Application of LbSNAP331 in salt resistance of plants

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of LbSNAP331 in salt resistance of plants. Specifically, a gene highly responsive to salt stress is screened from an LbSNARE gene family of limonium bicolor, and the gene is named as LbSNAP331. Researches show that the gene is positioned on salt glands, and the gene regulates the number of the salt glands and the secretion rate of a single salt gland, reduces the Na < + >, Na < + > / K < + > content and active oxygen content of the limonium bicolor and resists oxidative stress, so that the salt resistance and salt gland development conditions of the limonium bicolor are positively regulated. The invention lays a foundation for explaining the plant salt resistance mechanism and improving the utilization efficiency of the saline-alkali soil, so that the application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of LbSNAP331 in plant salt resistance. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Soil salinization is a global environmental problem. Screening and cultivating salt-tolerant crops can improve the economic output of saline-alkali land. Salinized soils are an important reserve land resource in my country and also contain rich salt-resistant resources. Therefore, conducting targeted research on salt-tolerance mechanisms and fully exploring and utilizing the salt-resistant gene resources of halophytes are of great significance for accelerating the development of new salt-tolerant crop varieties and the development and utilization of saline-alkali land.

[0004] Limonium bicolor ( Limonium bicolor Bunge and Kuntze adapt to saline environments by secreting salt through their salt glands. Research has shown that vesicle transport is involved in salt gland secretion, and SNARE proteins, as core components that facilitate vesicle-membrane fusion, play a crucial role in this process. SNARE proteins are transmembrane proteins that mediate fusion of vesicles with target membranes, forming SNARE complexes (composed of multiple SNARE proteins). The SNARE gene family has numerous members with diverse functions, necessitating further research. Summary of the Invention

[0005] In order to solve the deficiencies in the prior art, the present invention aims to provide an application of LbSNAP331 in plant salt resistance. LbSNARE In the gene family, a gene that is highly responsive to salt stress was screened and named LbSNAP331 Studies have shown that this gene is located in the salt glands and regulates the number of salt glands and the secretion rate of a single salt gland, while reducing the Na + 、Na + / K + The content and active oxygen content of Limonium bicolor can resist oxidative stress, thereby positively regulating the salt resistance and salt gland development of Limonium bicolor. Based on the above research results, the present invention was completed.

[0006] Specifically, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a gene named LbSNAP33 , the nucleotide sequence of the gene is as follows: (a1) the nucleotide sequence shown in SEQ ID NO. 1; (a2) a nucleotide sequence which encodes the same amino acid sequence as the nucleotide sequence of (a1) but differs in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence which has > 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (completely) sequence) identity to the nucleotide sequence shown in (a1) or (a2) and encodes the same functional protein; (a4) a nucleotide sequence complementary to any one of (a1) to (a3).

[0007] In a second aspect of the present application, a protein is provided, which is encoded by the above-mentioned gene.

[0008] The Lythrum salicaria protein has any one of the amino acid sequences shown in (b1) to (b3): (b1) the amino acid sequence shown in SEQ ID NO. 2; (b2) a protein derived from the amino acid sequence shown in SEQ ID NO. 2 by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function as the amino acid sequence shown in SEQ ID NO. 2; (b3) a protein encoded by another gene which has > 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (completely) sequence) identity to the amino acid sequence shown in SEQ ID NO. 2 and has the activity of the protein shown in SEQ ID NO. 2.

[0009] In a third aspect of the present application, a recombinant expression vector, a transgenic cell line, a host microorganism or a transgenic plant containing the above-mentioned gene is provided.

[0010] In a specific embodiment of the present application, the recombinant expression vector is obtained by effectively linking the above-mentioned Lythrum salicaria gene to an expression vector, which can be any one or more of a viral vector, a plasmid, a phagemid, a cosmid or an artificial chromosome, without being specifically limited herein.

[0011] The transgenic cell line can be isolated, ex vivo, cultured or, preferably, part of a plant.

[0012] The host microorganism can be eukaryotic or prokaryotic, such as fungi, bacteria, etc., without being specifically limited herein.

[0013] The transgenic plant can be a halophyte, and in a specific embodiment of the present application, the plant is Limonium bicolor.

[0014] In a fourth aspect of the present application, the above-mentioned gene LbSNAP33 , protein, recombinant expression vector containing the above-mentioned gene, transgenic cell line, host bacteria or transgenic plant are used in any one or more of the following: (c1) regulating the salt excretion ability of a plant; (c2) regulating the number of salt glands of a plant; (c3) regulating the Na + efflux rate of a plant; (c4) regulating a physiological index of a plant; (c5) regulating the expression of a salt gland development and ion transport related gene; (c6) regulating the salt tolerance of a plant; (c7) improving and / or breeding a salt-tolerant plant.

[0015] In the present application, the regulation can be positive regulation or negative regulation, that is, it can be manifested as promotion or inhibition.

[0016] In the (c4), the physiological index of a plant includes but is not limited to: the Na + , K + content in the leaves of a plant; the MDA, H2O2, O2 .- content, and the activity of antioxidant enzymes (including SOD, POD, CAT enzyme activity).

[0017] In the (c5), the salt gland development related gene includes but is not limited to LbSOS1 , LbNHX1 , LbNHX6 , LbHKT1 ; and the ion transport related gene includes but is not limited to LbSAD2 and LbTTG1 .

[0018] In a fifth aspect of the present application, a method for reducing the salt tolerance of a plant is provided, which comprises inhibiting the expression of the gene LbSNAP33 or protein of the plant.

[0019] The inhibition of the expression of the gene can be performed by using existing known methods, such as T-DNA insertion mutation, CRISPR / Cas9 gene editing or virus-mediated VIGS, and the inhibition of the expression of the protein can be performed by using an antibody, a small molecule antagonist, etc., which are not specifically limited herein.

[0020] In a sixth aspect of the present application, a method for improving the salt tolerance of a plant is provided, which comprises: increasing the expression of the above-mentioned geneLbSNAP33 the level or activity of the protein, or to LbSNAP33 Plant-expressed genes LbSNAP33 and / or egg whites.

[0021] The seventh aspect of the present invention provides a method for screening salt-resistant plants, the method comprising: detecting the gene of the plant LbSNAP33 The transcription level of the above proteins or the expression level or activity of the above proteins are detected.

[0022] In the present invention, the plant is preferably a halophyte, and more preferably Limonium bicolor.

[0023] Beneficial technical effects of one or more of the above technical solutions: The above technical solution reported for the first time a gene of Limonium bicolor LbSNAP33 , verified by experiments, and studies have shown that this gene is located on the salt gland, and this gene regulates the number of salt glands and the secretion rate of a single salt gland, while reducing the Na + 、Na + / K + content and active oxygen content, resisting oxidative stress, thereby positively regulating the salt resistance and salt gland development of two-color blood-replenishing grass, laying the foundation for explaining the plant salt resistance mechanism and improving the utilization efficiency of saline-alkali land, and therefore has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 In Example 1 of the present invention LbSNAP331 Full-length gene amplification.

[0026] Figure 2 In Example 1 of the present invention LbSNAP331 Amino acid sequence (A), secondary structure (B), and tertiary structure (C) of the gene.

[0027] Figure 3 In Example 1 of the present invention LbSNAP331 Promoter cloning and identification.

[0028] Figure 4 The results of the treatment of different hormones, NaCl and PEG6000 in Example 1 of the present invention are as follows: LbSNAP331 Express. A, 100 μM MeJA; B, 10 mg / L IBA; C, 5 mg / L ABA; D, 5 mg / L SA; E, 200 mMNaCl; F, 20% PEG6000.

[0029] Figure 5 A is the GUS staining result of Limonium bicolor in Example 1 of the present invention; control group; B, staining result of salt glands on leaves; arrows point to salt glands, scale bar = 50 μm.

[0030] Figure 6 In Example 1 of the present invention LbSNAP331 Full-length gene amplification.

[0031] Figure 7 This is the subcellular localization of LbSNAP331 in tobacco leaf epidermal cells in Example 1 of the present invention. Scale bar = 10 μm.

[0032] Figure 8 In Example 1 of the present invention VIGS Target gene fragment amplification electrophoresis results.

[0033] Figure 9 In Example 1 of the present invention LbSNAP331 Relative expression levels of genes in VIGS-silenced lines.

[0034] Figure 10 In Example 1 of the present invention LbSNAP331 Relative expression levels of genes in overexpression lines. A, seedling group; B, mature seedling group.

[0035] Figure 11 Effects of salt treatment on the biomass of the overexpression strain in Example 1 of the present invention; A, phenotype on the first day and phenotype on the twentieth day; B, number of roots; C, root length; D, total number of leaves; E, green leaf rate; F, yellow leaf rate; G, fresh weight; H, dry weight; I, water content.

[0036] Figure 12 VIGS detection by the disc method in Example 1 of the present invention LbSNAP331 Effects on the salt secretion capacity of leaves per unit area; A, Secretion of leaf discs of empty and silent strains; B, Volume of secretory vesicles; C, Na in secretory fluid + concentration.

[0037] Figure 13 The OE is detected by the disc method in Example 1 of the present invention. LbSNAP331 Effects on the salt secretion capacity of leaves per unit area; A, Secretion of leaf discs of wild type and overexpression strains; B, Volume of secretory vesicles; C, Na in secretory fluid + concentration.

[0038] Figure 14 The empty vector strain in Example 1 of the present invention and LbSNAP331 Observation and number statistics of salt glands of silenced strains; A, empty vector strain and LbSNAP331Silencing line salt gland observation; B, empty vector line and LbSNAP331 Silencing line leaf area; C, empty vector line and LbSNAP331 Silencing line salt gland number statistics. Scale = 50 μm.

[0039] Figure 15 For wild type and empty vector of Litorella dioica in example 1 of the present application, the leaf area was measured by the non-invasive micro-test technique. LbSNAP331 For wild type and empty vector of Litorella dioica in example 1 of the present application, the leaf area was measured by the non-invasive micro-test technique. LbSNAP331 For wild type and empty vector of Litorella dioica in example 1 of the present application, the leaf area was measured by the non-invasive micro-test technique. LbSNAP331 Silencing line leaf area; C, LbSNAP331 Silencing line salt gland number statistics. Scale = 50 μm.

[0040] Figure 16 For wild type and empty vector of Litorella dioica in example 1 of the present application, the leaf area was measured by the non-invasive micro-test technique. LbSNAP331 Effect of single salt gland Na + secretion rate; A, probe pointing to a single salt gland picture; B, Na + secretion rate of a single salt gland; C, Na + secretion rate trend over time.

[0041] Figure 17 Effect of 0 and 200 mM NaCl treatment on ion content in leaves of Litorella dioica silencing line and overexpression line in example 1 of the present application; A, B, C represent Na + , K + , Na + / K + in leaves of silencing line and empty vector line, respectively; D, E, F represent Na + , K + , Na + / K + in leaves of overexpression line and wild type, respectively.

[0042] Figure 18 MDA, H2O2, O2 .- content in leaves of silencing line and overexpression line in example 1 of the present application; A, B, C represent MDA, H2O2, O2 .- in leaves of silencing line and empty vector line, respectively; D, E represent MDA, H2O2, O2 .- in leaves of overexpression line and wild type, respectively.

[0043] Figure 19 SOD (A), POD (B) and CAT (C) activity in leaves of silencing line and empty vector line under 0 and 200 mM NaCl treatment in example 1 of the present application. LbSNAP331 SOD (A), POD (B) and CAT (C) activity in leaves of silencing line and empty vector line under 0 and 200 mM NaCl treatment in example 1 of the present application.

[0044] Figure 20In Example 1 of the present invention LbSNAP331 Expression of genes related to salt secretion and salt gland development in silenced and overexpression lines. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0046] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0047] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, the materials, reagents, vectors, strains, etc. used were all obtained from commercial sources unless otherwise specified.

[0048] Example 1 1 Experimental Materials The seeds of Limonium bicolor collected from the Yellow River Delta in Dongying City, Shandong Province were dried and stored in the school's germplasm resource bank at 4°C.

[0049] Reagents: Kanamycin (Kana, Kanamycin), Rifampicin (Rif, Rifampin), Acetosyringone (AS, Acetosyringone), LB medium, YEB medium, MS medium, NaCl, sucrose, glucose, agarose, 70% ethanol, 60% sodium hypochlorite, growth regulators such as 6-BA, NAA, IBA, hygromycin (Hyg, Hygromycin B), Piperacillin Sodium.

[0050] Strains: DH5α E. coli strain, EHA105, GV3101 Agrobacterium strain Vectors: TRV-RNA1, TRV-RNA2 vectors, pCAMBIA1300-35S-sGFP.

[0051] 2 Experimental methods 2.1 Real-time quantitative PCR Table 1 Primer sequences

[0052] With LbTUBLIN The gene was used as an internal reference, and the primers for real-time quantitative PCR of the internal reference gene and LbSNAPE331 were designed using BD software (Table 1). qPCR was performed according to the method of Pro Taq HS SYBR Green mixed qPCR kit (Accurate Biology, Hunan, China), and the expression of each gene under different treatments was quantified.

[0053] 2.2 LbSNAP331 Obtaining the full-length of the gene The cDNA of two-colored Limonium aureum was used as a template, and primers were designed upstream and downstream of the gene according to the full-length sequence of the gene in the genome. LbSNAP331

[0054] Table 2 LbSNAP331 Gene amplification primers

[0055] The PCR products were detected by 1% agarose gel electrophoresis, and the correct length of the PCR stock solution was selected for sequencing according to the electrophoresis results. The correct PCR stock solution was recovered and purified according to the Fast Pure® Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China).

[0056] 2.3 LbSNAP331 Promoter cloning and expression pattern analysis 2.3.1 Promoter cloning The sequence 2000 bp before the start codon was extracted from the genome, and Pst I and Nco I restriction enzyme sites were added at the 5' and 3' ends, respectively, by CE software. Primers were designed upstream and downstream of the promoter sequence, and the primer sequences are shown in Table 3. The CTAB method was used to extract the DNA of two-colored Limonium aureum as a template to clone the promoter sequence.

[0057] The PCR products were detected by 1% agarose gel electrophoresis, and the correct length of the band was selected for cutting according to the electrophoresis results, and the product was recovered and purified according to the Fast Pure® Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China). The recovered product was named prompter-cDNA.​

[0058] Table 3 LbSNAP331 Promoter amplification primer and vector ligation primer

[0059] 2.3.2 Vector DNA preparation The pCAMBIA3301 was double digested with Pst I and Nco I, and the PCR product was detected by 1% agarose gel electrophoresis. According to the electrophoresis results, the correct length of the band was selected, and the product was recovered and purified according to the Fast Pure® Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China). The recovered product was named Vector DNA.

[0060] 2.3.3 Connection of target fragment and vector, transformation and positive clone screening The LbSNAP331 vector was constructed using ClonExpress® II One Step Cloning Kit, and the reaction system and procedure were as follows:

[0061] Reaction procedure: 37°C, 30 min 2.3.4 Transformation of recombinant vector into E. coli, screening of positive clones and sequencing According to the transformation method of DH5a competent cells (Qingke Biology DH5a Chemically Competent Cell), the above-mentioned ligation product was transformed into E. coli. According to the electrophoresis results, the correct length of the band was selected for sequencing.

[0062] 2.3.5 Extraction of plasmid The sequencing correct bacterial liquid was extracted according to the method of FastPure® Plasmid Mini Kit DC201 (China, Vazyme) kit.

[0063] 2.3.6 Transformation of Agrobacterium, screening of positive clones and preservation of strains The recombinant plasmid was transformed into EH105 competent according to the method of EH105 Chemically Competent Cell.

[0064] 2.3.7 Analysis of promoter cis-acting elements The LbSNAP331 promoter sequence was analyzed for cis-acting elements using the PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / htmL / ) online tool.

[0065] 2.3.8 Genetic transformation experiment of the promoter of Limnanthes douglasii Using the successfully constructed pCAMBIA3301-p LbSNAP331 -GUS expression vector, Agrobacterium was used for the infection experiment.

[0066] 1. Shaking (1) The preserved pCAMBIA3301-p LbSNAP331 -GUS expression vector Agrobacterium strain was taken out from the -80°C refrigerator and placed in an ice-water mixture to melt.

[0067] (2) The melted bacterial solution was added to YEB culture medium containing Kan (100 μg / mL) and Rif (50 μg / mL) and incubated at 28°C on a shaking table to activate.

[0068] (3) 1 mL of activated bacterial solution was taken and added to 50 mL YEB culture medium containing 25 mL of Kan at a concentration of 100 μg / mL and 50 mL of Rif at a concentration of 50 μg / mL, and 10 μL of AS at a concentration of 100 mM and 5 μL of MES at a concentration of 1 mM were added, and incubated at 28°C on a shaking table for 16-18 h.

[0069] (4) The second activated pCAMBIA3301-pLbSNAP331-GUS bacterial solution was placed in a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min.

[0070] 2. Infection (1) Before infection, the young leaves and roots of the aseptic seedlings of Limnanthes douglasii cultured for 20 days were gently torn off with tweezers.

[0071] (2) The second activated Agrobacterium bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 6000 rpm for 10 min.

[0072] (3) The supernatant was discarded and the precipitate was resuspended in 50 mL MS liquid medium containing 10 μL of AS (100 mM) to make the OD value of the bacterial solution between 0.7 and 1.0.

[0073] (4) The torn aseptic seedling young leaves and roots were placed in the pCAMBIA3301-p LbSNAP331 -GUS resuspension solution and infected in the dark for 15-20 min.

[0074] (5) Use sterile filter paper to remove excess bacterial solution on the leaves and roots of the sterile seedlings, and transfer the leaves and roots of the sterile seedlings to the co-culture medium and shade them for 4 days.

[0075] (6) Prepare an appropriate amount of GUS staining working solution in a dark place, ensuring complete immersion. After staining for 12-16 hours, use anhydrous ethanol to elute until the plant material is colorless. Add a drop of transparent liquid to prepare a slide, and look for the blue GUS tissue location point under a dissecting microscope and microscope.

[0076] 2.4 LbSNAP331 Overexpression vector construction 2.4.1 LbSNAP331 Gene amplification Using Limonium bicolor cDNA as a template, clone the 933 bp sequence from the ATG to the stop codon. Primers were designed with BamH I and Sal I restriction sites at the 5' and 3' ends, respectively. The stop codon at the 3' end was removed, and 15-20 bp of vector sequence was added to both ends of the forward and reverse primers using seamless cloning. The primer sequences are shown in Table 4: Table 4 pCAMBIA 1300-35S -sGFP- LbSNAP331 Seamless cloning primers

[0077] The PCR products were detected by 1% agarose gel electrophoresis, and the fragments with the correct band length were selected according to the electrophoresis results for product recovery and purification.

[0078] 2.4.2 Vector DNA Preparation Double-digest the pCAMBIA1300-35S-sGFP plasmid with BamH I and Sal I. Detect the PCR product by 1% agarose gel electrophoresis. Select the fragment with the correct band length based on the electrophoresis results and recover the fragment according to the method in 3.2.6. Ligate the target fragment with the restriction enzyme-digested vector and transform into competent E. coli cells. Pick a single colony and sequence it. Extract the plasmid and transform it into competent A. stipedinium cells, and preserve the strain.

[0079] 2.5 LbSNAP331 subcellular localization analysis Will LbSNAP331 The gene was ligated into the pCAMBIA1300-35S-sGFP expression vector by homologous recombination, transformed into Agrobacterium tumefaciens GV3101, and transiently transformed into tobacco. The empty vector pCAMBIA1300-35S-sGFP was used as a control.

[0080] Two-photon laser scanning confocal microscopy observation: (1) Using tweezers to tear the lower epidermis of tobacco leaves to make temporary slides; (2) Observing the slides under a laser scanning confocal microscope.

[0081] 2.6 Construction of VIGS silencing vector 2.6.1 Cloning and enzyme digestion of VIGS silencing fragment Select to connect LbSNAP331 450 bp sequence in the CDS sequence of the gene into the pTRV2 vector. According to the enzyme digestion site of the TRV-RNA2 vector, the primers were designed using EcoR I (FD0274, Thermofisher, USA) and Kpn I (FD0524, Thermofisher, USA) were designed and double-digested, and T4 ligase was used for ligation.

[0082] Table 5 LbSNAP331 VIGS fragment cloning primers

[0083] PCR amplification and gel recovery were performed.

[0084] TRV is an RNA tobacco rattle virus containing both sense and antisense strands, and the target fragment can be inserted into TRV-RNA2 to play a gene silencing function, and TRV-RNA1 acts as an auxiliary viral vector to promote gene silencing.

[0085] 2.6.2 TRV-RNA2 vector enzyme digestion Select EcoR I (FD0274, Thermofisher, USA) and Kpn I (FD0524, Thermofisher, USA) as the double enzyme digestion site of the TRV-RNA2 vector, and the target fragment was ligated with the digested vector, transformed into E. coli competent cells, and single colony colonies were selected for sequencing, plasmid extraction, transformation of Agrobacterium competent cells, and strain preservation.

[0086] 2.6.3 VIGS infection of two-color bloodwort (1) Mix the TRV-RNA1 and TRV-RNA2 LbSNAP331 bacterial solution in a ratio of 1:1 as the experimental group, and mix the TRV-RNA1 and TRV-RNA2 bacterial solution in a ratio of 1:1 as the control group.

[0087] (2) Select six-leaf-stage two-color bloodwort treated with shade for 3 h as experimental material, and inject from the back of the leaf using a syringe, covering 2 / 3 of the leaf.

[0088] (3) After injection, the infected seedlings are dark treated for two days.

[0089] 2.7 Genetic transformation experiment of Limonium bicolor Agrobacterium containing the successfully constructed pCAMBIA1300- LbSNAP331 expression vector and Agrobacterium containing the control pCAMBIA1300 vector are used for infection experiments.

[0090] 2.8 Expression level verification of silenced lines The infected seedlings are cultured for about 20 days, and total RNA is extracted according to the method of Quick RNA Isolation Kit (Huayueyang, Beijing, China) kit. The reverse transcription is used as a qRT-PCR template, and the primers are shown in Table 6.

[0091] Table 6 Real-time quantitative PCR primers

[0092] The steps are shown in ChamQ Universal SYBR qPCR Master Mix Q711 (China, Vazyme).

[0093] 2.9 Expression level verification of overexpression lines The rooted sterile seedlings are sampled according to the method of Quick RNA Isolation Kit (Huayueyang, Beijing, China) kit, and total RNA is extracted. The reverse transcription is used as a qRT-PCR template, and the primers are shown in Table 7.

[0094] Table 7 Real-time quantitative PCR primers

[0095] The steps are shown in ChamQ Universal SYBR qPCR Master Mix Q711 (China, Vazyme).

[0096] 2.10 Determination of physiological indexes of overexpression lines and silenced lines 2.10.1 Determination of Na + , K + content of Limonium bicolor leaves 0.1 g of Limonium bicolor leaves is weighed and placed in a test tube, 6 mL of ultrapure water is added, and it is boiled for 3-4 h. After cooling, it is filtered with filter paper, filtered twice with a filter column before measurement, and made to 10 mL. The Na + , K + content is determined by a flame photometer.

[0097] 2.10.2 Malondialdehyde (MDA) content, hydrogen peroxide (H2O2), superoxide anion (O2 .- ) content determination Using Solebow MDA (BC0020), H2O2 (BC3590), O2 .- (BC1290) kit for determination.

[0098] 2.10.3 Determination of SOD, POD, and CAT Activities in Leaves of Transgenic and Silenced Lines The enzyme activity of SOD (BC0205), POD (BC0095), and CAT (BC0205) was determined using Solebo SOD (BC0205), POD (BC0095), and CAT (BC0205) enzyme activity kits.

[0099] 2.11 LbSNAP331 Detection of related gene expression Table 8 Fluorescence quantitative PCR primers for related marker genes

[0100] Silencing assayed by qPCR LbSNAP After the 331 gene was detected, the expression levels of genes related to salt resistance and salt gland development changed.

[0101] 3 Results and Analysis 3.1 LbSNAP331 Obtaining the full-length gene Using the cDNA of Limonium bicolor as a template, primers designed according to the sequence of the gene transcriptome were used to run PCR, and the PCR stock solution with the correct electrophoresis band was selected for sequencing. The results are as follows Figure 1 As shown, the cloned gene sequence is 933 bp in total, which is consistent with the sequence in the genome.

[0102] 3.2 Prediction and analysis of the primary, secondary, and tertiary structures and physicochemical properties of the LbSNAP331 protein from L. bicolor LbSNAP331 The gene is 936 bp long and encodes 311 amino acids. LbSNAP331 The primary structure of LbSNAP331 The protein formula of the gene is C 2788 H 4642 N936 O 1149 S 187, with a relative molecular mass of 75654.33, an isoelectric point (pI) of 5.11, an instability index of 40.15, and an overall average hydrophilicity (GRAVY) of 0.828, indicating that it is a hydrophilic protein. Figure 2 A). The secondary structure of LbSNAP331 protein was analyzed using the prabi online tool (Figure 2 B) The results show that among the secondary structure elements of this protein, α-helices account for 37.42%, β-turns 6.29%, random coils 49.17%, and extended chains 7.12%. The 3D structure of LbSNAP331 protein was predicted using Swiss-Model online software, as shown in Figure 2. Figure 2 As shown in C, the protein contains α-helix, β-turn, random coil and extended chain.

[0103] 3.3 Limonium bicolor LbSNAP331 Promoter cloning and expression vector construction The 2000 bp sequence before the start codon was extracted from the genome and amplified to obtain LbSNAP331 Promoter sequence ( Figure 3 ), the nucleotide sequence of which is shown in SEQ ID NO.3.

[0104] 3.4 Limonium bicolor LbSNAP331 Promoter element analysis and hormone treatment verification Use Plant CARE online tool to LbSNAP331 The promoter elements were analyzed, and the results are shown in Table 9. The promoter elements contain elements related to plant growth and development, response to adverse stress, response to hormones, and light response. This indicates that LbSNAP331 may be involved in the induced expression of various abiotic stresses such as salt, drought, and hormones, enabling plants to grow and develop in various adverse environments.

[0105] Table 9 LbSNAP331 Analysis of cis-acting elements in gene promoters

[0106] According to the analysis of hormone-responsive promoter elements, 7-day-old sterile seedlings were treated with 100 μM MeJA, 10 mg / L IBA, 5 mg / L LABA, 5 mg / L SA, 200 mM NaCl, and 20% PEG6000. The samples were collected at 0, 3, 6, 9, 12, and 24 hours after treatment, and RNA was extracted and reversed. LbSNAP331 Quantitative PCR was used to verify the Figure 4 ), the results showed that LbSNAP331 Response to methyl jasmonate, auxin, abscisic acid, salicylic acid, salt and drought stress. When Limonium bicolor seedlings were subjected to MeJA and PEG6000 stress, LbSNAP331 The expression level showed an overall upward trend, and the highest expression level was observed after 24 h of treatment. LbSNAP331The expression quantity presents a trend of first rising and then falling, wherein the expression quantity is the highest when the plants are treated by ABA, SA for 9 h, IBA for 6 h and NaCl for 12 h. LbSNAP331 The expression quantity is the highest.

[0107] 3.5 Construction LbSNAP331 Promoter expression vector infects Limonium bicolor After the infected Limonium bicolor seedlings are dyed by GUS dyeing reagent and decolorized by anhydrous ethanol, the Limonium bicolor seedlings are placed on a slide and observed under a microscope, and the blue region is LbSNAP331 Tissue localization of gene expression. As shown in the results, Figure 5 GUS staining is located on the salt glands of the leaves of Limonium bicolor.

[0108] 3.6 Construction LbSNAP331 Overexpression vector The vector and the gene are connected to construct a pCAMBIA 1300-35S-sGFP expression vector, and after the DH5α competent cells are inoculated, the single colony PCR is correct, and the test results are as shown in the following table. LbSNAP331 Figure 6

[0109] 3.7 Subcellular localization analysis of Limonium bicolor LbSNAP331 The temporary mount of tobacco lower epidermis is observed by using a two-photon confocal microscope, and according to the position of the GFP reporter gene, the encoded protein is expressed in the cell membrane, nucleus, endoplasmic reticulum and Golgi body, as shown in the following table. LbSNAP331 Figure 7

[0110] 3.8 Construction LbSNAP331 VIGS vector According to the virus-mediated gene silencing technology explored by Lu Chaoxia in Limonium bicolor, 450 bp is selected from the cDNA sequence of LbSNAP331 , connected to the RNA2 vector, as shown in the following table, and the function of Figure 8 in the plant is expressed. LbSNAP331

[0111] 3.9 LbSNAP331 Obtaining and identifying of the silencing strain According to the VIGS gene silencing technology, Limonium bicolor is infected, and the results are as shown in the following table. Compared with the empty strain, the expression quantity of Figure 9 in the silencing strain is obviously reduced, and the relative expression quantity of TRV: LbSNAP331 , TRV: LbSNAP331-2 and TRV: LbSNAP331-3 is the lowest, which is used for subsequent experiments. LbSNAP331-5 ​​​​​​

[0112] 3.10 Obtaining and identifying overexpression lines According to the genetic transformation of L. bicolor, the results are shown in Figure 10 Compared with the wild type, the expression of the overexpression lines is obviously increased, and the expression of OE-2, OE-3 and OE-5 is higher LbSNAP331 (B), so these three strains are selected for subsequent experiments. Figure 10

[0113] 3.11 Effect of salt treatment on biomass of overexpression lines The L. bicolor seedlings were cultured on 200 mM NaCl MS medium for 20 days, and the results are shown in Figure 11 Compared with the wild type, the overexpression lines grow better, have more roots and total leaves, have higher green leaf rate, fresh weight and dry weight, and have lower yellow leaf rate. The root length and water content of the overexpression lines have no significant difference from the wild type.

[0114] 3.12 Effect of silencing LbSNAP331 gene on salt excretion ability of L. bicolor As shown in Figure 12 Compared with the empty line, the silencing LbSNAP331 gene line has significantly reduced excretion bubble, significantly reduced excretion volume, and no significant difference in Na + concentration in the excretion liquid, which indicates that silencing LbSNAP331 gene can reduce the salt excretion ability of L. bicolor leaves.

[0115] 3.13 Effect of overexpression LbSNAP331 gene on salt excretion ability of L. bicolor As shown in Figure 13 Compared with the wild type, the overexpression line has significantly increased excretion bubble, significantly increased excretion volume, and no significant difference in Na + concentration in the excretion liquid, which indicates that overexpression LbSNAP331 gene can enhance the salt excretion ability of L. bicolor leaves.

[0116] 3.14 Effect of silencing LbSNAP331 gene on the number of salt glands of L. bicolor The silencing and empty lines with no significant difference in leaf area were selected for salt gland observation, and the results are shown in Figure 14 Compared with the empty line, the structure of the salt gland does not change, but the number of salt glands in a single field of view of the silencing line is significantly less than that of the empty line.

[0117] 3.15 Effect of overexpression LbSNAP331 gene on the number of salt glands of L. bicolor ​Overexpression lines and wild type with no significant difference in leaf area were selected for salt gland observation. Figure 15 As shown, the structure of the salt gland did not change compared with the wild type, but the number of salt glands in a single field of view of the overexpression strain was significantly more than that of the wild type.

[0118] 3.16 Effect of salt treatment on the Na expression of a single salt gland in the silenced strain + Effect of outflow rate Effect of salt treatment on the Na + The effect of outflow rate is as follows Figure 16 As shown, under control conditions, compared with the empty vector strain, the single salt gland Na + The efflux rate was significantly reduced. Under the condition of 200 mM NaCl treatment, the Na + The efflux rate increased significantly, but there were significant differences between different strains. Compared with the empty strain, the single salt gland Na + The outflow rate was significantly reduced.

[0119] 3.17 Effects of salt treatment on physiological parameters of silenced and overexpression lines 3.17.1 Effects of salt treatment on Na in leaves of silenced and overexpression lines + , K + Effect of content Under salt stress conditions, excessive Na + It accumulates in the plant body, causing plant ion imbalance, so the level of ion content represents the salt tolerance of the plant. + , K + The effect of content Figure 17 The results showed that under control conditions, the Na + Content, K + Content, Na + / K + The results were basically the same as those of the empty vector strain, with no significant difference. Under 200 mM NaCl treatment conditions, the Na + Content, Na + / K + The value increased significantly, K + The content of Na was significantly reduced, but there were significant differences between different strains. Compared with the empty strain, the Na content of the silenced strain was + Content, Na + / K + Both increased significantly, K + Similarly, under control conditions, the Na + Content, K + Content, Na+ / K + The Na + content, Na + / K + value of the wild type and the overexpression lines were significantly increased, and the K + content was significantly decreased, but there were significant differences between different lines. Compared with the wild type, the Na + content, Na + / K + of the overexpression lines were significantly decreased, and the K + content had no significant difference.

[0120] 3.17.2 Effect of salt treatment on the contents of MDA, H2O2, O2 .- in the leaves of the silenced lines and the overexpression lines The effects of salt stress on the contents of MDA, H2O2, O2 .- in the leaves of the overexpression and the silenced lines are shown in Figure 18 , which shows that under the control condition, the contents of MDA, H2O2, O2 .- in the silenced lines were basically consistent with those in the empty vector lines, with no significant difference. Under the 200 mM NaCl treatment condition, the contents of MDA, H2O2, O2 .- in the empty vector lines and the silenced lines were significantly increased, but there were significant differences between different lines. Compared with the empty vector lines, the contents of MDA, H2O2, O2 .- in the silenced lines were significantly increased. Similarly, under the control condition, the contents of MDA, H2O2, O2 .- in the overexpression lines were basically consistent with those in the wild type, with no significant difference. Under the 200 mM NaCl treatment condition, the contents of MDA, H2O2, O2 .- in the wild type and the overexpression lines were significantly increased, but there were significant differences between different lines. Compared with the wild type, the contents of MDA, H2O2, O2 .- in the overexpression lines were significantly decreased.

[0121] 3.17.3 Effect of salt treatment on the activities of antioxidant enzymes in the leaves of the silenced lines The effects of salt stress on the activities of SOD, POD, CAT enzymes in the leaves of the silenced lines are shown in Figure 19 , which shows that under the control condition, the activities of SOD, POD, CAT enzymes in the silenced lines were basically consistent with those in the empty vector lines, with no significant difference. Under the 200 mM NaCl treatment condition, compared with the empty vector lines, the activities of SOD, POD, CAT enzymes in the silenced lines were significantly decreased.

[0122] 3.19 Silencing and overexpression LbSNAP331Effects on the expression of genes related to salt gland development and ion transport Silencing and overexpression LbSNAP331 Effects on the expression of genes related to salt gland development and ion transport are seen Figure 20 The results show that the expression of LbSOS1 , LbNHX1 , LbNHX6 , LbHKT1 genes in the leaves of the silencing lines is significantly reduced compared to the empty vector lines. Similarly, the expression of LbSOS1 , LbNHX1 , LbNHX6 , LbHKT1 genes in the leaves of the overexpression lines is significantly increased compared to the wild type. Silencing LbSNAP331 genes leads to a decrease in the expression of positively regulated ion transport genes, which can be one of the reasons for the weakened salt tolerance of Limonium bicolor; overexpression of LbSNAP331 genes leads to an increase in the expression of positively regulated ion transport genes, which can be one of the reasons for the enhanced salt tolerance of Limonium bicolor.

[0123] The genes LbSAD2 and LbTTG1 that can be involved in salt gland development were selected for quantitative analysis. The results show that the expression of LbSAD2 gene in the leaves of the silencing lines is significantly reduced compared to the empty vector lines, LbTTG1 gene is significantly increased. Similarly, the expression of LbSAD2 gene in the leaves of the overexpression lines is significantly increased compared to the wild type, LbTTG1 gene is significantly reduced. This shows that LbSNAP331 gene can have certain relevance to genes related to salt gland development.

[0124] Nucleotide / amino acid sequences appearing in the examples LbSNAP331 Gene sequences ATGATTGGGATGAAGAAATCCCCAATGAGGCACAGAATGTTCAAGCCTAGCGTTGCTGATCCCCAAATTTCAAGCAAAAAAAGCAGCTCCAACCCCTTTGATTCAGACGATGAATCAGACACCAAAACTCTTGCATCCTCAGAAAGAGTCTCATCTGAACTGAAGCAGAATGCATCAAATTCTGATACCAATGTCTCCGATTATTACAATGATGTTGGGAGCAGCGAGATGACAAATACATCCTCTAAACACTCGTACTCTTATGAACAAAGAAGGAGATACAAGGATGATTTTCGCCAGTCAGGAGGAGTAGATAGTCAGAATGTGCAGGAATTGGAAAACTATGCTGTGTTTAAGTCTGAGGAGACAACAAAGTCTGTGAACAACTGTGTAAGAATTGCCGAGGATATAAGAGAGGATGCTACAAAGACACTCGTCACTTTGCATCAGCAGGGTGAGCAGATCACGAGGACACACATTGCAGCTGCTGATATTGATCATGATCTAAGTCGGAGTGAGAAACTGCTTGGAAGTCTCGGTGGAATATTTTCTAGGACTTGGAAGCCAACGAAGAACCGCCCCATCACTGGGCCAACTATCTCAAGAGATGACCCGGTGAGATCAAGGGCCAGTCATCTTGAGCAGAGAGGGAAGTTGGGATTGGCTCCTCTACCCAATGGGAAACCTAAATCGCTAACACCTCCACCTGAATCAGCAGATGCGTACCAAAGAGTTGAGTATGAAAAATCAAAGCAAGATGATGGTTTGGATAATTTGAGCAGCCTTTTAGGAGAACTAAAGGAGATGGCTGTTGACATGGGCTCTGAGATTGAACGACAAAACAAAGCATTGGATCCTCTTGGTGATGACGTGGATGAACTAGGCTTCAGGGTCCAAAATGCCAATCGACGTGGTCGTAGGCTGCTGGGTAAATAA (SEQ ID NO. 1) LbSNAP331 Amino acid sequence of the encoded protein MIGMKKSPMRHRMFKPSVADPQISSKKSSSNPFDSDDESDTKTLASSERVSSELKQNASNSDTNVSDYYNDVGSSEMTNTSSKHSYSYEQRRRYKDDFROSGGVDSQNVQELENYAVFKSEETTKSVNNCVRIAEDIREDATKTLVTLHQQGEQITRTHIAAADIDHDLSRSEKLLGSLGGIFSRTWKPTKNRPITGPTISRDDPVRSRASHLEORGKLGLAPLPNGKPKSLTPPPESADAYORVEYEKSKQDDGLDNLSSLLGELKEMAVDMGSEIERQNKALDPLGDDVDELGFRVONANRRGRRLLGK (SEQ ID NO. 2) LbSNAP331 Promoter sequence It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is explained in detail with reference to the examples, the technical solutions of the present application can be modified or equivalently replaced according to the needs without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A gene, named LbSNAP33, characterized in that: The nucleotide sequence of the gene is as follows: (a1) the nucleotide sequence shown in SEQ ID NO. 1; (a2) a nucleotide sequence that encodes a protein having the same amino acid sequence as the nucleotide sequence of (a1) but differs in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes the same functional protein; (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

2. A protein, characterized in that The protein is obtained by encoding the gene according to claim 1; Furthermore, the protein has any one of the amino acid sequences (b1) to (b3): (b1) the amino acid sequence shown in SEQ ID NO. 2; (b2) a protein derived from the amino acid sequence shown in SEQ ID NO. 2, wherein one or more amino acid residues are substituted and / or deleted and / or added and which has the same function as the amino acid sequence shown in SEQ ID NO. 2; (b3) proteins encoded by other genes that have an amino acid sequence identity of more than 90% with the amino acid sequence shown in SEQ ID NO. 2 and have the activity of the protein shown in SEQ ID NO.

2.

3. A recombinant expression vector, transgenic cell line, host bacteria or transgenic plant containing the gene according to claim 1.

4. Use of the gene of claim 1, the protein of claim 2, and / or the recombinant expression vector, transgenic cell line, host bacteria, or transgenic plant of claim 3 in any one or more of the following: (c1) regulating the salt secretion capacity of plants; (c2) regulating the number of plant salt glands; (c3) Regulate plant salt gland Na + outflow rate; (c4) regulating plant physiological indicators; (c5) regulates salt gland development and ion transport-related gene expression; (c6) regulating plant salt tolerance; (c7) Improving and / or breeding salt-tolerant plants; The plant is preferably a halophyte, and more preferably Limonium bicolor.

5. The use according to claim 4, characterized in that In (c4), plant physiological indicators include but are not limited to: Na in plant leaves + , K + Content; MDA, H2O2, O2 .- content, antioxidant enzyme activity (including SOD, POD, and CAT enzyme activity).

6. The use according to claim 4, characterized in that In (c5), the salt gland development-related genes include LbSOS1, LbNHX1, LbNHX6, and LbHKT1; the ion transport-related genes include LbSAD2 and LbTTG1.

7. A method for reducing the salt resistance of plants, characterized in that: The method comprises inhibiting the expression of the gene according to claim 1 or the protein according to claim 2 in a plant; the plant is preferably a halophyte, and more preferably Limonium bicolor.

8. The method according to claim 7, wherein The expression of the gene is inhibited by T-DNA insertion mutagenesis, CRISPR / Cas9 gene editing or virus-mediated VIGS; the expression of the protein is inhibited by antibodies and small molecule antagonists.

9. A method for improving the salt resistance of plants, the method comprising: The level of the endogenous gene LbSNAP33 or the activity of the protein in the plant is increased, or a plant not containing the gene LbSNAP33 is made to express the gene LbSNAP33 and / or protein; the plant is preferably a halophyte, and more preferably Limonium bicolor.

10. A method for screening salt-resistant plants, characterized in that: The method comprises: detecting the transcription level of the gene according to claim 1 or detecting the expression level or activity of the protein according to claim 2 in a plant; the plant is preferably a halophyte, and more preferably Limonium bicolor.

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