Small peptide for improving abiotic stress tolerance of crops and application thereof

Abstract

The invention belongs to the technical field of biotechnology and botany, and particularly relates to a small peptide for improving abiotic stress tolerance of crops and application of the small peptide, and the small peptide is GhIDL02 and / or GhPIPL7. The amino acid sequence of the GhIDL02 is as shown in SEQ ID NO. 1; the amino acid sequence of the GhPIPL7 is as shown in SEQ ID NO. 2. According to the method, the small peptide GhIDL02 or GhPIPL7 is exogenously applied, so that the hormone-like effect is quickly exerted, the salt tolerance of the cotton is remarkably improved, and the adverse effect of a chemical regulating agent on the environment is avoided. Through a research technology combining bioinformatics and molecular biology, the positive regulation effect of the GhIDL02 and GhPIPL7 small peptides under salt stress is confirmed for the first time, and by activating an MAPK signal channel, the steady state of cotton sodium and potassium ions is maintained, the salt tolerance of cotton is improved, and a theoretical basis and technical support are provided for green development of an agricultural biotechnology.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology and botany, and specifically relates to a small peptide for improving the tolerance of crops to abiotic stress and application thereof. Background Art

[0002] Abiotic stress is becoming increasingly serious, among which drought, floods, extreme temperatures, high salt and heavy metal pollution are common abiotic stress factors. The mainstream methods to improve crop tolerance to abiotic stress include molecular breeding technology, such as transgenic and gene editing, and the use of chemical regulators. Although these methods show great potential in theory, they still face many limitations in practical applications. The shortcomings of existing technologies are mainly reflected in the following aspects:

[0003] (1) Long and time-consuming cycle: Traditional molecular breeding techniques, including conventional hybrid breeding and molecular marker-assisted breeding, usually require multiple generations of breeding and selection, and the breeding cycle may be as long as 10 to 15 years, making it difficult to quickly promote and apply. Even transgenic and gene editing technologies, although they can accurately modify the genome, also require complex screening, field trials, approval and promotion steps. Therefore, the entire breeding process is still relatively long and cannot quickly meet the needs of large-scale agricultural production.

[0004] (2) Limitations in promotion and application: Although gene editing technology has made breakthroughs in precision, the public's acceptance of genetically modified and gene-edited crops is low, which limits the commercialization progress and application scale of these technologies.

[0005] (3) Potential ecological safety concerns: Molecular breeding technologies, especially transgenic and gene editing, may cause problems such as gene drift or non-target effects by directly changing the genome of crops. Although relevant safety assessment mechanisms are currently available, since the long-term impact of these technologies on ecosystems has not yet been fully clarified, there are still potential ecological safety risks, especially under different environmental and ecological conditions, which may lead to unforeseen consequences.

[0006] (4) Limitations of chemical regulators: Although chemical regulators, such as plant hormones or exogenous small molecule compounds, can be used to improve crop stress resistance in the short term, their effects are often temporary and difficult to sustain, and when widely used, they may lead to environmental pollution or the accumulation of harmful chemicals in the soil. The long-term use of these chemical products may also have a negative impact on soil microbial communities and ecosystems, increasing the environmental burden. In addition, a production model that relies too much on regulators is difficult to meet the development needs of sustainable agriculture.

[0007] In summary, it is an urgent need to find an efficient and environmentally friendly crop tolerance enhancer. Summary of the invention

[0008] The purpose of the present invention is to provide a small peptide for improving the tolerance of crops to abiotic stress and its application, which solves the problem that the prior art requires a long period of time for improving the tolerance of crops and is not environmentally friendly.

[0009] The technical solution adopted by the present invention is:

[0010] The present invention provides a small peptide for improving the tolerance of crops to abiotic stress. The small peptide is GhIDL02 and / or GhPIPL7. The amino acid sequence of GhIDL02 is shown in SEQ ID NO.1; the amino acid sequence of GhPIPL7 is shown in SEQ ID NO.2.

[0011] The present invention also provides an application of the small peptide, wherein the small peptide is used for improving the tolerance of crops to abiotic stress.

[0012] Preferably, the abiotic stress is at least one of salt stress, drought stress, flood stress, temperature stress, heavy metal stress and radiation stress.

[0013] Preferably, the salt concentration of the salt stress is not higher than 500mM; the relative soil moisture content of the drought stress is between 30% and 40%; the temperature of the temperature stress is lower than 0°C or higher than 35°C; the heavy metal stress is at least one of lead stress, mercury stress, cadmium stress and chromium stress; the radiation stress is at least one of ionizing radiation stress, ultraviolet radiation stress and electromagnetic radiation stress.

[0014] Preferably, the working concentration of the small peptide is 100 nM to 10 μM, and the application amount of the small peptide is 2 mL to 5 mL per seedling, or 16 L to 18 L per acre of crops.

[0015] Preferably, the small peptide is applied by spraying or irrigation.

[0016] Preferably, the small peptide is applied during the 3-4 leaf stage of the crop.

[0017] Preferably, the crop comprises at least one of cotton, rice, wheat and corn.

[0018] The present invention also provides a biological pesticide prepared by using the small peptide of the present invention.

[0019] Preferably, the biopesticide further comprises an auxiliary material; the auxiliary material is at least one of an emulsifier, a dispersant, a wetting agent and a thickener.

[0020] Preferably, the emulsifier includes at least one of sodium lauryl sulfate, sodium stearate, Tween and lecithin.

[0021] Preferably, the dispersant includes at least one of sodium lignin sulfonate, sodium polyacrylate and potassium alkyl phosphate.

[0022] Preferably, the wetting agent includes at least one of alkylbenzene sulfonate, sodium sulfate of fatty alcohol polyoxyethylene ether, saponin and fatty alcohol polyoxyethylene ether.

[0023] Preferably, the thickener comprises at least one of pectin, gelatin, carbomer, chitin and chitosan.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a small peptide for improving the tolerance of crops to abiotic stress, wherein the small peptide is GhIDL02 and / or GhPIPL7; the amino acid sequence of the GhIDL02 is shown in SEQ ID NO.1; the amino acid sequence of the GhPIPL7 is shown in SEQ ID NO.2. The present invention rapidly exerts a hormone-like effect by exogenously applying small peptides, significantly improves the salt tolerance of cotton, and avoids the adverse effects of chemical regulators on the environment. Through the research technology combining bioinformatics and molecular biology, the present invention confirms for the first time the positive regulatory effect of GhIDL02 and GhPIPL7 small peptides under salt stress, and improves the salt tolerance of cotton by activating the MAPK signaling pathway, thereby maintaining the homeostasis of sodium and potassium ions in cotton, providing a theoretical basis and technical support for the green development of agricultural biotechnology.

[0026] Based on overcoming the limitations of the prior art, the present invention has the following unique advantages:

[0027] (1) Rapidly improve crop salt tolerance: Compared with transgenic and gene editing technologies, the present invention can quickly induce cotton salt tolerance within two days by exogenously applying GhIDL02 and GhPIPL7 peptides. The present invention does not require complex genome modifications and long breeding cycles, and can trigger the plant's salt stress response mechanism in a relatively short period of time, allowing crops to quickly adapt to adverse saline-alkali environments. This feature allows the present invention to be used to quickly respond to sudden or severe salt stress situations.

[0028] (2) Possessing broad application prospects: GhIDL02 and GhPIPL7 peptides not only show significant effects in improving salt tolerance, but also have broad application potential in plant growth regulation and various stress responses. Therefore, the present invention is not only applicable to cotton, but can also be extended to other crops, with great application and commercial prospects, especially playing an important role in precision agriculture and targeted stress management.

[0029] (3) No genome modification, environmentally friendly and safe: The GhIDL02 and GhPIPL7 small peptides used in the present invention are endogenous molecules of plants, which avoid direct intervention in the crop genome through transgenic or gene editing, thereby eliminating the long-term ecological safety risks associated with genome modification, such as gene drift or non-target effects. In addition, small peptides, as biological molecules naturally present in plants, are easily degraded and will not remain in the environment, avoiding environmental pollution problems that may be caused by chemical regulators.

[0030] (4) Strong specificity and high regulatory efficiency: The GhIDL02 and GhPIPL7 small peptides of the present invention can accurately regulate the salt stress response of cotton by activating the plant MAPK signaling pathway. This regulatory mechanism has a high degree of specificity and does not interfere with other growth and development processes of cotton, ensuring that the normal physiological functions of crops are not affected while improving salt tolerance. Compared with traditional molecular breeding or chemical regulators, the small peptide molecules of the present invention have higher regulatory efficiency and can efficiently mobilize the endogenous stress resistance mechanism of plants, providing a precise and effective solution for improving crop salt tolerance.

[0031] The present invention can effectively overcome the problems of long breeding cycle, limited promotion and potential ecological safety hazards in the prior art, and provide a green, safe and efficient technical path for improving cotton salt tolerance. The present invention not only has the ability to quickly improve salt tolerance, but also meets the needs of modern agriculture for efficient and sustainable crop improvement technology through environmentally friendly molecular regulation means, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The relative expression levels of representative GhIDLs and GhPIPLs members of upland cotton under ddH2O, simulated drought stress and salt stress conditions. A to F are the relative expression levels of GhIDL02, GhIDL17, GhIDL24, GhPIPL2, GhPIPL7 and GhPIPL10, respectively.

[0033] Figure 2Figure 3 Effects of GhIDL02 and GhPIPL7 polypeptide precursor gene interference expression on cotton salt and drought tolerance. A: Phenotypes of two parallel transformation lines of pYL156-GhCLA1; B and C: qRT-PCR analysis of GhIDL02 and GhPIPL7 expression levels after VIGS; D: Analysis of growth phenotype differences of pYL156 negative control, pYL156-GhIDL02 and pYL156-GhPIPL7 cotton plants under salt and drought stress; E and F: Quantitative analysis of seedling fresh weight and chlorophyll content corresponding to Figure D; G: Analysis of chlorophyll fluorescence characteristics of pYL156, pYL156-GhIDL02 and pYL156-GhPIPL7 plants under salt and drought stress; H and I: F corresponding to Figure G. v / F m Ratio and evaluation results of effective photochemical efficiency of photosystem II [Y(II)].

[0034] Figure 3 Exogenous synthetic peptides GhIDL02 and GhPIPL07 enhanced the salt stress tolerance of cotton seedlings. A and F respectively: the growth phenotype observation of cotton seedlings under salt stress after treatment with exogenous synthetic GhIDL02 and GhPIPL07 peptides; B and G respectively: the effects of exogenous synthetic GhIDL02 and GhPIPL07 peptides on the fresh weight of cotton leaves under salt stress; C and H respectively: the effects of exogenous synthetic peptides GhIDL02 and GhPIPL07 peptides on the chlorophyll content of cotton leaves; D and E respectively: the effects of exogenous synthetic GhIDL02 peptides on the photosynthetic fluorescence parameters of cotton leaves under salt stress. v / F m and Y(II); I and J: Effects of exogenous GhPIPL07 peptide treatment on photosynthetic fluorescence parameters F in cotton leaves under salt stress v / F m and the influence of Y(II).

[0035] Figure 4 Figure 2 Effects of synthetic peptide GhPIPL7 on salt stress tolerance of cotton seedlings. A: Phenotype of cotton seedlings treated with different concentrations of GhPIPL7 peptide under salt stress conditions; B: Fresh weight analysis of cotton seedlings in Figure A; C: Chlorophyll content analysis of cotton seedlings in Figure A; D and E: Atomic absorption spectrometry AAS was used to determine the content of sodium and potassium ions in cotton leaves in Figure A; F: Na + / K + Ratio analysis.

[0036] Figure 5Figure 3 Effects of synthetic peptide GhIDL02 on salt stress tolerance of cotton seedlings. A: Phenotype of cotton seedlings treated with different concentrations of GhPIPL7 peptide under salt stress conditions; B: Fresh weight analysis of cotton seedlings in Figure A; C: Chlorophyll content analysis of cotton seedlings in Figure A; D and E: Atomic absorption spectrometry AAS was used to determine the content of potassium and sodium ions in cotton leaves in Figure A; F: Na + / K + Ratio analysis.

[0037] Figure 6 Analysis of the transcriptional regulatory network of GhPIPL7-mediated upland cotton under salt stress, A: DEGs volcano diagram; B~I: qPCR verification of transcriptome data of Ghir_A01G5055, Ghir_A07G37527, Ghir_A03G13985, Ghir_A06G33624, Ghir_A07G38413, Ghir_D05G22180, Ghir_D03G12631 and Ghir_A12G68353.

[0038] Figure 7 GhPIPL7 regulates cotton salt tolerance by activating MPK3 and MPK6. A: Analysis of the activation status of MPK3 and MPK6 under salt stress; B: Analysis of the activation status of MPK3 and MPK6 under GhPIPL7 treatment; C: Analysis of the inhibition status of MPK3 / 6 activation under GhPIPL7 interference; D: qRT-PCR analysis of the expression level of GhMAPK3 after VIGS; E: qRT-PCR analysis of the expression level of GhMAPK6 after VIGS; F: Growth phenotype observation of pYL156, pYL156-GhMAPK3 and pYL156-GhMAPK6 seedlings under salt stress and GhPIPL7 peptide treatment and untreated conditions; G: Fresh weight of cotton leaves corresponding to Figure F; H: Chlorophyll content of cotton leaves corresponding to Figure F.

[0039] Figure 8 Figure 2 is a GhPIPL7-mediated salt stress tolerance model. A: GhPIPL7 enhances cotton salt tolerance by regulating physiological and molecular mechanisms; B: Under normal growth conditions, the wild type WT shows good growth and development; C: Under salt stress conditions, the GhPIPL7 polypeptide is significantly upregulated, and it activates the MPK3 and MPK6 signaling pathways by binding to an unknown cell surface receptor, promoting salt stress signal transduction, and regulating Na + / K +transport to maintain cellular ion homeostasis, thereby improving the salt tolerance of plants; D: In pYL156-GhPIPL7 gene-silenced plants, the expression of the GhPIPL7 polypeptide gene was significantly downregulated, and the MPK3 / 6 signaling pathway was not activated, which could not effectively regulate Na + / K + balance, ultimately leading to a significant decrease in the plant's salt tolerance. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0041] The inventive concept of the present invention is as follows:

[0042] The mainstream methods for improving crop tolerance to abiotic stress include molecular breeding technology and the use of chemical regulators, but the above methods have the disadvantages of long cycle, limited promotion and application, potential ecological safety concerns, etc. Based on this, the present invention provides a small peptide for improving crop tolerance to abiotic stress and its application, the small peptide is GhIDL02 and / or GhPIPL7; the amino acid sequence of GhIDL02 is shown in SEQ ID NO.1; the amino acid sequence of GhPIPL7 is shown in SEQ ID NO.2.

[0043] The present invention is aimed at the stress resistance function analysis of GhIDL02 and GhPIPL7 polypeptides, mainly for salt and drought stress. The present invention adopts virus-induced gene silencing VIGS technology to silence the GhIDL02 and GhPIPL7 polypeptide precursor genes in cotton to verify whether they regulate the salt tolerance of cotton. The specific operation steps are: inserting a specific fragment of the target gene into the TRV vector pYL156, the fragment is designed to specifically silence GhIDL02 and GhPIPL7 without affecting the expression of other genes. The constructed vector is introduced into the Agrobacterium GV3101 strain by electroporation. Subsequently, the OD 600 An Agrobacterium suspension with a value of 1.0 was injected into the cotyledons of cotton seedlings to induce gene silencing.

[0044] After obtaining the above gene-silenced plants, the expression levels of GhIDL02 and GhPIPL7 peptide precursor genes were first analyzed by RNA extraction and qRT-PCR technology. The specific steps are: extract total RNA from cotton leaf samples of the treatment group and the control group to ensure that the purity and integrity of the RNA meet the experimental requirements. Subsequently, the extracted RNA was reverse transcribed into cDNA using reverse transcriptase as a template for subsequent qRT-PCR analysis. In the qRT-PCR reaction, the SYBR Green dye method was used to detect the relative expression levels of GhIDL02 and GhPIPL7. In order to ensure the accuracy of quantitative analysis, the cotton Actin gene was selected as an internal reference to correct the differences between samples. By comparing the qRT-PCR results of the gene silencing treatment group and the control group, the silencing efficiency of the GhIDL02 and GhPIPL7 peptide precursor genes was determined.

[0045] In order to evaluate the salt tolerance of cotton under salt stress, the present invention adopts Na + and K + The cotton seedlings of the treatment group and the control group were treated with 500mM NaCl solution for 3 days, and leaf samples were collected after the treatment. The leaves were dried at 60℃ to constant weight and then ground into powder. The samples were soaked in 0.1M HNO3 solution and diluted for determination after high temperature digestion. Atomic absorption spectrometer AAS was used to determine Na + and K + content, thereby analyzing the changes in ion balance under salt stress conditions and determining the role of GhIDL02 and GhPIPL7 peptides in regulating ion balance.

[0046] The present invention also evaluates the light energy absorption and utilization efficiency of cotton by measuring the chlorophyll content. In the experiment, the cotton leaves were cut into pieces and placed in a mortar, and 1 mL of 96% ethanol solution was added and ground to a homogenous state, and then 1 mL of 96% ethanol was added and continued to grind until the mixture was bleached. The mixture was transferred to an empty graduated tube, and the mortar was rinsed with 4 mL of 96% ethanol solution. After that, centrifuged at 4000 r / min for 10 minutes, and the supernatant was aspirated. The chlorophyll content was calculated by using a spectrophotometer to measure the absorbance of the extract at 649 nm and 665 nm, respectively, according to the standard formula:

[0047] Chl=[(13.95×D665-6.88×D649)+(24.96×D649-7.32×

[0048] D665)] × (volume of extract) / fresh weight);

[0049] Among them, Chl: chlorophyll content, unit is mg / g, FW.

[0050] D665: absorbance of the extract at 665 nm.

[0051] D649: absorbance of the extract at 649 nm.

[0052] Extraction volume: the volume of ethanol used, in L.

[0053] Fresh weight: the weight of cotton leaves, in grams.

[0054] This step is used to determine the effects of GhIDL02 and GhPIPL7 peptides due to silencing expression or peptide treatment on photosynthetic pigment absorption and growth status.

[0055] In addition, the photosynthesis efficiency was further evaluated by a chlorophyll fluorescence meter purchased from IMAGING-PAM, Shanghai Zequan. The treated cotton seedlings were adapted to dark conditions for 30 min to eliminate the effect of instantaneous photosynthesis. Subsequently, the initial fluorescence F was measured using a chlorophyll fluorescence meter. o and maximum fluorescence F m , calculate F v / F m The value, that is, the maximum photochemical efficiency of photosystem II, was compared and analyzed to analyze the effect of salt stress on the efficiency of photosystem II, so as to evaluate the health of cotton photosynthesis.

[0056] In order to study the exogenous application effects of GhIDL02 and GhPIPL7 peptides, the present invention obtains these two small peptides by chemical synthesis. The synthesized small peptides are purified to ensure their biological activity. The small peptides are dissolved and sprayed on cotton leaves, or used in detached leaf experiments. By monitoring the growth and physiological indicators of the small peptide treatment group under salt stress, such as Na + / K + content, chlorophyll content, etc., to determine the regulatory effects of GhIDL02 and GhPIPL7 peptides on cotton salt tolerance.

[0057] In order to explore the molecular mechanism of GhIDL02 and GhPIPL7 peptides on cotton salt tolerance, the present invention also performs transcriptome sequencing analysis. In the experiment, cotton samples treated with salt stress and small peptides were collected, total RNA was extracted and quality tested. The transcriptome of the samples was sequenced and analyzed using the Illumina high-throughput sequencing platform. The differentially expressed gene DEGs were screened, combined with GO and KEGG pathway enrichment analysis, to further clarify the mechanism of action of GhIDL02 and GhPIPL7 peptides in regulating cotton salt tolerance.

[0058] The present invention also detects the phosphorylation level of the MPK3 / 6 signaling pathway to verify whether the GhPIPL7 small peptide regulates the salt tolerance of cotton by activating the MAPK signaling pathway. After the cotton seedlings are treated, the samples are quickly frozen and the total protein is extracted. Western blot analysis is performed using anti-phosphorylated MPK3 and MPK6 antibodies to detect changes in phosphorylation levels. By comparing the MPK3 / 6 phosphorylation levels of the treatment group and the control group, it is verified whether the small peptide regulates salt tolerance through the MAPK signaling pathway. At the same time, the present invention also synchronously constructs pYL156:GhMAPK3 and pYL156:GhMAPK6 cotton strains, performs gene silencing on MPK3 and MPK6, and cooperates with the exogenous application of PIPL07 to observe the phenotypic analysis of salt sensitivity of different materials. The present invention finally confirms that the PIPL07 peptide can improve the salt tolerance of cotton by activating MPK3 and MPK6 components.

[0059] Through the above technical scheme, the present invention comprehensively explains the molecular regulatory mechanism of GhIDL02 and GhPIPL7 in cotton salt tolerance, and provides an innovative and effective technical path for improving cotton salt tolerance.

[0060] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0061] Example 1

[0062] Application of a small peptide for improving crop tolerance to abiotic stress

[0063] Experiment 1: Analysis of the expression patterns of polypeptide precursor genes of GhIDLs and GhPIPLs under abiotic stress, as follows:

[0064] The present invention comprehensively analyzes the expression patterns of polypeptide precursor genes of GhIDLs and GhPIPLs in cotton leaves to determine the dynamic expression response patterns of these genes under abiotic stresses, such as drought, salt, cold and heat stress. The experiment uses RNA sequencing technology RNA-seq and qRT-PCR verification to show the changing trends of the expression levels of these genes under specific stress conditions. The experimental results show that Figure 1, under drought and salt stress, the expression of GhIDL02 and GhPIPL7 peptides was significantly upregulated. This upregulation dynamic became more significant with the extension of stress time, among which GhIDL02 maintained a continuous high level of expression under continuous salt and drought stress treatment. GhPIPL7 also showed a significant upregulation trend in various time periods of salt stress. 20% PEG treatment was used to simulate drought, and 20% PEG treatment was equivalent to 30% to 40% relative soil water content. These experimental results clearly indicate that GhIDL02 and GhPIPL7 may play a key regulatory role in the process of cotton coping with drought and salt stress. The correlation between the high expression of GhIDL02 and GhPIPL7 peptides and stress response provides new molecular targets and ideas for improving cotton tolerance to abiotic stress.

[0065] Figure 1 The relative expression levels of representative GhIDLs and GhPIPLs members under ddH2O, simulated drought stress, and salt stress conditions were validated using qRT-PCR data. The error represents the standard deviation SD, and the values ​​are the mean ± SD of three biological replicates, each of which contained three technical replicates.

[0066] Experiment 2: Silencing of the polypeptide precursor genes of GhIDL02 and GhPIPL7 inhibited the tolerance of cotton to salt and drought stress as follows:

[0067] Based on the previous analysis of peptide precursor expression patterns, the present invention further explored the specific functions of GhIDL02 and GhPIPL7 in improving cotton tolerance to salt and drought stress. To this end, the virus-induced gene silencing VIGS technology was used to silence the two background polypeptide precursor genes of cotton, and evaluate its ability to tolerate salt and drought stress in cotton. In the experiment of the present invention, the precursor gene coding sequences of GhIDL02 and GhPIPL7 were constructed and inserted into the pYL156 silencing vector to obtain two recombinant vectors pYL156-GhIDL02 and pYL156-GhPIPL7, which were transformed into Agrobacterium, respectively, and then the transformed Agrobacterium was injected into cotton cotyledons. The gene silencing efficiency was detected by qRT-PCR, and the results are shown in Figure 2. Figure 2. It was shown that on the 14th day after inoculation, pYL156-GhCLA1 was transformed. The CLA1 gene is involved in chloroplast development. The loss of CLA1 function resulted in an albino phenotype on the true leaves, providing an excellent visual marker for silencing efficiency. Cotton seedlings transformed with pYL156-GhIDL02 and pYL156-GhPIPL7 vectors showed significant bleaching and yellowing symptoms, and the expression levels of GhIDL02 and GhPIPL7 peptide precursor genes decreased significantly, verifying the effectiveness of VIGS technology in silencing these genes. These experimental data showed that the GhIDL02 and GhPIPL7 polypeptide genes were successfully silenced in cotton. Further experimental results showed that under normal irrigation conditions, cotton seedlings in all treatment groups did not show obvious stress symptoms. However, under salt and drought stress conditions, seedlings of GhIDL02 and GhPIPL7 polypeptide gene-silenced plants showed more obvious stress symptoms, mainly manifested in yellowing and wilting of leaves. Further physiological measurement results showed that the fresh weight, chlorophyll content and photosynthetic activity of GhIDL02 and GhPIPL7 silenced plants decreased significantly under salt and drought stress conditions. These results indicate that GhIDL02 and GhPIPL7 play an important role in cotton's resistance to salt and drought stress. Silencing GhIDL02 and GhPIPL7 by VIGS technology significantly inhibited cotton's tolerance to salt and drought abiotic stress, proving that the expression of GhIDL02 and GhPIPL7 peptide precursor genes has a positive regulatory effect on cotton stress response. This study provides a theoretical basis and experimental support for the development of polypeptide molecular regulation technology to improve cotton's salt and drought tolerance.

[0068] Experiment 3: Exogenous application of GhIDL02 and GhPIPL7 polypeptides enhanced cotton salt stress tolerance, as follows:

[0069] To further clarify the functions of GhIDL02 and GhPIPL7 polypeptides in regulating cotton salt stress tolerance, the present invention provides a conserved polypeptide motif derived from GhIDL02 and GhPIPL7 polypeptide precursor genes prepared by chemical synthesis technology, and the amino acid sequences of GhIDL02 and GhPIPL7 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0070] GhIDL02, SEQ ID NO. 1: PKGFVPPSGSSPCHN.

[0071] GhPIPL7, SEQ ID NO. 2: GIKDNSGPSTSGPGH.

[0072] In the experiment, the above polypeptide was sprayed on cotton seedlings at concentrations of 100nM, 1μM and 10μM. The salt stress experiment included two treatment schemes:

[0073] Protocol 1: Salt stress analysis of detached leaves under 200 mM NaCl stress conditions, see Figure 3 ;

[0074] Scheme 2: Under the condition of irrigating 500mM NaCl in the soil, the whole cotton plant was sprayed with peptides, and the spraying amount for each cotton plant was 2mL. Figure 4 and Figure 5 .

[0075] Through these two treatment schemes, the regulatory effects of GhIDL02 and GhPIPL7 peptides on cotton salt stress response were systematically evaluated. The results showed that cotton detached leaves without peptide application showed obvious growth inhibition under 200mM NaCl stress conditions, accompanied by leaf yellowing and wilting. Figure 3 In contrast, the detached leaves treated with GhIDL02 and GhPIPL7 peptides showed significant salt tolerance, especially at a concentration of 1 μM, where the leaves remained fresh, and their fresh weight and chlorophyll content were significantly higher than those of the control group. The above results indicate that exogenous application of GhIDL02 and GhPIPL7 peptides can effectively reduce the damage of cotton to salt stress. In addition, based on the results of chlorophyll fluorescence measurement, the F v / F m The ratio and Y(II) values ​​were significantly higher than those of the control group, indicating that these peptides can effectively improve the photosynthetic efficiency of cotton and reduce photooxidative damage. Under normal growth conditions, no significant difference was found between the treatment group and the control group, which further indicates that these peptides mainly exert their functions under salt stress.

[0076] The present invention further sprayed different concentrations of GhIDL02 and GhPIPL7 polypeptides using Scheme 2, and measured the Na in the leaves of cotton plants after spraying. + and K + Changes in content, see Figure 4 and Figure 5 The results showed that compared with the control group CK, cotton seedlings treated with GhPIPL7 peptide showed higher fresh weight and chlorophyll content, significantly improving the salt tolerance of cotton. At the same time, after spraying the peptide, the Na + The accumulation of K + The content increased significantly, thus effectively reducing the Na + / K + Ratio. + / K + The reduction of the ratio is an important physiological indicator for plants to maintain ion balance and improve salt tolerance under salt stress conditions.

[0077] The results showed that compared with the control group CK, cotton seedlings treated with GhIDL02 peptide showed higher fresh weight and chlorophyll content under salt stress conditions, significantly improving the salt tolerance of cotton. + The accumulation of K + The content increased significantly, thus effectively reducing the Na + / K + These results indicate that exogenous application of GhIDL02 and GhPIPL7 polypeptides can significantly enhance the adaptability of cotton to salt stress by regulating the balance of sodium and potassium ions.

[0078] Based on the above data, the present invention proves that exogenous application of GhIDL02 and GhPIPL7 polypeptides can significantly improve the salt tolerance of cotton through multiple mechanisms such as enhancing the photosynthesis efficiency of cotton, reducing photooxidative damage, and improving ion balance. The present invention provides a potential molecular tool for improving economic crops such as cotton under salt stress conditions, and has important agricultural application prospects and practical value.

[0079] Experiment 4: Analysis of transcriptional regulatory network of GhPIPL7-mediated upland cotton under salt stress, as follows:

[0080] In order to reveal the molecular regulatory mechanism of GhPIPL7 polypeptide in cotton salt stress tolerance, the present invention systematically analyzed the transcriptional regulatory network of cotton after GhPIPL7 polypeptide treatment using transcriptome sequencing technology RNA-seq. Figure 6 The experiment was divided into a control group CK treated with 500mM NaCl, a group T1 treated with 500mM NaCl, and a group treated with 1μM GhPIPL7 peptide. Figure 6The results showed that GhPIPL7 polypeptide treatment significantly changed the transcriptional regulation pattern of cotton, and the treatment group showed obvious transcriptional differences compared with the single salt treatment group. The results of DEG analysis of differentially expressed genes showed that under salt stress conditions, the expression of 1997 genes changed significantly, of which 1281 genes were upregulated and 716 genes were downregulated. These differentially expressed genes are involved in multiple biological processes. In order to further explore the molecular response mechanism under the regulation of GhPIPL7 polypeptide, the present invention performed KEGG pathway analysis on differentially expressed genes. The results showed that GhPIPL7 polypeptide treatment significantly affected key biological pathways such as plant-pathogen interaction, MAPK signaling pathway and plant hormone signaling. In the MAPK signaling pathway, there were 42 differentially expressed genes, most of which showed an up-regulated trend after GhPIPL7 polypeptide treatment. The key components of the MAPK signaling pathway include mitogen-activated protein kinase MAPK, LRR receptor-like serine / threonine protein kinase, serine / threonine protein kinase, and WRKY transcription factor. The significant expression changes of these genes indicate that the GhPIPL7 polypeptide precisely regulates the salt stress response of cotton through the MAPK signaling pathway. Previous studies have shown that these genes play an important role in plant response to adverse stress, indicating that the GhPIPL7 polypeptide can enhance the plant's ability to respond to salt stress by activating these genes.

[0081] In addition, the expression of genes related to plant hormone signaling in different hormone pathways also showed significant differences. Specifically, genes involved in hormone signaling pathways such as abscisic acid ABA, brassinolide BR, gibberellin GA, jasmonic acid JA and indole-3-acetic acid IAA showed complex regulatory patterns under GhPIPL7 polypeptide treatment. For example, jasmonic acid-related genes showed upregulation under the co-treatment of GhPIPL7 polypeptide and salt stress, while the expression pattern of auxin-related genes was more diverse, indicating that different hormone signaling pathways played different regulatory roles in the salt stress response mediated by GhPIPL7 polypeptide. In order to verify the accuracy of RNA sequencing results, the present invention selected some differentially expressed genes for qRT-PCR verification. Except that the expression pattern of Ghir_D03G12631 gene was different from the sequencing data, the qRT-PCR verification results of other genes were highly consistent with the RNA sequencing data, further proving the reliability of RNA sequencing results.

[0082] Based on these research results, the present invention proves that the GhPIPL7 polypeptide plays a key regulatory role in the process of cotton coping with salt stress by activating the MAPK signaling pathway and regulating multiple plant hormone signaling pathways. In particular, the GhPIPL7 polypeptide can improve the physiological state of cotton and significantly improve its salt tolerance by precisely regulating gene expression. This discovery provides an important theoretical basis and technical support for further using the GhPIPL7 polypeptide to improve crop salt tolerance.

[0083] Experiment 5: GhPIPL7 regulates cotton salt tolerance by activating MPK3 / 6, as follows:

[0084] The results of the present invention show that a considerable portion of the differentially expressed gene DEGs regulated by GhPIPL7 are significantly enriched in the MAPK signaling pathway. The MAPK signaling pathway is the core regulatory hub of plant stress response, in which MPK3 and MPK6, as key regulatory components, usually play an important role downstream of peptide signals and are crucial to the transmission of stress signals. In order to verify the role of MPK3 and MPK6 in GhPIPL7 regulating salt tolerance in upland cotton, the present invention evaluated the phosphorylation status of MPK3 and MPK6 in wild WT and pYL156-GhPIPL7 seedlings under salt stress conditions.

[0085] Figure 7 The results showed that under salt stress, the phosphorylation levels of MPK3 and MPK6 in WT cotton were significantly increased. Subsequently, 1 μM synthetic GhPIPL7 peptide was applied to WT seedlings, and the phosphorylation status of MPK3 and MPK6 was evaluated at specific time points. It was found that the exogenous application of GhPIPL7 peptide significantly induced the phosphorylation levels of MPK3 and MPK6. In contrast, after 200 mM NaCl treatment, the phosphorylation levels of MPK3 and MPK6 in the seedlings of the pYL156-GhPIPL7 interference strain did not increase significantly, indicating that the interference expression of GhPIPL7 significantly inhibited the activation of MPK3 and MPK6 under salt stress. These results suggest that GhPIPL7 may play an important regulatory role under salt stress conditions by regulating the MPK3 and MPK6 signaling pathways.

[0086] To further explore the regulatory role of MPK3 and MPK6 in GhPIPL7-mediated cotton salt tolerance, the present invention constructed pYL156-GhMPK3 and pYL156-GhMPK6 gene silenced cotton lines and conducted salt sensitivity analysis. Figure 7. The results showed that under normal growth conditions, after spraying different concentrations of GhPIPL7 peptide, there was no significant difference in the fresh weight and chlorophyll content of seedling leaves of pYL156, pYL156-GhMPK3 and pYL156-GhMPK6 lines. However, under 500mMNaCl treatment, pYL156 plants sprayed with different concentrations of GhPIPL7 peptide showed significant salt tolerance, while the seedling fresh weight and chlorophyll content of pYL156-GhMPK3 and pYL156-GhMPK6 gene silencing lines were significantly reduced compared with the pYL156 line, and were insensitive to the treatment of GhPIPL7 peptide. These results indicate that the GhPIPL7 peptide-mediated cotton salt tolerance mechanism depends on the regulation of MPK3 and MPK6 signaling pathways, further emphasizing the key role of the MAPK signaling pathway in GhPIPL7-mediated salt tolerance.

[0087] The present invention proposes a molecular regulation technology for cotton salt tolerance based on GhIDL02 and GhPIPL7 small peptides. Through the virus-induced gene silencing VIGS technology, the polypeptide precursor genes of GhIDL02 and GhPIPL7 are targeted and silenced to verify their key role in regulating cotton salt tolerance.

[0088] By measuring Na + , K + Physiological and biochemical parameters such as ion content, chlorophyll content and photosynthesis efficiency were used to evaluate the effects of gene-silenced plants or exogenous small peptide treatment on cotton salt tolerance. In addition, by exogenously applying GhIDL02 and GhPIPL7 small peptides and combining transcriptome sequencing analysis, the molecular mechanism of these small peptides in regulating cotton salt tolerance was further revealed. The present invention also verifies whether GhIDL02 and GhPIPL7 small peptides regulate cotton salt tolerance by activating the MAPK signaling pathway by detecting the phosphorylation levels of the MPK3 and MPK6 signaling pathways. The experimental design process is shown in Figure 8 This invention provides a new research idea for molecular breeding of cotton salt tolerance and has broad application prospects.

[0089] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0090] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A small peptide for improving the tolerance of crops to abiotic stress, characterized in that: The small peptide is GhIDL02 and / or GhPIPL7; The amino acid sequence of GhIDL02 is shown in SEQ ID NO.1; The amino acid sequence of GhPIPL7 is shown in SEQ ID NO.

2.

2. The use of the small peptide according to claim 1, characterized in that: The small peptide is used for improving the tolerance of crops to abiotic stress.

3. The use of the small peptide according to claim 2, characterized in that: The abiotic stress is at least one of salt stress, drought stress, flood stress, temperature stress, heavy metal stress and radiation stress.

4. The use of the small peptide according to claim 3, characterized in that: The salt concentration of the salt stress is not higher than 500 mM; The relative water content of the drought-stressed soil is 30% to 40%; The temperature of the temperature stress is lower than 0°C or higher than 35°C; The heavy metal stress is at least one of lead stress, mercury stress, cadmium stress and chromium stress; The radiation stress is at least one of ionizing radiation stress, ultraviolet radiation stress and electromagnetic radiation stress.

5. The use of the small peptide according to claim 2, characterized in that: The working concentration of the small peptide is 100 nM to 10 μM, and the application amount of the small peptide is 2 mL to 5 mL per seedling, or 16 L to 18 L per acre of crops.

6. The use of the small peptide according to claim 2, characterized in that: The small peptide is applied by spraying or irrigation.

7. The use of the small peptide according to claim 2, characterized in that: The small peptide is applied during the 3-4 leaf stage of the crop.

8. The use of the small peptide according to claim 2, characterized in that: The crop includes at least one of cotton, rice, wheat and corn.

9. A biological pesticide prepared using the small peptide according to claim 1.

10. The biopesticide according to claim 9, characterized in that: The biopesticide also includes auxiliary materials; The auxiliary material is at least one of an emulsifier, a dispersant, a wetting agent and a thickener.

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