative expression levels of fungal biomass in Arabidopsis overexpressing GhEG45 were detected, and the results showed that the accumulation of fungal biomass in WT was higher than that in transgenic Arabidopsis ( Figure 6 C), indicating that GhEG45 has a clear anti-V. dahliae function in Arabidopsis thaliana, and its overexpression can significantly reduce the severity of the disease, indicating that this gene plays an active regulatory role in the plant disease resistance mechanism and has potential application value in breeding.

[0078] Example 4: GhEG45 gene regulates Na + and K + Ion homeostasis

[0079] In order to determine the effect of GhEG45 gene on plant endogenous Na + and K + The Na content of GhEG45 gene-silenced cotton plants and GhEG45 gene-transfected Arabidopsis plants was determined. + and K + The specific operation is as follows: the sample is dried in an oven, then ground into a fine powder, and an appropriate amount of sample is weighed and added to ultrapure water for cation extraction. Methanesulfonic acid (12 mM) is used as the mobile phase. Ion chromatograph (ICS-1500, Thermofisher scientific), chromatographic column: Dionex IonPacTM CS12ARFICTM (4×250 mm), mobile phase: 12 mM methanesulfonic acid, flow rate: 1.0 mL min -1 , injection volume: 25 μL, column temperature: 35.0 ° C, suppressor model: AERS_4mm, suppressor current: 25 mA, detector type: conductivity detector, run time: 12 min, retention time: Na + About 5.9 minutes, K + About 8.6min, standard Na + and K + The solution (5 ppm) was used to calibrate and calculate the correct ion concentration of the leaf sample solution, and the results were statistically analyzed using the chromatograph.

[0080] GhEG45 is involved in the regulation of ion homeostasis in plants. The Na+ and K + Concentration and K + / Na + Ratio. Under the condition of no inoculation, Na + The content was lower than that of the control ( Figure 7 A), K + Content and K + / Na + There was no significant difference in the ratio ( Figure 7 B, C); after V.dahliae infection, K + The content of Na + The accumulation level increases, leading to K + / Na + The ratio was reduced in silenced plants, indicating that GhEG45 affects Na + Accumulation level affects ion balance.

[0081] To understand the mechanism of GhEG45 in plant disease resistance, the Na + and K + The concentration of K + / Na + Ion chromatography analysis showed that the Na + The content was significantly higher than that of wild type, while K + There was no significant difference in the content ( Figure 7 D, E), calculate K + / Na + The ratio was found to be significantly decreased by GhEG45 overexpression ( Figure 7 F), GhEG45 may regulate Na + and K + Homeostasis, regulating plant growth and disease resistance.

[0082] Example 5: Expression and preparation of GhEG45 recombinant protein

[0083] This example provides a method for recombinant expression and purification of the plant peptide signaling molecule GhEG45.

[0084] First, based on the GhEG45 gene sequence cloned from upland cotton (e.g. Figure 1 Specific primers were designed using the plasmid (shown in Figure A). PCR amplification was performed and then ligated into the pET-22b(+) expression vector containing NdeI and SalI restriction sites to construct the expression vector pET-22b-GhEG45. This plasmid was transformed into E. coli DH5α for amplification, verified by restriction enzyme digestion and sequencing, and then transformed into the BL21(DE3) expression strain.

[0085] The designed specific primers:

[0086] eGhEG45-F:5'-GGAATTCCATATGGATCAGG GGAAGGCTGTTTT-3'

[0087] eGhEG45-R:5'-ACGCGTCGACAACCTCATTAAATTCAACTTGG-3'.

[0088] Under the induction condition of 1mM IPTG, the GhEG45 protein was induced to express at low temperature (16-30℃). The bacteria were resuspended in PBS and then ultrasonically broken. The lysate was purified by Ni 2 The His-tagged recombinant GhEG45 protein was purified by affinity chromatography. After the tag was removed by enzymatic cleavage, the target protein was inactivated by high-temperature treatment at 80°C and freeze-dried to produce a peptide powder for exogenous administration.

[0089] Alternatively, the peptide signaling molecule GhEG45 can be obtained through chemical synthesis and purified by HPLC to ensure its structural integrity and stable biological activity. The resulting short peptide can be directly dissolved in water and sprayed at different stages of plant growth for use in plant growth regulation and disease resistance research.

[0090] Example 6: Exogenous application of GhEG45 to regulate plant growth and disease resistance

[0091] Considering that peptide signaling molecules in Arabidopsis act as plant cytokines to regulate immune and biological responses, it is speculated that the cotton homolog peptide signaling molecule GhEG45 may also play an important role in regulating plant growth and disease resistance.

[0092] To test this hypothesis, the peptide signaling molecule GhEG45 was exogenously applied to upland cotton plants, and the growth and development of the cotton, as well as the symptoms of infection with Verticillium wilt, were observed. GhEG45 powder was dissolved in distilled water to a working concentration of 10-100 μM. Normally grown upland cotton plants under greenhouse conditions were sprayed with the peptide signaling molecule at the seedling stage, spraying once every three days for two consecutive sprays. This served as the treatment group, while a control group sprayed with plain water served as the control group. The cotton varieties and other growing conditions of the control and treatment groups remained the same.

[0093] The evaluation method for cotton Verticillium wilt phenotype in the above experiment is as follows:

[0094] The highly pathogenic strain V991 of V. dahliae was used as the inoculum to infect the plants. The conidia suspension (about 1×10 7spores / mL) were inoculated into the roots of upland cotton and moisture was maintained for 2 days to ensure normal V. dahliae infection. The spread of V. dahliae in upland cotton was assessed by counting symptomatic plants 7, 9, 11, 13, 15, 17, and 21 days after inoculation. The incidence of V. dahliae was assessed when true leaves of the control and treatment groups turned yellow and wilted, and the disease index was recorded. Disease grading criteria: Grade 0: Healthy plant, no symptoms; Grade 1: 1-2 cotyledons diseased; Grade 2: 1 true leaf diseased; Grade 3: 2 or more true leaves diseased or fallen, leaving only the heart leaf; Grade 4: All true leaves turned yellow or necrotic, and the plant was bare or dead. Disease indexes were calculated for identification materials.

[0095] Disease index = [Σ(number of diseased plants at each level × corresponding disease level) / total number of plants surveyed × highest disease level] × 100.

[0096] The experimental results showed that the leaves of upland cotton treated with water had more obvious wilting and yellowing, and the vascular bundles in the stems had more severe browning; while the upland cotton plants treated with the peptide signal molecule GhEG45 had significantly reduced disease symptoms, and the plants were in a healthy growth state overall ( Figure 8 ).

[0097] In addition, the peptide signaling molecule GhEG45 not only regulates plant cell Na + With K + Homeostasis and disease defense, it also has the physiological function of significantly promoting plant growth and development. Exogenous application of the synthetic and purified peptide signal molecule GhEG45 to upland cotton seedlings ( Figure 9 ), and found that the plant height, biomass, and leaf area of ​​the treated group were better than those of the water-treated control group, showing obvious characteristics of strong plants, accelerated growth, and good physiological state, indicating that GhEG45 also has the effect of promoting plant growth and enhancing activity under non-stress conditions.

[0098] In summary, GhEG45 is a natural and efficient multifunctional peptide signaling molecule that regulates plant Na + With K + Steady state, with the function of regulating plant growth and development and disease resistance, is used in plant stress resistance, yield increase and green cultivation systems in the agricultural field, and has extremely high promotion and development value.

[0099] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. As long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope of this specification.

[0100] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A peptide signal molecule GhEG45, characterized in that The gene coding sequence of the peptide signal molecule GhEG45 is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The peptide signaling molecule GhEG45 according to claim 1, characterized in that The preparation method of the peptide signal molecule GhEG45 is as follows: Specific primers were designed based on the GhEG45 gene sequence cloned from upland cotton. After PCR amplification, the gene was ligated into the pET-22b(+) expression vector with NdeI and SalI restriction sites to construct the recombinant plasmid pET-22b-GhEG45. This plasmid was transformed into Escherichia coli DH5α for amplification, and after verification by restriction enzyme digestion and sequencing, it was further transformed into the BL21 expression strain. Under IPTG induction conditions, the GhEG45 protein was induced to express; the cells were resuspended in PBS and then ultrasonically disrupted, and the resulting lysate was purified by chromatography to obtain the recombinant GhEG45 protein containing the His tag; after the tag was removed by enzymatic cleavage, the target protein was treated with high temperature to inactivate the enzyme and freeze-dried to prepare a powder; The specific primer sequences are: eGhEG45-F:5'-GGAATTCCATATGGATCAGG GGAAGGCTGTTTT-3' eGhEG45-R:5'-ACGCGTCGACAACCTCATTAAATTCAACTTGG-3'.

3. A genetically engineered product, characterized in that: The genetic engineering product is formed by recombination of the GhEG45 gene, and the cDNA sequence of the GhEG45 gene is shown in SEQ ID NO.

1.

4. The genetically engineered product according to claim 3, characterized in that The genetic engineering products include recombinant vectors and recombinant strains.

5. Use of the peptide signal molecule GhEG45 according to claim 1 or the genetically engineered product according to claim 3 in regulating plant growth and disease resistance.

6. The use according to claim 5, characterized in that The plant is a dicotyledonous plant.

7. The use according to claim 5, characterized in that The disease resistance refers to cotton Verticillium wilt caused by Verticillium dahliae.

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