Application of CLE11 small peptide gene in regulation and control of plant salt tolerance

Abstract

The invention discloses application of a CLE11 small peptide gene in regulation and control of plant salt tolerance, and belongs to the technical field of biological materials. According to the invention, foxtail millet is used as a research material, a SiPROCLE11 gene (CLE11 small peptide gene) is cloned, a SiPROCLE11 overexpression and silent foxtail millet material is constructed, an artificially synthesized small peptide SiCLE11p is externally applied, and the effect of the small peptide SiCLE11p on enhancing the salt tolerance of foxtail millet is determined. Furthermore, it is found that the Setaria italica can promote lateral root development of Setaria italica and reduce Na < + > accumulation in root systems, and a foundation is laid for digging of salt-tolerant related genes of Setaria italica and salt-tolerant breeding of

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the CLE11 small peptide gene in regulating plant salt tolerance. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Currently, large areas of land are severely affected by salinization. Salt stress refers to the negative effects of high concentrations of soluble salts on plant growth and development. Under salt stress, plants suffer from osmotic, ionic, and secondary stress, which affects their normal growth and development and adversely impacts physiological processes such as photosynthesis, seed germination, flowering, and fruiting.

[0004] Millet ( Setaria italica As one of the world's oldest domesticated diploid C4 spike crops, millet originated in the Yellow River basin of China during the Neolithic Age and is the earliest domesticated dryland crop in my country. Compared with other C4 plants, millet has advantages such as a short life cycle, a small diploid genome (approximately 490 Mb), a small number of chromosomes, abundant germplasm resources, an efficient transformation system, and outstanding tolerance to abiotic stresses. Therefore, millet has become an ideal model for studying plant physiological, biochemical, and molecular traits. In recent years, this typical C4 crop has demonstrated unique advantages in plant function research and has been widely used in research fields such as crop stress resistance mechanisms.

[0005] Plant peptides, a class of bioactive signaling molecules with a length of less than 20 amino acids, play an important role in regulating plant growth, development, and stress responses. Among them, CLE peptides have been shown to participate in regulating cell division and play a key role in various stress responses, including drought and temperature. The functions of CLE peptides in model plants such as Arabidopsis thaliana have been reported, but the salt tolerance regulatory mechanism of millet CLE peptides remains unexplored. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of the CLE11 small peptide gene in regulating plant salt tolerance. This invention uses millet as the research material and clones... SiPROCLE11 Genes, construction SiPROCLE11 Overexpression and silencing of millet materials, exogenous application of the synthetic peptide SiCLE11p, and its role in enhancing millet salt tolerance were determined. Further investigation revealed that it can promote lateral root development and reduce Na+ in the roots. + This accumulation of knowledge laid the foundation for the discovery of salt-tolerant genes in millet and for salt-tolerant millet breeding.

[0007] The application takes millet variety Ci846 as a material, and preliminarily explores the action mechanism of the gene through a series of experimental research and analysis, thereby providing a new idea and insight for crop salt tolerance improvement. SiPROCLE11 The application provides a CLE11 small peptide gene from millet

[0008] In order to achieve the above-mentioned purpose, the technical scheme of the application is as follows: In a first aspect, the application provides a CLE11 small peptide gene or a coded protein thereof. SiPROCLE11 The gene has a nucleotide sequence as shown in SEQ ID NO: 1, and the coded protein has an amino acid sequence as shown in SEQ ID NO: 2.

[0009] The CLE11 small peptide gene is a gene from millet SiPROCLE11 The CDS sequence of the gene (SETIT_007678mg) is 249 nucleotides in length.

[0010] The amino acid sequence of the protein as shown in SEQ ID NO: 2 is 82 amino acids in length.

[0011] In a second aspect, the application provides a CLE11 small peptide, which is a small peptide SiCLE11p having an amino acid sequence as shown in SEQ ID NO: 3.

[0012] RLSPGGPNPQHH (SEQ ID NO: 3).

[0013] The small peptide SiCLE11p is a functional fragment in the protein as shown in SEQ ID NO: 2.

[0014] In a third aspect, the application provides a biological material comprising the above-mentioned gene or coded protein thereof or the above-mentioned CLE11 small peptide.

[0015] The biological material includes an expression cassette, a recombinant expression vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, a transgenic plant tissue, a transgenic plant, a tissue culture produced by regenerable cells of the transgenic plant, or a protoplast produced by the tissue culture.

[0016] The transgenic cell line can be isolated, ex vivo, cultured, or preferably part of a plant.

[0017] The recombinant microorganism can be a eukaryotic fungus (such as a fungus, specifically a yeast fungus, etc.) or a prokaryotic fungus (such as a bacterium, specifically an Escherichia coli, a Bacillus, an Agrobacterium, etc.).

[0018] The plant can be a crop, and more specifically a cereal crop, such as millet.

[0019] The recombinant expression vector is obtained through the above-mentioned millet. SiPROCLE11 Genes are obtained by effectively ligating them into an expression vector, which may be any one or more of a viral vector, plasmid, phage, granule, or artificial chromosome.

[0020] according to SiPROCLE11 Gene design cloning primers are used to perform PCR amplification with the nucleotide sequence of a gene as a template. The cloned product is then ligated to an expression vector. After correct sequencing, the expression vector is transformed into plant cells using Agrobacterium-mediated transformation to obtain transgenic plants.

[0021] Fourthly, the present invention provides an application of the above-mentioned gene or its encoded protein, or the above-mentioned CLE11 peptide, or the above-mentioned biological material in any one or more of the following: (a1) Application in regulating plant salt tolerance; (a2) Application in screening, identifying, and cultivating salt-tolerant plants or improving salt-tolerant plant germplasm resources.

[0022] Fifthly, the present invention provides the application of the above-mentioned gene or its encoded protein, or the above-mentioned CLE11 peptide, or the above-mentioned biological material as a target for improving plant salt tolerance modification, thereby promoting the above-mentioned SiPROCLE11 The expression of the aforementioned genes or proteins can enhance the salt tolerance of plants. Specifically, overexpressing the genes or proteins in plants can improve their salt tolerance, while knocking down, eliminating, or inhibiting their expression will decrease their salt tolerance.

[0023] In a sixth aspect, the present invention provides the application of increasing the expression level of the above-mentioned gene or its encoded protein or the above-mentioned CLE11 peptide or the above-mentioned biological material in improving plant salt tolerance.

[0024] The functions that improve plant salt tolerance include any one or more of the following: (b1) Regulate the fresh weight, dry weight, root length and aboveground part length of plants under salt stress; (b2) Regulating Na+ in plants under salt stress + K + content; (b3) Regulate the chlorophyll content of plants; The chlorophyll includes chlorophyll a and chlorophyll b.

[0025] The salt stress is carried out under 100~200mM NaCl, preferably 150 mM NaCl.

[0026] Under salt stress, the overexpressed plants can realize significant increase of fresh weight, dry weight, root length and length of aboveground part of the plants; can realize lower Na + content, higher K + content, and higher chlorophyll content. + + The method can also realize lower Na

[0027] The seventh aspect of the present application provides a method for improving salt tolerance of plants, which promotes expression of the above-mentioned gene or its encoded protein or the above-mentioned CLE11 small peptide or the above-mentioned biological material in the plants.

[0028] The plants are cereal crops, preferably millet.

[0029] The method specifically comprises promoting expression of the millet SiPROCLE11 gene or protein in the plants by using genetic engineering means. The genetic engineering means comprises: introducing an expression vector carrying a target gene into plant cells by using Ti plasmid, plant virus vector or direct DNA transformation technology.

[0030] The eighth aspect of the present application provides a method for screening, identifying and cultivating salt-tolerant plants or improving salt-tolerant germplasm resources of plants, which detects expression level of the above-mentioned gene or its encoded protein or the above-mentioned CLE11 small peptide or the above-mentioned biological material in the plants, and judges salt tolerance of the plants according to the expression level: The expression level of the above-mentioned gene or its encoded protein or the above-mentioned CLE11 small peptide or the above-mentioned biological material is positively correlated with salt tolerance of the plants.

[0031] The plants are cereal crops, preferably millet.

[0032] Further, the reagent for detecting the expression level of the gene comprises a forward primer shown in SEQ ID NO: 4 and a reverse primer shown in SEQ ID NO: 5.

[0033] One or some of the above technical solutions have the following advantages or beneficial effects: The present application provides a gene SiPROCLE11 for regulating salt tolerance function of millet. By using a stable overexpression transformation system and a VIGS gene silencing system of millet, it is proved that the gene SiPROCLE11 plays a positive regulation role under salt stress, and can enhance salt tolerance of the millet. The present application provides a gene resource for studying salt tolerance of plants, and has important theoretical significance and application value.

[0034] The present application provides a gene SiPROCLE11 for regulating salt tolerance function of millet. By externally applying an artificial synthetic small peptide SiCLE11p, it is proved that the small peptide can reduce Na​+ accumulation, and enhancing the salt tolerance of millet. The present application provides a gene resource for studying plant salt tolerance, and has important theoretical significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0035] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the present application, and their

[0036] Figure 1 The salt stress expression analysis of the millet CLE gene (SiCLE11) and the subcellular localization analysis of the millet CLE protein (SiCLE11 protein) are shown in FIG. 2; wherein A is the expression analysis of the millet CLE gene under different salt stress times, and B is the subcellular localization of the millet CLE protein. SiPROCLE11 The salt stress expression analysis of the millet CLE gene (SiCLE11) and the subcellular localization analysis of the millet CLE protein (SiCLE11 protein) are shown in FIG. 2; wherein A is the expression analysis of the millet CLE gene under different salt stress times, and B is the subcellular localization of the millet CLE protein. Figure 2 The screening of transgenic lines positive seedlings is shown in FIG. 3; wherein (A) is hygromycin identification, (B) is DNA level identification, and (C) is RNA level identification. Figure 3 The phenotype and biomass analysis of wild type and transgenic millet under salt stress is shown in FIG. 4; wherein (A) is the phenotype of each line of the control group millet, (B) is the phenotype of each line of the salt treatment group millet, (C) is the overall phenotype of each line of the millet under salt treatment, (D) is total fresh weight, (E) is total dry weight, and (F) is root length. Figure 4 The physiological index determination of overexpression lines under salt stress is shown in FIG. 5; wherein (A) is Na + content, (B) is K + content, (C) is Na + / K + , (D) is chlorophyll a, (E) is chlorophyll b, and (F) is total chlorophyll. Figure 5 The physiological index determination of overexpression lines under salt stress is shown in FIG. 5; wherein (A) is relative conductivity, (B) is MDA content, (C) is DAB staining, and (D) is NBT staining. Figure 6 The effect of salt treatment on the proton secretion of millet is shown in FIG. 6; wherein (A) is the original proton secretion graph, and (B) is the sharpened proton secretion graph. Figure 7 The phenotype and biomass determination of gene silencing lines under salt stress is shown in FIG. 7; wherein (A) is RNA level identification, (B) is a phenotype graph, (C) is total fresh weight, (D) is root length, and (E) is the length of the aboveground part. SiPROCLE11 The physiological index determination of gene silencing lines under salt stress is shown in FIG. 8; wherein (A) is NBT staining, and (B) is the determination of relative conductivity. Figure 8 SiPROCLE11 The physiological index determination of gene silencing lines under salt stress is shown in FIG. 8; wherein (A) is NBT staining, and (B) is the determination of relative conductivity. ​Figure 9 Effects of SiCLE11p exogenous leaves on salt tolerance of foxtail millet seedlings under salt stress; wherein, (A) overall phenotype, (B) local phenotype, (C) total fresh weight, (D) root length, (E) total dry weight, (F) root fresh weight. DETAILED DESCRIPTION

[0037] TERMS: ABA refers to: abscisic acid.

[0038] 6-KT refers to: one of the cell division factors.

[0039] JA refers to: jasmonic acid.

[0040] BF refers to: bright field.

[0041] GFP refers to: green fluorescent protein.

[0042] Merge refers to: merged image.

[0043] 35S:GFP refers to: 35S promoter and green fluorescent protein.

[0044] 35S: SiPROCLE11 -GFP refers to: 35S promoter and CLE11 gene and green fluorescent protein.

[0045] WT refers to: Wild Type, wild type.

[0046] OE refers to: Overexpression, overexpression strain.

[0047] OE1 refers to: overexpression strain 1.

[0048] OE2 refers to: overexpression strain 2.

[0049] OE3 refers to: overexpression strain 3.

[0050] pTRV:00 refers to: gene silencing vector without inserting any target gene fragment.

[0051] pTRV: SiPROCLE11 refers to: gene silencing vector inserted SiPROCLE11 with genes.

[0052] CK-pTRV:00 refers to: control group of pTRV:00 plants with empty vector.

[0053] CK-pTRV: SiPROCLE11 refers to: control group of plants with silenced SiPROCLE11 genes.

[0054] ST-pTRV:00 means: salt treatment group of the plants of empty vector pTRV:00.

[0055] ST-pTRV: SiPROCLE11 means: salt treatment group of the plants of the silenced SiPROCLE11 gene.

[0056] In the present application, if no special description, other test materials and instruments and equipment are conventional test materials in the art, which can be purchased through commercial channels.

[0057] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0058] Example 1: SiPROCLE11 CDS sequence of the gene ATGAAGCTCGTCCTGTGCTTGTGCATCTGCGTCGTCCTAGTCATCGCTTCGTCTCCGGTCCCACTCTCGGATAGCAGCCCTTCTCTGGGGTGGCGCTGGCTGCAGGACGCTGTCATCGTCGCCAACGGCCCAGCGGCGAACACAACCACTTCCTTTGGCCCCGCTTGGTCCCAGGAGCCACCGGCGGAGGTACCCTGCGACGGGTCCAAGAGGCTCAGCCCCGGCGGTCCGAACCCGCAGCATCACTGA (SEQ ID No: 1).

[0059] Example 2: Amino acid sequence of SiPROCLE11 protein MKLVLCLCICVVLVIASSPVPLSDSSPSLGWRWLQDAVIVANGPAANTTTSFGPAWSQEPPAEVPCDGSKRLSPGGPNPQHH (SEQ ID No: 2).

[0060] Example 3: SiPROCLE11 Gene cloning and expression vector construction (1) Millet total RNA extraction and cDNA synthesis In order to obtain SiPROCLE11 the complete nucleotide fragment encoded by the gene, the present application uses the Novagen RNA extraction kit to extract plant RNA, and the specific operation steps are operated according to the instruction manual. The total RNA of the young leaf of millet is extracted. According to the Evo M-MLV reverse transcription premix reagent kit instruction manual (Sains Technology Co., Ltd.), the cDNA of millet is synthesized.

[0061] (2) SiPROCLE11 Gene cloning The forward and reverse specific primers were designed using the NCBI Primer-BLAST online website, the primer names were SiCLE11-F2 and SiCLE11-R2, and the primer sequences were as follows: SiCLE11-F2: ATGAAGCTCGTCCTGTGC (SEQ ID No: 4).

[0062] SiCLE11-R2: GTGATGCTGCGGGTTCGGAC (SEQ ID No: 5).

[0063] The specific primers SiCLE11-F2 and SiCLE11-R2 were used for amplification with the millet cDNA as a template, and the coding region of the gene, i.e., the CDS sequence, was isolated and cloned by the method of PCR. SiPROCLE11

[0064] Table 1

[0065] (3) VIGS silencing vector construction SiPROCLE11 The gene was used as a template, and the following specific primers were used for amplification.

[0066] The VIGS silencing strain used pTRV2 as a vector. The PCR amplification product and the double enzyme digestion product were connected by using a homologous recombinase; 10 μL of the connection system was as follows: 2×CE II Buffer: 2 μL; Exnase II: 1 μL; double enzyme-digested vector: 3 μL; PCR amplification product: 2 μL; ddH2O: 2 μL; 37℃ reaction for 30 min, then reduced to 4℃ or immediately placed on ice for cooling. The DH5α competent cells were thawed on ice, and the above 5 μL recombinant product was added to 100 μL of the competent cells, mixed uniformly by flicking the tube wall, and placed on ice for 30 min. After 42℃ water bath heat shock for 45 s, immediately placed on ice for cooling for 2 min. 700 μL of LB medium without antibiotics was added, and the bacteria were shaken at 200 rpm and 37℃ for 1 h. The LB solid medium containing kanamycin resistance was plated in a 37℃ incubator. Centrifuged at 5000 rpm for 3 min, and the supernatant was discarded. The bacterial cells were resuspended, and a sterile spreader was used to evenly spread on the plate containing kanamycin resistance. Inverted culture in a 37℃ incubator for 12 h. The colonies that were positive in the colony PCR were inoculated into liquid LB medium containing kanamycin and cultured overnight, and the plasmid was extracted and sequenced.

[0067] Example 4: Millet SiPROCLE11 Expression pattern analysis of the gene ​SiActin-F and SiActin-R as internal reference primers, SiCLE11-F1 and SiCLE11-R1 as gene quantitative primers, and the roots of foxtail millet treated with 150 mM NaCl salt for different time periods (0 h, 6 h, 12 h, 24 h, and 48 h) as templates, the experimental method referring to the Eco Real quantitative kit, and 3 replicates for each treatment. Foxtail millet SiPROCLE11 The relative expression amount of the gene under different salt stresses was calculated according to 2 --ΔΔCt Graphpad prism 8 software was used for plotting.

[0068] SiActin-F: CGCATATGTGGCTCTTGACT (SEQ ID No: 10); SiActin-R: GGGCACCTAAATCTCTCTGC (SEQ ID No: 11); SiCLE11-F1: CGTCCTAGTCATCGCTTCGT (SEQ ID No: 12); SiCLE11-R1: AGAAGGGCTGCTATCCGAGA (SEQ ID No: 13).

[0069] The expression of the gene in foxtail millet was analyzed by RT-qPCR technology under salt treatment for 0 h, 6 h, 12 h, and 24 h, SiPROCLE11 The expression of the gene in foxtail millet was analyzed by RT-qPCR technology under salt treatment for 0 h, 6 h, 12 h, and 24 h, Figure 1 A). The quantitative analysis results show that under the condition of salt stress, SiPROCLE11 showed a typical stress response characteristic, and the expression amount was significantly up-regulated at the initial stage of salt stress, and then showed a steady downward trend. This indicates SiPROCLE11 may be a positive regulatory factor in the regulation of foxtail millet salt tolerance response process.

[0070] Example 5: SiPROCLE11 Subcellular localization analysis of the gene 10 μL of the recombinant expression vector pCAMBIA1300- SiPROCLE11-GFP, empty vector pCAMBIA1300-GFP, P19 plasmid were added into 100 μL Agrobacterium competent cells respectively, mixed gently by blowing and sucking, and then sequentially placed on ice for 5 min, in liquid nitrogen for 5 min, at 37 °C for 5 min, and on ice for 5 min; in the clean bench, 900 μL sterile LB liquid medium was added into a centrifugal tube, which was cultured in a 28 °C Agrobacterium incubator for 2.5 h; centrifuged at 5000 rpm for 2 min, the volume of the supernatant was 100 μL, and 30 μL of the resuspended bacterial solution was evenly coated on the medium containing the corresponding resistance after resuspension by blowing; and cultured at 28 °C for 12-16 hours. The successfully transformed pCAMBIA1300- SiPROCLE11 GV3101 Agrobacterium of GFP and pCAMBIA1300-GFP, and GV3101 Agrobacterium containing P19 plasmid.

[0071] The successfully transformed pCAMBIA1300- SiPROCLE11 GV3101 Agrobacterium of GFP and pCAMBIA1300-GFP, and GV3101 Agrobacterium containing P19 plasmid were inoculated in LB liquid medium, and kanamycin and rifampicin were added to the medium; and cultured at 28 °C overnight until turbid; after the bacterial solution was turbid, it was transferred to 50 mL of new LB liquid medium containing kanamycin and rifampicin resistance and 10 μL of 20 μM acetosyringone (AS); and cultured in a 28 °C incubator; MMA solution was prepared: 10 mM MgCl2, 10 mM MES, and the pH value was adjusted to 5.6, then 100 μM AS was added; when the OD 600 =0.9-1, centrifuged at 3000 g for 10 min, the supernatant was discarded, and the bacterial solution was resuspended with 20 mL of MMA; after being placed in the dark for 3 h, it was mixed with P19 at a ratio of 1:1; the tobacco was watered and placed in the dark for 8 h, then injected from the back of the leaf, and the whole leaf was injected. After the injection was completed, the culture was continued for 36-48 h, and the fluorescence signal was observed using a two-photon laser confocal microscope (Leica TCS SP8 MP, Germany).

[0072] To explore the biological function of SiPROCLE11 , the subcellular localization of SiCLE11 protein was analyzed. Previous studies have shown that CLE small peptides belong to a class of post-translationally modified secretory peptides, which are usually located in the extracellular space. The subcellular localization results showed that SiCLE11 protein may be located on the outside of the cell membrane (Fig. 1). Figure 1B). Based on the results of physicochemical property analysis of SiCLE11 protein, it is speculated that SiCLE11 protein may be secreted to the extracellular space through the endoplasmic reticulum-Golgi secretory pathway, and then play its biological function as an extracellular signaling molecule through post-translational modification processes such as proteolytic processing and proline hydroxylation.

[0073] Example 6: Stable transformation SiPROCLE11 Gene The foxtail millet young embryos were isolated, disinfected with 70% alcohol and 12% (v / v) bleach solution, and then washed thoroughly with sterile water. Subsequently, the disinfected foxtail millet young embryos were heat-treated for 3 min at 43°C in a medium containing Agrobacterium solution for infection. The infected young embryos were transferred to co-cultivation solution and stored in the dark for 2-3 d, and then transferred to a dormancy medium for further culture. After the young embryos formed callus, they were transferred to MS medium for continuous culture until germination. Subsequently, they were transferred to rooting medium to promote root growth, and after the callus grew healthy roots, the transgenic plants were transplanted to pots with mixed substrate for hardening-off. Subsequently, the identified positive lines were multiplied and purified in an artificial climate chamber, and seeds were collected, with the specific culture conditions being: 28-30°C (16 h) during the day and 20-23°C (8 h) at night, with a relative humidity of 70%.

[0074] The harvested seeds were sown in a culture dish containing hygromycin, and the germination rate was counted after 7 d of culture in a 25°C light incubator. Seven positive transformation lines with a germination rate of ≥ 95% were obtained by screening (e.g., OE1, OE2, OE3, OE4, OE5, OE6, and OE7). Figure 2 A), the DNA of the seven lines obtained by screening was used as a template for PCR amplification with the gene forward primer F / gene reverse primer R, and the gene forward primer F / vector GFP region reverse primer R, respectively, and the amplified bands are shown in Fig. Figure 2 B). As can be seen from the figure, all seven lines are positive. These results confirm that SiPROCLE11 the gene was successfully overexpressed in foxtail millet. In addition, the transgenic materials were analyzed at the transcriptional level by RT-qPCR technology. The results showed that the expression levels of all overexpression lines were significantly up-regulated compared with WT (e.g., Fig. SiPROCLE11 C). Figure 2

[0075] Based on the results of hygromycin, DNA, and RNA identification, the OE1, OE2, and OE3 lines with moderate expression levels and good genetic stability were selected for subsequent experiments.

[0076] Example 7: SiPROCLE11 Determination of growth indicators of overexpression and silencing lines of foxtail millet The phenotypes of the lines were recorded by taking photos after 7 d of treatment with 150 mM NaCl.​

[0077] As Figure 3 and Figure 7 shown, under salt stress, the growth of the overexpression lines is better than that of the wild type, and the fresh weight, dry weight and root length are significantly increased; under salt stress, the growth of the silencing lines is more obviously inhibited, and the fresh weight, root length and aboveground height are lower than those of the wild type.

[0078] Example 8: SiPROCLE11 The Na + , K + content of the overexpression lines of Setaria italica was determined 0.3 g of sample was weighed in a test tube, 10 mL of water was added to each test tube, and the mouth of each test tube was sealed with a sealing film. A small hole was made on the sealing film with a toothpick. The test tube was placed in an electromagnetic oven and boiled for 2-3 h. After cooling to room temperature, the liquid in the test tube was filtered into a 25 mL volumetric flask using a funnel, and water was added to make the volume to 25 mL. The solution was filtered into a 10 mL centrifuge tube through a 0.22 μm filter column using a 1 mL syringe, and the flame spectrophotometer was preheated before determining the Na + , K + content. Ion content (mmol / g FW) =

value × constant volume (0.025 L) × dilution multiple

atomic mass of the element (M) × sample mass (g)

[0079] The results show that, as Figure 4 (A-C) shown, after salt stress treatment, the Na + content is lower, the K + content is higher, the Na + / K + is lower, which can effectively maintain ion homeostasis and improve the salt tolerance of Setaria italica.

[0080] Example 9: SiPROCLE11 Determination of chlorophyll content of overexpression lines of Setaria italica After fresh Setaria italica leaves were cut and mixed evenly, 0.1 g was weighed in a 1.5 mL centrifuge tube, and the sample was broken by a sample machine. 1 mL of 80% acetone was added, mixed well, and placed in a 28-degree shaking bed for 12 h of incubation. After incubation, centrifugation was performed at 4°C, 12000 rpm, for 15 min. The supernatant was taken and diluted 5 times with 80% acetone. 80% acetone was used as a blank control, and the absorbance at 645 nm and 663 nm was determined by ultraviolet spectrophotometry.

[0081] Total chlorophyll Ct (mg / L) = (8.02 A663+20.21 A645) × constant volume (L) / fresh weight (g) Chlorophyll content (mg / g FW) = (Ct x total volume of extract x dilution factor) / fresh weight of sample As Figure 4 As shown in SiPROCLE11 The overexpression lines have higher chlorophyll content.

[0082] Example 10: SiPROCLE11 Conductivity determination of overexpression and silencing lines of Setaria italica Fresh Setaria italica leaves were cut into equal length fragments and mixed uniformly, and 0.3 g was weighed in a clean test tube. After standing for 4 h, the original conductivity of the Setaria italica leaves was determined using a conductivity meter preheated for more than 30 min. The total conductivity was determined after boiling for 5 min. Relative conductivity = original conductivity / total conductivity x 100%.

[0083] As Figure 5 (A) and Figure 8 (B) show that under salt stress, compared with the wild type, the relative conductivity content of the overexpression lines is lower, and the salt tolerance is improved; under salt stress, the silencing lines have higher relative conductivity, and the salt tolerance is significantly reduced.

[0084] Example 11: MDA content determination The MDA content determination in this study used the Malondialdehyde (MDA) content detection kit (upgrade version) of Solabio Company, with product number BC6410.

[0085] As Figure 5 (B) show that under salt stress, compared with the wild type, the MDA content of the overexpression lines is lower, indicating that the oxidative stress of the overexpression lines is lower than that of the wild type, and they are more salt-tolerant.

[0086] Example 12: DAB and NBT staining Both of the two drugs used for staining were purchased from Coolaber Company, with CAS number 868272-85-9 for DAB (3'3-diaminobenzidine tetrahydrochloride) and CAS number 298-83-9 for NBT (chloronitro tetrazolium blue).

[0087] DAB staining: 1 g / L DAB staining solution was prepared, pH=5.8, and the same position and size of the leaves of each line after treatment with 0 mM NaCl and 150 mM NaCl for 7 days were cut. The leaves were completely immersed in the staining solution, and after 12 h of 28°C in the dark, 80% ethanol was used for decolorization until the leaves were green, and then photographed and recorded.

[0088] NBT staining: 0.5 g / L NBT staining solution was prepared, and NBT was dissolved in PBS (pH 7.2-7.4) with 0 mM NaCl and 150 mM NaCl, respectively. The same position and size of the leaves of each strain after 7 days of treatment were taken. The leaves were completely immersed in the staining solution, and after 3 h of dark incubation at 28°C, 80% ethanol was used for decolorization until the leaves were green. Photographs were taken for recording.

[0089] It was found by DAB and NBT staining that the staining degree of the three overexpression strains was lighter than that of WT under salt stress condition Figure 5 C-D), indicating that the overexpression strains have stronger ability to scavenge reactive oxygen species. The NBT staining result of the silencing strain is deeper than that of the wild type Figure 8 A), and the above indicates that SiPROCLE11 the gene has the ability to scavenge reactive oxygen species, making the plant more salt-tolerant.

[0090] Example 13: Proton secretion detection Hygienic paper and filter paper were sequentially laid on a culture dish, 20 mL of deionized water was added, and then millet seeds were placed on the culture dish for culture for about 14 d. After two leaves grew out, the millet seeds were treated with 50 mM NaCl for 3 days. Then the millet seedlings were washed clean and pressed into the bromocresol purple acid-base indicator medium. The composition of the medium was 0.75%(W / V) Agar, 0.006%(W / V) bromocresol purple, 1 mmol / L K2SO4, and pH 5.6-6.0. After 5 h of culture in the dark, the color change of the medium was observed and photographed.

[0091] The proton secretion experiment showed that under the condition of 50 mM NaCl treatment, the proton secretion rate of millet seedlings decreased significantly, however, the proton secretion rate of the overexpression strain was still significantly higher than that of the wild type Figure 6 .

[0092] Example 14: Identification of millet salt tolerance by SiCLE11p small peptide leaf spraying method When the length of the above-ground part of the millet was about 6-7 cm, the seedlings with consistent growth state were selected for the experiment, and two groups of non-salt treatment and 150 mM NaCl treatment were set. 1 µM small peptide SiCLE11p was sprayed on the leaves of the millet, and the control group was sprayed with the same amount of sterile water. Each group was treated in triplicate.

[0093] The small peptide SiCLE11p (RLSPGGPNPQHH) was synthesized by Beijing Qikexin Biotechnology Co., Ltd. Qingdao Branch.

[0094] Phenotypic observation results showed that under normal growth conditions, the growth status of millet plants treated with SiCLE11p was not significantly different from that of the control. Under salt stress conditions, the growth status of millet plants treated with SiCLE11p was significantly better than that of the control group. Figure 9 (AB), indicating that SiCLE11p can alleviate the inhibitory effect of salt stress on millet growth. Biomass analysis results showed that under normal growth conditions, SiCLE11p treatment increased the fresh weight, dry weight, and root fresh weight of millet to some extent, while root length had no significant effect. However, under 150 mM NaCl treatment, compared with the control group, SiCLE11p treatment significantly increased the fresh weight of millet (AB). Figure 9 C) Dry weight ( Figure 9 E), Root length ( Figure 9 D) and fresh root weight ( Figure 9 F).

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A CLE11 small peptide gene or its encoded protein, characterized in that, The gene is millet SiPROCLE11 a gene whose nucleotide sequence is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO:

2.

2. A CLE11 small peptide, characterized in that, The CLE11 small peptide is a small peptide SiCLE11p, the amino acid sequence of which is shown as SEQ ID NO:

3.

3. A biomaterial, characterized by, The CLE11 small peptide gene or the encoded protein thereof of claim 1 or the CLE11 small peptide of claim 2.

4. The biomaterial of claim 3, wherein, The biological material includes an expression cassette, a recombinant expression vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, a transgenic plant tissue, a transgenic plant, a tissue culture produced by regenerable cells of a transgenic plant, or a protoplast produced by a tissue culture; Preferably, the transgenic cell line is isolated, ex vivo, cultured, or preferably part of a plant. Preferably, the plant is a crop, preferably a cereal crop, and further preferably millet. Preferably, the expression vector can be any one or more of a viral vector, a plasmid, a phagemid, a cosmid, or an artificial chromosome.

5. Use of the CLE11 small peptide gene of claim 1 or the encoded protein thereof or the CLE11 small peptide of claim 2 or the biological material of claim 3 or 4 in any one or more of the following: (a1) use in regulating salt tolerance of a plant; (a2) use in screening, identifying, breeding, or improving salt-tolerant plants or plant salt-tolerant germplasm resources.

6. Use of the CLE11 small peptide gene of claim 1 as a target for improving plant salt tolerance modification, characterized in that, Promote expression of the CLE11 small peptide gene, thereby improving salt tolerance of a plant.

7. Use of the CLE11 small peptide gene of claim 1 or the encoded protein thereof or the CLE11 small peptide of claim 2 or the biological material of claim 3 or 4 in any one or more of the following: (b1) use in regulating fresh weight, dry weight, root length, and aboveground height of a plant under salt stress; (b2) modulating the Na + , K + content of a plant under salt stress; (b3) use in regulating chlorophyll content of a plant; (b4) use in improving salt tolerance of a plant.

8. A method for increasing salt tolerance in plants, characterized by, Promote expression of the CLE11 small peptide gene of claim 1 or the encoded protein thereof or the CLE11 small peptide of claim 2 or the biological material of claim 3 or 4 in a plant.

9. A method for screening, identifying, breeding salt-tolerant plants or improving salt-tolerant germplasm of plants, characterized in that, Detect expression level of the CLE11 small peptide gene of claim 1 or the encoded protein thereof or the CLE11 small peptide of claim 2 or the biological material of claim 3 or 4 in a plant, and determine salt tolerance of the plant according to the expression level. The expression level of the CLE11 small peptide gene of claim 1 or the encoded protein thereof or the CLE11 small peptide of claim 2 or the biological material of claim 3 or 4 is positively correlated with salt tolerance of a plant.

10. The method of claim 9, wherein, The reagent for detecting the expression level of the gene includes a forward primer shown as SEQ ID NO: 4 and a reverse primer shown as SEQ ID NO: 5.

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