CN113528533A - Limonium bicolor gene LbRSG and application thereof - Google Patents

Abstract

The invention provides a Limonium bicolor gene LbRSG and application thereof. The gene LbRSG is obtained by cloning from limonium bicolor of a salivation halophyte, the total length of mRNA of the gene LbRSG is 669bp, 222 amino acids are coded, and the gene LbRSG shows high expression in the growth period of the salt gland. In situ hybridization shows that LbRSG can be directly related to the development of saline glands, and the function of LbRSG genes is further verified by using knockout and overexpression technologies. The results show that the knock-out of LbRSG effectively causes the number of light-emitting points of the salt gland to be reduced to 3 compared with 4 light-emitting points of the salt gland possessed by the wild type; the over-expression strain shows 8-9 luminous points, which shows that the LbRSG participates in the differentiation of the salt gland and plays a positive regulation role in the development process of the salt gland. The invention lays a foundation for the research of the LbRSG gene participating in the bicolor limonium salt gland development and the salt resistance mechanism.

Description

Limonium bicolor gene LbRSG and application thereof

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a Limonium bicolor gene LbRSG and application thereof.

Background

The salinization of soil seriously threatens grain production, the area of saline-alkali soil is increased year by year due to factors such as drought, unreasonable irrigation and the like, and the development and utilization of the saline-alkali soil to ensure the safety of grains and energy becomes a hotspot and a difficulty of the current world research. Practice shows that exploring the molecular mechanism of plant salt resistance and utilizing modern biotechnology to cultivate salt-tolerant crops is a fundamental strategy for developing and utilizing saline-alkali soil. The halophytes are divided into three types of salt accumulation, salt rejection and salt secretion according to a salt-resistant strategy, wherein the salt secretion halophytes have a unique salt secretion structure (salt glands) and can be morphologically distinguished from other halophytes and all non-halophytes, and excessive Na accumulated in the plants can be removed⁺Directly discharged out of the body, thereby effectively avoiding salt damage. Therefore, the molecular mechanism for revealing the salt gland development is of great significance for heterogeneously transforming non-halophytes, particularly crops, and improving the salt resistance of the non-halophytes.

Disclosure of Invention

The invention aims to provide a Limonium bicolor gene LbRSG and application thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides limonium bicolor gene LbRSG, which is a gene encoding the following protein (a) or (b):

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO; or

(b) 1, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.

According to the three-generation full-length transcription group information, the LbRSG mRNA obtained by cloning from Limonium bicolor has the full length of 669bp, codes 222 amino acids, shows high expression in the salt gland development period, and onion subcells show that the LbRSG is positioned on a cell plasma membrane. Bioinformatics analysis shows that the gene encodes a hydrophilic protein, a double-transmembrane domain exists, the outer part of the membrane occupies 64 percent (141 amino acids) to form 1 large extracellular loop, the continuous negatively charged domain possibly participates in ion response, and the C end in the membrane has phosphorylation sites of threonine and serine, possibly leads to enzyme-linked reaction in cells through phosphorylation. The gene is directly positioned on the salt gland by in situ hybridization, which indicates that the gene is possibly closely related to the development of the salt gland.

In a second aspect, the present invention provides a limonium bicolor gene LbRSG promoter, the sequence of which is: i) a nucleotide sequence shown as SEQ ID NO. 2; or ii) a nucleotide sequence with one or more nucleotides substituted, deleted and/or added and the same function as the nucleotide sequence shown in SEQ ID NO. 2; or iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO. 2 under stringent conditions with hybridization at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution and washing the membrane with the solution, and having the same function; or iv) a nucleotide sequence having more than 90% homology with the nucleotide sequence of i), ii) or iii) and having the same function.

In a third aspect, the invention provides a biological material containing said gene LbRSG or said promoter, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or non-regenerable plant parts.

In a fourth aspect, the invention provides an application of the promoter in regulating and controlling the expression of downstream genes, wherein the downstream genes include but are not limited to genes LbRSG, reporter genes GUS, GFP and the like.

In a fifth aspect, the invention provides the use of the gene LbRSG, the promoter or a biological material containing the gene LbRSG or the promoter in the preparation of a transgenic plant.

In a sixth aspect, the present invention provides any one of the following uses of the gene LbRSG or a biological material containing the gene LbRSG: (1) used for regulating and controlling the growth and development of salt glands of salt secreting halophytes; (2) is used for plant variety improvement.

Preferably, the plant is Limonium bicolor.

The modulation is positive modulation, such as positive modulation of the diameter of the salt gland, the number of salt gland cells, and the like.

In a seventh aspect, the invention provides a method for reducing the diameter of limonium bicolor salt glands, reducing the number of salt gland cells and abnormal salt gland development, which utilizes a genetic engineering means to weaken a limonium bicolor gene LbRSG to obtain a genetic weakening strain; the attenuation includes knocking out or reducing expression of the gene.

Preferably, the genetic engineering means may be selected from one of mutagenesis, site-directed mutagenesis, homologous recombination, and the like.

Further, a sgRNA sequence based on CRISPR/Cas9 is designed aiming at a target gene LbRSG in limonium bicolor, a DNA fragment containing the sgRNA sequence is connected into a vector carrying the CRISPR/Cas to transform the limonium bicolor, and then a transgenic plant with the gene function deletion is obtained.

Preferably, the nucleotide sequence of the sgRNA action site is 5'-AGTATCGATGCAGTGAAAC-3'.

In an eighth aspect, the present invention provides a method for promoting the development of plant salt gland (including the enlargement of the diameter of salt gland and the increase of the number of salt gland cells), which is selected from the following (i) or (ii): making the plant express the protein coded by the gene LbRSG; ② over-expressing the gene LbRSG in the plant.

Preferably, the plant is a salivation plant, more preferably limonium bicolor.

The mode of overexpression is selected from the following 1) to 5), or an optional combination:

1) by introducing a plasmid having the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by altering the promoter sequence of said gene on the plant chromosome;

4) by operably linking a strong promoter to the gene; 5) by introducing an enhancer.

In a ninth aspect, the present invention provides any one of the following uses of the transgenic plant obtained according to the above method: i. for plant breeding; and ii, planting in saline-alkali soil.

Such breeding methods include, but are not limited to, transgenics, crosses, backcrosses, selfs, or asexual propagation.

The gene LbRSG is cloned from Limonium bicolor for the first time and the biological function of the gene LbRSG is analyzed, the gene is highly expressed in the salt gland development period, in-situ hybridization shows that the LbRSG can be directly related to the salt gland development, and the function of the LbRSG gene is further verified by adopting knockout and overexpression technologies. An LbRSG knockout carrier is constructed by using a CRISPR/Cas9 gene editing technology, LbRSG silent mutant LbRSG is obtained through a Limonium bicolor genetic transformation system, and the development condition of the LbRSG saline gland is determined by adopting a method that the saline gland has blue autofluorescence under ultraviolet excitation light. Compared with wild four-light-point salt gland, the LbRSG silent line LbRSG has a large number of three-light-point salt glands, which indicates that the salt gland dysplasia is effectively caused by the LbRSG knockout. Meanwhile, a 35S over-expression vector is constructed, and after the over-expression vector is infected by agrobacterium, an over-expression strain OE is obtained by screening, the over-expression strain is expressed by 8-9 luminous points, which indicates that LbRSG participates in the differentiation of salt glands and plays a positive regulation role in the development process of the salt glands. Through the phenotype observation of LbRSG gene knockout strains, the LbRSG is determined to play a positive regulation role in the salt gland development process.

Drawings

FIG. 1 shows the result of PCR amplification of LbRSG gene in a preferred embodiment of the present invention, wherein 1-8 is LbRSG gene, M: DNA Marker 2000.

FIG. 2 shows the primary structure of LbRSG protein in a preferred embodiment of the invention.

FIG. 3 is a diagram showing the analysis of the transmembrane domain of LbRSG protein in a preferred embodiment of the present invention.

FIG. 4 shows the prediction of the phosphorylation sites of LbRSG protein in a preferred embodiment of the invention.

FIG. 5 shows the relative expression levels of LbRSG gene in Limonium bicolor treated at different times in a preferred embodiment of the invention. 3 replicates were set for each experimental group, with different lower case representations differing significantly at the p-0.05 level.

FIG. 6 is an electrophoresis diagram of 1st, 2nd and 3rd PCR products using LbRSG 5SPs and APs as primers in a preferred embodiment of the present invention. The inner circle is the destination strip. M1, λ -Hind III digest; 1-3, AP 11 st 2nd 3rd PCR product; 4-6, AP 21 st 2nd 3rd PCR product; 7-9, AP 31 st 2nd 3rd PCR product; 10-12, AP 41 st 2nd 3rd PCR product; 13-15, positive control 1st 2nd 3rd PCR product; m2, DL2,000DNA marker.

FIG. 7 shows the LbRSG gene promoter sequence in a preferred embodiment of the invention. The black part is the promoter sequence and the grey part is the CDS sequence.

FIG. 8 shows the analysis of LbRSG promoter elements in a preferred embodiment of the present invention.

FIG. 9 shows the result of agarose gel electrophoresis in a preferred embodiment of the invention. Wherein, (A) the double-restriction enzyme digestion recovery result is M2: DL15000DNA Marker; 1: pCAMBIA 3301; 2, purifying a product after enzyme digestion of Hind III/Nco I; m1: DNA Marker 2000; (B) an electrophoretogram of LbRSG gene promoter clone, M1: DL5,000DNA Marker; (C) PCR result of Escherichia coli colony of pCAMBIA3301-LbRSG promoter, M: DL5,000DNA Marker; (D) pCAMBIA3301-LbRSG promoter Agrobacterium colony PCR, M: DL5,000DNA Marker.

FIG. 10 is a diagram of the selection of Arabidopsis thaliana positive seedlings by the herbicide BASTA in a preferred embodiment of the present invention.

FIG. 11 is an electrophoresis diagram of PCR products using heterologous expression strain DNA as a template in a preferred embodiment of the present invention. M: DNA Marker with standard molecular weight of 2000.

FIG. 12 shows GUS color analysis results of LbRSG promoter at different positions of seedling in the preferred embodiment of the present invention. (A) GUS color analysis results of young leaf promoters; (B) GUS color analysis result of the vein promoter of the young leaf; (C) GUS color development analysis results of promoters in the root hair generation region of young leaves; (D, E, F) GUS color analysis of the promoter at the root-tuber junction; (G) GUS color development analysis results of young leaf root hair promoters; (H) GUS color analysis results of the young leaf main root tip promoter; (I) and (5) performing color analysis on GUS (glucuronidase) of the root tip promoter of the young leaf side root.

FIG. 13 is a diagram of a sequence of an exon defining a primer that does not span an intron in a preferred embodiment of the invention.

Fig. 14 shows agarose gel electrophoresis results of CRISPR vector construction and agrobacterium transformation in a preferred embodiment of the invention.

FIG. 15 shows the preferred embodiment of the present invention, wherein Agrobacterium infects Limonium bicolor leaf and CRISPR-Cas9 knockdown vector infects leaf. Wherein, B, 7 days after the bicolor limonium major root is infected; C. and D, screening the limonium bicolor regenerated buds by the hygromycin.

FIG. 16 shows the regenerated adventitious shoots and adventitious roots after 20 days of the selection culture in the preferred embodiment of the present invention. Wherein, (A) knocking out the carrier and screening the regenerated adventitious bud and adventitious root after 20 days of culture; (B) the new buds are screened and killed by hygromycin; (C) transgenic regeneration lines.

FIG. 17 shows the salt gland phenotype of the knockout and overexpression lines of Limonium bicolor Lbrsg1 in a preferred embodiment of the invention. WT: 10 μm. Lbrsg1, 5 μm for ruler; OE: the number of salt gland component cells of limonium bicolor (LbRSG1) after Lbrsg1 is knocked out by the CRISPR is obviously reduced, the number of salt gland component cells of an over-surface strain (OE) is obviously increased, the salt gland is in a sole shape, an arrow indicates the position of a luminous point, and the luminous point is consistent with the number of salt secreting holes.

FIG. 18 shows the salt gland phenotype of Limoid Limonium bicolor Lbrsg1 overexpression strain in a preferred embodiment of the invention. Wherein, (A)5 luminous point salt gland; (B)7 luminous spot saline gland; (C)8 light-emitting point saline gland.

FIG. 19 is a diagram showing the result of subcellular localization analysis of onion epidermal cells of 35S: LbRSG-GFP in the preferred embodiment of the present invention. The GFP-LbRSG fusion was expressed in the cytoplasmic membrane, as well as in the control cytoplasm. 35S, GFP is an empty vector. DAPI with blue fluorescence specificity indicates the location of the nucleus, and FM4-64 with red fluorescence specificity indicates the location of the plasma membrane. (Bar 75 μm in the left panel and 100 μm in the right panel).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Statistical analysis methods used in the following examples: statistical significance of P0.05 (Duncan multiple range test) was determined using SPSS. Analysis of variance and orthogonal control and mean comparison programs were used to detect significant differences between different treatments.

Example 1 full-Length cloning of LbRSG Gene and bioinformatic analysis

Through the second generation transcriptome information of early salt gland development, 1 transmembrane protein gene Lb1G01054 with unknown function is screened, has high expression in the salt gland development period, and the function of the gene in the bichromal limonium salt gland development is researched. The experiment shows that the Salt gland of the CRISPR knockout limonium bicolor mutant is obviously reduced, so the gene is named LbRSG (reduced Salt Gland).

And (3) cloning to obtain the LbRSG gene cDNA full length by taking the true leaves of the Limonium bicolor at the A period and the B period as materials by referring to the full length sequence of the third generation genome of the Limonium bicolor. Meanwhile, bioinformatics analysis is carried out by using various bioinformatics software, the protein sequence, the secondary structure, the hydrophile-hydrophobicity, the transmembrane domain, the conserved region, the phosphorylation site and the like of the bioinformatics software are predicted, the promoter is analyzed, and the charged amino acid condition of the transmembrane structure is analyzed according to the prediction result.

1 test materials and reagents

1.1 plant Material

Bicolor hematinic seeds are collected in saline-alkali soil of yellow river delta in Dongying City of Shandong province, dried and stored in a germplasm resource library of Shandong university at 4 ℃.

1.2 main chemical reagents and instruments: 1) culture medium: LB medium, YEB medium, MS medium (Murashige et al) 1962). 2) Antibiotics: ampicillin Ampicillin, Kanamycin Kanamycin (60615, Sigma, USA), rifampicin Rifampin (R350, Sigma, USA). 3) The kit comprises: FastPure Gel DNA Extraction Mini Kit (DC301, Nanking Vazyme), FastPure Plasmid Mini Kit (Nanking Vazyme, China),

Plant Total RNA Isolation Kit(Nanjing Vazyme，China)。4)

Max Super-Fidelity DNA Polymerase(Nanjing Vazyme)。5)Sal I(P213-AA，Nanjing Vazyme)。6)

II One Step Cloning Kit (C112, Nanjing Vazyme). 7) SYBR Green real-time fluorescent quantitative PCR premix (Gene Star, China). 8) Competence: DH5 alpha (Nanjing Vazyme), GV3101(Nanjing Vazyme). 9) Carrier: pMD18-T Vector, pCAMBIA1300-35S-sGFP (TaKaRa). OthersReagents, e.g. agarose, sodium hypochlorite, ethanol, AS (acetosyringone), ddH₂O is commercially available

Reagents required for in situ hybridization: DEPC (amresco), 4% paraformaldehyde DEPC water (Servicebio), paraffin (Sakura), xylene (national chemical group chemical agents limited). PBS buffer (DEPC)20 XSSC eluent, BSA, protease K, DAPI, anti-fluorescence quenching blocking tablet hybridization buffer, anti-DIG-488 (ServiceBio).

2 method of experiment

2.1 two-color Limonium bicolor material acquisition in A and B stages: (1) and (4) selecting bicolor hematinic seeds with full seeds and uniform sizes. The bicolor hematinic seeds are firstly placed in 70% ethanol for surface disinfection and fully shaken for 5 min. (2) Shaking with 6% sodium hypochlorite (as prepared) in shaking table for 15-20 min. (3) By autoclaving of ddH₂Washing the Limonium bicolor seeds for 5-6 times by O, and then standing the seeds in water for 30 min. (4) After 2-3 times of washing the seeds with sterile water, uniformly sowing the seeds in an MS culture medium, and completely absorbing water. (5) Laying at 26/22 deg.C (day/night), with photoperiod of 16/8 hours (day/night) and light intensity of 600 μmol/m²And/s culturing in a tissue culture room. (6) When culturing for 4-5 days, taking the leaf of A period; and for 6-7 days, the true leaves in stage B can be taken. Taking the first true leaf under a dissecting mirror by using a pointed forceps, and quickly freezing and enriching in liquid nitrogen. (7) Storing the plant material in a refrigerator at-80 deg.C, and extracting plant RNA when the material is sufficiently enriched.

2.2 extraction and reverse transcription of total RNA of plants: and (3) taking the frozen leaves of the Limonium bicolor at different periods to extract total RNA, wherein the experimental steps are carried out according to the instruction of the Novozapine RNA extraction kit, and the extracted RNA can be directly subjected to reverse transcription to synthesize cDNA.

2.3 full-Length cloning of LbRSG Gene: primers were designed according to the information of the full length of the LbRSG gene in the third generation sequencing and Primer premier 5 software, the sequences of the primers are shown in Table 1, and the full length sequence of the gene was cloned using the above cDNA as a template.

TABLE 1 LbRSG Gene amplification primers

And (3) PCR system: primer mix 12.5. mu.l, LbRSG-S0.2. mu.M, LbRSG-A0.2. mu.M, 300ng cDNA, ddH₂The volume of O is filled to 25 μ l. PCR reaction procedure: 3min at 95 ℃; 15sec at 95 ℃,20 sec at 51-55 ℃, 3min at 72 ℃ and 34 cycles; 10min at 72 ℃; storing at 4 ℃.10 μ l of PCR product was subjected to 1% agarose gel electrophoresis, and gel recovery was carried out if a band of interest was detected.

2.4 ligation of the recovered fragment to the pMD18-T vector

The linking system is as follows: 1. mu.l of pMD18-T vector, 1. mu.l of the DNA fragment of interest, ddH₂O3. mu.l, Solutioni 5. mu.l. After brief centrifugation of the above system, the mixture was incubated for 16h at 16 ℃.

2.5 transformation

(1) DH 5. alpha. competent cells were removed from the freezer at-80 ℃ and thawed in ice bath (20 min);

(2) mixing the ligation product and competent cells according to the volume ratio of 1:10, flicking and uniformly mixing, and standing on ice for 30 min;

(3) adjusting the temperature of the water bath to 42 ℃ in advance, standing on ice for 30min, thermally shocking the water bath for 45s, immediately placing the water bath on ice for 2-3min, immediately standing in ice bath for 2 min;

(4) adding 900 mul of LB liquid culture medium without antibiotic into a clean bench, and performing shake culture at 37 ℃ and 200rpm for 1 h;

(5) centrifuging at 8000rpm for 2min at normal temperature. Removing the supernatant, leaving a small amount of supernatant, suspending on a clean bench, taking 20 μ l of the supernatant, coating the supernatant on an LB solid culture medium containing Amp antibiotics, inverting the supernatant in a 37 ℃ incubator, and culturing overnight;

(6) streaking, colony PCR, and selecting the correct colony with band for sequencing.

Colony PCR system: primer mix 12.5. mu.l, LbRSG-S0.2. mu.M, LbRSG-A0.2. mu.M, DNA template (single colony), ddH₂The volume of O is filled to 25 μ l.

PCR reaction procedure: 3min at 95 ℃; 15sec at 95 ℃,20 sec at 51-55 ℃, 3min at 72 ℃ and 34 cycles; 10min at 72 ℃; storing at 4 ℃.

(7) And (4) selecting a bacterial colony with a correct sequence according to a sequencing result, and shaking the bacterial colony in an LB (lysogeny broth) culture medium. The Plasmid extraction was carried out on the correctly sequenced bacterial solution according to the instructions of the FastPreplasmid Mini Kit from Vazyme.

2.6 bioinformatics analysis

Bioinformatics analysis the base sequence and amino acid sequence of a target gene are calculated and searched using biotechnology software, and biological information of the gene is analyzed. In order to better understand the characteristics of genes and encoded proteins, various bioinformatics means are used to analyze gene-encoded proteins in various ways.

After determining the nucleotide sequence encoding the gene of interest, the gene is subjected to bioinformatic analysis: (ii) a gene encoding an amino acid sequence by a Translate Tool (https:// web. expask. org/Translate /); ProtParam (https:// web. expasy. org/protpara) primary structure analysis; the secondary structure of the target gene is analyzed by prabi (https:// NPSA-prabi. ibcp.fr/cgi-bin/NPSA _ Automat.plpage ═ NPSA/NPSA _ sopma. html); carrying out tertiary structure prediction on a target gene by robeta (http:// new. robeta. org/results. phpid ═ 43056# pdb _ format); (ii) a hydrophilicity prediction of a ProtScale (https:// swissmodel. expasy. org) encoded protein; TMHMM (http:// www.cbs.dtu.dk/services/TMHMM /) encodes a prediction of the transmembrane domain of the protein; conserved domain prediction of SMART (http:// SMART. embl-heidelberg. de) encoded protein; server (http:// www.cbs.dtu.dk/services/SignalP /) predicts whether the encoded protein contains a signal peptide; NetPhos2.0Server (http:// www.cbs.dtu.dk/cgi-bin/webface2.) encodes a phosphorylation site prediction of the protein; homology analysis of BLAST (https:// blast.ncbi.nlm.nih.gov/blast.cgi) encoded protein; the evolutionary tree was constructed using MEGA-X software.

3 results and analysis of the experiments

3.1 obtaining the full Length of LbRSG Gene

The result of the full-length amplification of LbRSG gene is shown in FIG. 1.

And comparing the full-length gene clone sequencing result with the full-length sequence of the third generation transcriptome LbRSG by using DNAMAN software.

3.2 analysis of Primary Structure and physicochemical Properties of LbRSG sequences

The 669bp open reading frame is cloned according to the PCR technology of the leaf development transcriptome data of Limonium bicolor, and 222 amino acids are coded (figure 2). According to ExPASY analysis, the LbRSG gene encodes a protein with the relative molecular mass of 55341.84Da and the isoelectric point (pl) of 5.15, and the molecular formula of the encoded protein is C₂₀₂₆H₃₃₈₈N₆₆₆O₈₅₁S₁₄₅And 7076 atoms in total. The coding amino acid sequence has 22 negatively charged amino acids (Asp + Glu) and 27 positively charged amino acids (Arg + Lys).

3.3 LbRSG Secondary Structure prediction

And (5) performing secondary structure prediction on the protein by utilizing the prabi. As shown in the figure, the protein contains alpha helix, random coil and extension chain. The proportion of each element of the secondary structure in the secondary structure is 31.98 percent of alpha helix, 50.90 percent of random coil and 17.12 percent of extended chain.

3.4 prediction of the tertiary structure of LbRSG protein

The 3D structure of LbRSG protein was predicted de novo using ESyPred 3D Web Server tool. From this prediction, the protein contains an alpha helix, random coil and extended chain.

3.5 LbRSG amino acid sequence hydrophilicity and hydrophobicity analysis

The gene is known to code 666bp nucleotide sequence and 222 amino acids, and the Prot-Scala tool in ExPASy software is used for analyzing the hydrophilicity and hydrophobicity of the amino acid sequence, wherein the lower the value of the amino acid is, the stronger the hydrophilicity is. The results show that the hydrophilic regions are larger than the hydrophobic regions, and the distribution of negative values is predominant, so that the entire polypeptide chain appears hydrophilic, being a hydrophilic protein.

3.6 LbRSG transmembrane structure prediction analysis

TMHMM software is used for carrying out transmembrane structure prediction on the protein of the gene, wherein 1-12 parts are intramembrane proteins, 13-35 parts are transmembrane domains, 36-176 parts are extramembrane proteins, 177-199 parts are transmembrane domains, and 200-222 parts are intramembrane proteins (figure 3).

3.7 analysis of conserved domains of proteins encoded by LbRSG Gene

SMART software was used to predict the conserved domain of LbRSG, with transmembrane domains present at 13-35 and 177-199.

3.8 LbRSG Signal peptide analysis

The singal4.1 software is used for detecting a signal peptide, and the protein is analyzed and found to have the signal peptide, and the signal peptide is supposed to be possibly related to the membrane location for inducing the protein.

3.9 LbRSG phosphorylation site analysis

Phosphorylation and dephosphorylation are important modes of intracellular signaling, and phosphorylation site analysis was performed using the program netphos2.0Server (http:// www.cbs.dtu.dk/cgi-bin/webface2.) on CBS Server of danish university of technology (DTU). NetPhos2.0Server program is based on neural network algorithm, and predicts the phosphorylation sites that three amino acid residues, Ser, Thr and Tys, in the protein sequence may become. The predicted results are shown in FIG. 4, from which we can see that there are 21 sites at which the protein is likely to be phosphorylated.

3.10 schematic representation of LbRSG transmembrane protein

Analysis of the charge of the amino acids on the outside of the membrane for analysis, the amino acids with negative charge outside the membrane, namely aspartic acid and glutamic acid (Asp (D)) + Glu (E)), are 21 amino acids with negative charge in total; extramembranous positively charged amino acids, namely arginine and lysine (arg (r) + lys (k)) for a total of 18 negatively charged amino acids; at this point, there are net 3 negative charges outside the membrane.

3.11 relative expression of LbRSG at different times

A high-sensitivity fluorescent quantitative PCR method is adopted to detect the relative expression quantity of unknown LbRSG genes in 11 different development stages (stage A-E, maturation stage) of Limonium bicolor leaves, different tissues (roots and stems) and different treatments (NaCl, GA and ABA) (figure 5). The Real-time PCR results showed that LbRSG was highly expressed during the early stage of stage A, stage B, and stage C leaf development and during salt treatment.

Since no saline gland structure is reported in any of the currently sequenced species, and no homologous gene is aligned in the Nr database, we determined that it is probably a new unknown functional gene specific to limonium bicolor and speculated that it is highly likely to participate in the saline gland development of limonium bicolor.

According to a second generation transcriptome database, LbRSG genes highly expressed in early salt gland development stage are screened, and after the full-length sequences of the LbRSG genes are cloned, the amino acid sequences coded by the genes are subjected to bioinformatics analysis. The protein is a transmembrane protein, and a peptide chain at the outer side of a membrane accounts for a large part, and analysis shows that the net 3 negative charges of amino acid outside the membrane are presumed to have a certain relation with the combination of certain positive salt ions, so that the protein has certain salt resistance. The C-segment has serine and threonine phosphorylation sites, and may be involved in intracellular reactions caused by cascade amplification signals.

According to the judgment of the relative expression quantity of the LbRSG gene in different periods, parts and treatments of Limonium bicolor leaves, the expression quantity is obviously increased after NaCl treatment, GA treatment is not obviously changed after ABA treatment, and the expression quantity in roots is the least. The expression level was high at B, C, and it was assumed that the protein might be involved in the development of salt gland. The in situ hybridization verifies that the gene is expressed in the saline gland and the stomata, and particularly the expression level of the gene is higher in the saline gland part. Constructing a pCAMBIA 1300-35S-sGFP-LbRSG gene expression vector for subcellular localization, selecting pCAMBIA1300-35S-sGFP no-load as a control, and combining marker gene localization of cell nucleus and plasma membrane to show that the gene is expressed on cell membrane. The experimental process has a great relationship with the freshness of the onions, the concentration of the bacterial liquid and the culture time, and the light emitting conditions of GFP, DAPI and FM4-64 are observed by using a two-photon confocal microscope after the surface bacterial liquid needs to be cleaned when the temporary slices are loaded.

The embodiment defines the basic structure of the LbRSG and performs the credit generation analysis, and lays a foundation for the subsequent deep development of the functional research of the LbRSG. On the one hand, it is clear that LbrSG is a basic transmembrane protein, but unlike most transmembrane proteins, LbrSG has a very large extracellular loop, possibly involved in ionic response and signaling, and in addition, the continuous negative charge region of the extracellular loop suggests whether LbrSG can be involved in cations (such as Na)⁺、Ca²⁺) The intracellular signal cascade amplification is caused by the phosphorylation of the C terminal, which is used for explaining the characteristic of Lbrsg participating in plant salt resistance and laying a foundation for further research on Lbrsg participating in the development of salt glandsAnd (6) determining a foundation.

Example 2 LbRSG promoter cloning and functional analysis

This example clones the promoter of a gene by chromosome walking and analyzes the elements of the promoter. Constructing an expression vector containing a gene specific promoter, transforming arabidopsis thaliana, screening homozygote, and then positioning the expression position of the gene through GUS staining for explaining the possible salt resistance function of LbRSG.

1 test materials and reagents

1.1 plant Material

Plant material: columbia ecotype (Col-0) Arabidopsis plants.

The culture method comprises the following steps:

(1) seed disinfection: shaking with 75% ethanol for 3 times, 4min each time, shaking with 95% ethanol for 3 times, 1min each time, and washing with sterile water for 3 times;

(2) vernalizing Arabidopsis seeds on 1/2MS culture medium at 4 deg.C for 2 d;

(3) transferring to an arabidopsis culture box, and culturing for about 7 days;

(4) transplanting the seedlings into a 10cm small flowerpot (nutrient soil is used as a culture medium) for continuous culture.

1.2 Main chemical reagents and instruments

TaKaRa Tks Gflex DNA Polymerase (Code No. R060A); TaKaRa Genome Walking Kit (Code No. 6108); DNA Ligation Kit (Code No. 6022); takara MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0(TaKaRa Code No. 9762); In-Fusion HD Cloning Kit (Clontech Code No. 639648); coli component Cell JM109(TaKaRa Code No. 9052); hind III (TaKaRa Code No. 1060A); nco I (TaKaRa Code No. 1160A); DL15000DNA Marker (TaKaRa Code No. 3582A); GL DNA Marker 5000 (Code No. AG11906, Ex Bio-engineering Co., Ltd., Hunan).

2 method of experiment

2.1 acquisition of the DNA flanking sequences of Limonium bicolor Lbrsgamum Lbrsg gene

Primers for cloning the sequences flanking the DNA at the 5' end of the gene were designed based on the DNA sequence of the LbRSG gene (Table 2).

TABLE 2 primers used for cloning of DNA flanking sequence at 5' end of LbRSG gene

The primers used for colony PCR verification of LbRSG gene are shown in Table 3.

TABLE 3 primers used for PCR verification of LbRSG Gene colonies

The leaf Genome DNA was used as a template, and TaKaRa Genome Walking Kit (Code No.6108) was used for the reaction, which was as follows:

(1)1st PCR reaction:

a first PCR reaction was carried out using P1(AP2, AP3, AP4 performed simultaneously) as the upstream primer and 5SP1 as the downstream primer.

Firstly, preparing 1st PCR reaction solution according to the following components:

1st PCR reaction conditions are as follows:

(2)2nd PCR reaction:

taking 1 mul of diluted 1st PCR reaction solution as a template of 2nd PCR reaction, and using an upstream primer P1; the downstream primer 5SP 2.

The 2nd PCR reaction system is as follows:

② 2nd PCR reaction conditions are as follows:

(3)3rd PCR reaction:

taking 1 mu l of diluted 1st PCR reaction solution as a template of 3rd PCR reaction, and using an upstream primer P1; the downstream primer 5SP 3.

Preparing 3rd PCR reaction solution according to the following components:

② 3rd PCR reaction conditions are as follows:

(4) taking 5 mu l of each PCR reaction solution, and carrying out electrophoresis by using 1% agarose gel;

(5) after cutting and recovering the gel, sequencing a PCR product by taking 5SP3 Primer as a Primer;

(6) taking 5 mu l of each PCR reaction solution, and carrying out electrophoresis by using 1% agarose gel;

(7) and cutting and recovering the gel, transforming the recovered product into a pMD19T cloning vector, transforming escherichia coli Trans-T1 competence, selecting a colony with a correct band after colony PCR, extracting a plasmid for sequencing, and performing DNA sequencing on a PCR product by taking pMD 18F, P1, pMD 18R and 5SP3 Primer as primers.

The batch identification system is as follows:

the reaction conditions were as follows: 1min at 94 ℃; 10sec at 98 ℃, 15sec at 50 ℃, 1min at 68 ℃ and 34 cycles.

2.2 LbRSG promoter element analysis

The promoter sequence cis-acting elements of the LbRSG gene were analyzed using the on-line tool on the plantaCARE (Lescot et al 2002) and GSDS2.0(Hu et al 2015).

2.3 replacement of the pCAMBIA 330135S promoter by the LbRSG promoter

In order to clarify the expression site of LbRSG, the 35S promoter of the pCAMBIA3301 vector is replaced by the LbRSG promoter, and the GUS gene expression position is determined after the Arabidopsis is infected.

2.3.1 pCAMBIA3301 Linear vector production

The plasmid pCAMBIA3301-35S-GUS was digested with HindIII/NCoI.

The enzyme digestion reaction system is as follows:

reaction conditions are as follows: at 37 ℃ for 2 h.

2.3.2 cloning of promoter fragments

2.3.2.1 extraction of Limonium bicolor DNA

Extracting the bicolor limonium DNA by a CTAB method: (1) taking a small amount of Limonium bicolor leaves, grinding by liquid nitrogen, and adding into a centrifuge tube; (2) adding 800 μ l CTAB, water-bathing at 65 deg.C for 30min, continuously reversing and mixing, centrifuging at 13000rpm for 5min at normal temperature by centrifuge; (3) transferring the supernatant to a new centrifuge tube, adding 500 μ l chloroform, mixing, and standing for 5 min; centrifuging at 13000rpm for 5 min; (4) transferring the supernatant to a new centrifuge tube, adding isopropanol with the same volume, mixing uniformly, standing at-20 ℃ for 2h, and then centrifuging at 13000rpm at 4 ℃ for 10 min; (5) removing supernatant, adding 700 μ l of 75% ethanol, washing, and repeating once; (6) add 50. mu.l of RNase-containing ddH₂O, DNA is dissolved.

2.3.2.2 primers for LbRSG gene promoter cloning were designed (Table 4).

TABLE 4 primers for LbRSG Gene promoter cloning

Amplification of 2.3.2.3 target product

The reaction system is as follows:

the reaction conditions were as follows: 1min at 94 ℃; 10sec at 98 ℃, 15sec at 55 ℃, 2min at 68 ℃ and 40 cycles.

2.3.3 ligation of Linear vector and PCR product into recombinant plasmid

The linking system is as follows:

reaction conditions are as follows: 15min at 50 ℃.

2.4 infection of wild type Arabidopsis thaliana

2.4.1 Arabidopsis culture and treatment: and (3) cutting off fruit pods and flowers which are already opened when the arabidopsis thaliana in the incubator grows to about 10cm, and fully watering for later use. The conditions of the incubator are light intensity of 120-²The photoperiod was 16/8h (day/night), the culture temperature was 22 ℃/18 ℃ (day/night), and the relative humidity was 75%.

2.4.2 preparing bacterial liquid: (1) melting Agrobacterium containing target gene in ice bath, transferring to YEB liquid culture medium, adding Kan and Rif (final solubility is 50 μ g/ml), culturing at 28 deg.C in incubator at 180rpm overnight; (2) taking 1-2ml of activated bacterium liquid, carrying out secondary shake bacterium in YEB culture medium containing Kan, Rif and AS (final solubility is 10 mu g/ml), and culturing for 6-8 h; (3) transferring the activated bacterial liquid into a 50ml large centrifuge tube, centrifuging at 4 ℃ and 6000rpm for 5min, and removing the supernatant; (4) resuspending the pellet with 5% sucrose solution, measuring absorbance with UV spectrophotometer, and adjusting OD₆₀₀The value is in the range of 0.8-1.0; (5) the suspended bacterial solution was separately filled in 1.5mL portions0.1 percent of surfactant Silwet L-77 is added into each Ep tube and is fully mixed.

2.4.3 infection of Arabidopsis inflorescences: sucking the prepared bacterial liquid, dip-dyeing the arabidopsis inflorescences until each flower is fully dip-dyed by the bacterial liquid, placing the flower in an incubator for dark culture for 24 hours, then normally culturing, infecting the flower once every 7 days or so until no new inflorescences grow out, and totally infecting the flower three times.

2.5 screening and identification of Arabidopsis Positive lines: 2.5.1 screening of the positive lines was carried out according to the resistance of the pCAMBIA3301-35S-sGUS vector using the herbicide BASTA (from Coolaber, the main component being glufosinate), the specific experimental procedure being as follows: (1) drying the harvested seeds after inflorescence infection, removing impurities, then placing the seeds in a 5mL centrifuge tube, adding a proper amount of sterile water, vernalizing the seeds in a refrigerator at 4 ℃ for 2 days, then planting the seeds in a big white basin filled with nutrient soil, covering a layer of preservative film on the basin, and placing the basin in an incubator for culture; (2) when the arabidopsis grows to the six-leaf stage, 0.1 percent herbicide BASTA is sprayed, and the spraying is carried out for 2 to 3 times in a week; (3) the surviving plants were transferred to new pots for cultivation.

2.5.2 CTAB method for rapidly extracting genome DNA of Arabidopsis thaliana

(1) Shear 2mm²Positive seedling leaves are placed in a small Ep tube; (2) add 20. mu.l TPS buffer; (3) mashing the leaves with toothpick; (4) reacting at 95 deg.C for 15min, immediately placing on ice for 3-5 min;

(5) add 50. mu.l ddH₂And O, uniformly mixing the mixture to be used as a template for PCR reaction.

2.5.3 identification of positive lines using pCAMBIA3301-S and LbRSG-A as primers.

TABLE 5 primers used for screening of heterologously expressed plants

After positive strains are verified by DNA level, positive seedlings are selected and cultured to T3 generation.

2.6 GUS staining to observe the location of the gene tissue

GUS staining analysis was performed according to previously reported methods (Troll et al, 1955; Lin et al, 2015).

The specific method comprises the following steps: (1) pretreatment: the screened homozygous arabidopsis seeds are planted in a culture medium, and after the seeds grow for 5 days, the whole arabidopsis seedling is taken and the culture medium is washed by distilled water; (2) dyeing: putting a proper amount of prepared GUS staining working solution into a 1.5mL Ep tube to completely immerse the plant material, wrapping the plant material with tin foil paper at 28 ℃ and standing overnight; (3) and (3) elution: eluting with 70% ethanol for 1 hr each time until the leaves become white by naked eye, and eluting for 3 times; (4) and (4) observation: and (4) dropwise adding a transparent liquid to prepare a temporary mounting piece, and observing a blue GUS expression site under a microscope.

3 results and analysis of the experiments

3.1 acquisition of the DNA flanking sequences of Limonium bicolor LbRSG Gene

PCR was carried out using the TaKaRa Genome Walking Kit using Limonium bicolor leaf Genome DNA as a template and nested primers LbrSG 5SP1, LbrSG 5SP2, LbrSG 5SP3 and degenerate primers AP1, AP2, AP3, AP4 as primers, and 5. mu.l of each of the first, second and third PCR products was subjected to 1% agarose gel electrophoresis, and the bands obtained in the three times are shown in FIG. 6. Using Takara miniBEST Agarose Gel DNA Extraction Kit Ver.4.0(Code No.9762) to cut rubber and recover an amplification product band with the size of about 0.8kbp of 3rd AP4, connecting the recovery product of the target band to a PMD19T cloning vector, transforming escherichia coli, using LbRSG YF1 and LbRSG YR1 as primers for colony PCR identification of genes, selecting the identified strain to be put into a liquid LB culture medium, adding Amp for bacteria shaking, extracting plasmids, using a general primer pMD 18F, pMD 18R for sequence determination, and a sequencing result shows that the obtained DNA fragment corresponds to the LbRSG gene and a gene sequence part can be found in the sequencing result. Finally, the sequence 3030bp of the 5' end of the LbRSG gene is obtained (figure 7).

3.2 promoter element analysis: predicted from LbRSG gene promoter, it was found to have necessary promoter elements, and some elements (ABRE, GARE-motif, AAAC-motif) responding to abscisic acid, gibberellin, auxin, and in addition some action elements (LTR, MBS, G-Box) responding to low temperature, drought, and light response, and the specific elements and positions are shown in FIG. 8.

3.3 pCAMBIA3301-LbRSG Promoter-sGUS vector construction: the products obtained by digestion were subjected to 1% agarose gel electrophoresis using appropriate amounts of the gel, and the results are shown in FIG. 9A: and cutting and recycling the rubber. And (3) performing PCR (post-electrophoresis) on the target gene (FIG. 9B), recovering glue, connecting with a linear expression vector, transforming to escherichia coli (FIG. 9C), transforming to agrobacterium after correct sequencing, and selecting the No. 2 strain for storage, wherein the result is shown in FIG. 9D.

3.4 screening and identification of Arabidopsis heterologous expression positive strains: after regular spraying of the herbicide BASTA (from Coolaber, main ingredient glufosinate), it was found that most arabidopsis thaliana were screened lethal in the first screening generation (fig. 10). DNA of each surface strain of Arabidopsis thaliana after herbicide screening is extracted, primers are designed for PCR amplification, and the electrophoresis detection result of the PCR product is shown in FIG. 11.

3.5 GUS staining result observation: in order to understand the tissue location of the LbRSG gene in Arabidopsis, an expression vector is constructed by using a gene-specific promoter, and GUS staining is used for observing the expression of the gene in different tissues.

In order to more intuitively display the expression pattern of LbRSG in different tissues of Arabidopsis, the expression distribution in LbRSG plant cells was examined by staining the LbRSG promoter-GUS transgenic line. GUS coloration indicated that LbRSG was significantly expressed in arabidopsis young leaves, floral organs, roots and pods, but rarely expressed in cotyledons. GUS visualization also showed that LbRSG was significantly expressed in epidermal hair, root hair and veins of mature leaves (fig. 12).

In order to investigate the tissue localization of the LbRSG gene, this example obtained 3030bp of promoter sequence before the coding region of the gene by means of chromosome walking. Three specific primers (SP primers) with high homology and specificity are respectively designed according to CDS sequences of genes, and are subjected to thermal asymmetric PCR reaction with four uniquely designed degenerate primers with lower annealing temperatures, namely AP1, AP2, AP3 and AP 4.

By analyzing the promoter sequence, it is found that the promoter of the gene has some elements (ABRE, GARE-motif and AAAC-motif) responding to abscisic acid, gibberellin and auxin and some elements (LTR, MBS and G-Box) responding to low temperature, drought and light response besides some necessary cis-acting elements (TATA-Box and CAAT-Box) (Twyman,2003), which may have a certain relation with the function of the gene. Provides important information for the subsequent research on gene functions.

The pCAMBIA3301-35S-GUS is used for constructing a gene specific promoter expression vector, a promoter of a gene per se is required to replace a 35S strong promoter of an original vector, the vector contains a GUS reporter gene, the constructed expression vector can be used for infecting arabidopsis thaliana, screening T3 generation homozygous transgenic arabidopsis thaliana, and observing the expression of the gene on different tissues by staining, so that the gene is mainly expressed at a vein, a root and rhizome combination part, a root and root hair part of the arabidopsis thaliana, particularly the staining at the root and rhizome combination part is deeper, and a certain relation is possibly formed between the formation of the root and the occurrence of the root hair. Secondly, it was found that the expression is more pronounced in the mature region of the primary root, but is weaker or even absent at the root tip, especially in the root apical meristematic region.

After the arabidopsis thaliana is transformed after a gene-specific promoter is constructed and connected with a GUS vector by promoter cloning and replacing a 35S promoter, the LbRSG1 is mainly positioned in a conduction tissue, such as veins, a root-tuber junction and root hair, and the connection of the gene and the absorption and response of ions can be suggested.

Example 3 phenotypic analysis of Limonium bicolor Gene LbRSG silencing lines, overexpressing transgenic plants

Construction of Limonium bicolor gene LbRSG silent line

1 main chemical reagents and instruments

1) Culture medium: LB medium, YEB medium, MS medium. 2) Antibiotics: ampicillin Ampicillin, Kanamycin Kanamycin (60615, Sigma, USA), rifampicin Rifampin (R350, Sigma, USA). 3) The kit comprises: FastPure Gel DNA Extraction Mini Kit (DC301, Nanjing Vazyme), FastPusmid Mini Kit (DC201, Nanjing Vazyme Biotech, China),

plant Total RNA Isolation Kit (Vazyme, Nanjing, China). 4) Enzyme: nco I (NEB), BsaI (NEB), T4 Ligase (NEB),2XTaq Plus Master MixⅡ(P213-AA，Nanjing Vazyme)，

Max Super-Fidelity DNAPolymerase(P505-d1/d2/d3，Nanjing Vazyme)，

II One Step Cloning Kit (C112, Nanjing Vazyme). 5) SYBR Green real-time fluorescent quantitative PCR premix (Gene Star, China). 6) Competence: DH5 alpha (Nanjing Vazyme), GV3101(Nanjing Vazyme). 7) Carrier: pCAMBIA3301-35S-sGUS (TaKaRa), pHEC401-CRISPR/Cas9(TaKaRa), pCBC-DT1T2 (TaKaRa). Other reagents, e.g. agarose, glycerol, AS (acetosyringone), ddH₂O and the like are all commercially available.

2 method of experiment

2.1 construction of LbRSG knockout vectors

Gene knockout primers were designed using CRISPR/Cas9 gene knockout technology (Ma Y, z.l., Huang x.2014 Dec5186-93 (2018) Genome modification by CRISPR/case 9.febs j.bmc Plant Biol,281(23), 86-93).

The CRISPR/Cas9 gene knockout technology is utilized, introns need to be avoided when primers are designed, the genomic DNA of limonium bicolor is used as a template, 2DT1-BsF, 2DT1-F0, 2DT2-R0 and 2DT2-BSR are used as primers, and a four-primer method is used for PCR amplification to obtain corresponding DNA sequences. Cas9 can target a specific gene sequence through a 20bp guide sequence on the sgRNA, the only requirement for selection of the Cas9 target site is the presence of a PAM sequence at the 3' end of the 20bp target sequence. Therefore, based on the obtained gene DNA sequence, knockout primers were designed in the exon region, the length of the primers was about 20bp, and the primers were searched 20bp before the PAM sequence, and the method was referred to (Cong L, R.F.A., Cox D, et al (2013) Multiplex genome engineering using crisopr/cas systems, science,339 (6121)). Information of primers used for constructing the CRISPR-Cas9 vector is shown in table 6.

Table 6 primers for construction of CRISPR-Cas9 vector

The nucleotide sequence of the sgRNA action site is 5'-AGTATCGATGCAGTGAAAC-3'.

2.1.1 PCR amplification

Firstly, carrying out four-primer PCR amplification by taking a pCBC-DT1T2 intermediate vector as a template to obtain an intermediate vector pCBC-DT1T2 containing two target site sequences, wherein the fragment length is 626bp, and the CRISPR-Cas9 expression vector fragment is obtained. The following components were added to a microcentrifuge tube to form a 50. mu.l system:

the reaction conditions were as follows: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 56 ℃, 1min at 72 ℃ and 34 cycles; storing at 4 ℃.

2.1.2 purification and recovery of PCR products, the procedure is as in example 2.

2.1.3 the following enzyme digestion-ligation system was established: the intermediate vector pCBC-DT1T2 containing the two target site sequences was ligated to the vector pHEC401 (the vector pHEC401 was awarded by Liu flare light professor of south China university) using BsaI (NEB) for enzymatic cleavage, using T4 Ligase (NEB), to construct the pHEC401-2gR-LbRSG expression vector, in the following system:

the reaction system is as follows:

the reaction conditions were as follows: 5h at 37 ℃; 5min at 50 ℃; 10min at 80 ℃.

Second, construction of overexpression LbRSG transgenic strain

Construction of 1pCAMBIA1300-LbRSG vector

1.1 cloning of the LbRSG fragment of interest: by utilizing a seamless cloning technology, SalI restriction enzyme cutting sites are selected, and homologous recombination primers for amplifying target genes are designed through CE Design V1.04 software, wherein the primers are shown in a table 7:

TABLE 7 pCAMBIA1300-35S-sGFP homologous recombination primers

PCR amplification reaction System:

reaction conditions are as follows: 3min at 95 ℃; 95 ℃ 15sec, 68 ℃ 15sec, 72 ℃ 90sec, 34 cycles; 5min at 72 ℃; storing at 4 ℃.

1.2 recovery of fragments of interest

1.3 pCAMBIA1300 vector DNA preparation: the pCAMBIA1300-35S-sGFP expression vector containing hygromycin resistance gene and GFP reporter gene is used for subcellular localization of the gene.

1) Enzyme digestion reaction

Reaction system:

reaction conditions are as follows: 30min at 37 ℃.

2) And (5) recovering the carrier.

1.4 ligation of the Gene of interest to pCAMBIA1300-35S-sGFP vector DNA: using the seamless cloning technique

II One Step Cloning Kit, (C112), the recovered product of the gel was ligated with the linearized pCAMBIA1300-35S-sGFP expression vector, and the ligated product was named pCAMBIA 1300-LbRSG.

A connection system:

connection conditions are as follows: 30min at 37 ℃.

2 plasmid transformation of Escherichia coli

1) Taking out the competent cell DH5 alpha from-80 refrigerator, and thawing on ice for 20 min; 2) pipetting 5-10 μ l of the ligation product with a pipette, adding into 30-50 μ l of competent cells, blowing, mixing, and standing on ice for 30 min; 3) heating in water bath at 42 deg.C for 45s, immediately placing on ice for 2 min; 4) the pipette is used for sucking 900 mul of LB liquid medium (without adding antibiotics) and adding the LB liquid medium into the mixture, and shaking the mixture for 1h at 37 ℃ (the rotation speed is 200 and 250 rpm); 5) pouring the plate in 1h of shaking the bacteria, heating and melting LB solid culture medium in a microwave oven, cooling to about 60 ℃, adding corresponding antibiotic (Kana or Amp with the final concentration of 50 mug/mL), uniformly mixing and pouring into a plate; 6) shaking the bacteria for 1h, centrifuging at 5000rpm in a centrifuge for 5min, discarding the supernatant, lightly blowing the residual liquid culture medium with a pipette to resuspend the bacteria, sucking 20 μ l of the resuspended bacteria into a solid culture medium (the residual bacteria can be temporarily stored in a refrigerator at 4 ℃), and lightly coating the bacteria on a flat plate with a sterile coating rod (the coating rod rotates in one direction in the coating process); 7) after the bacterial liquid is coated evenly, sealing a plate with a sealing film, putting the plate into a 37 ℃ incubator for inverted culture for 12-16h, and screening positive clones; 8) after the culture, a monoclonal product can grow on the transformed plate (at the moment, if a sterile colony grows out, the residual bacterial liquid can be used for secondary coating), and a single colony is picked for lineation and propagation; 9) colony PCR is carried out by using the primers on the carrier, and colonies with correct bands are selected for sequencing.

3 transformation of Agrobacterium with vector

1) After sequencing, comparing, selecting corresponding bacterial colony shake bacteria without mismatch, extracting plasmids such as the plasmid in the connection of the target fragment amplified in the second chapter 2.5 and the pMD18-T vector; 2) taking out the agrobacterium-infected state from a-80 refrigerator, and unfreezing the agrobacterium-infected state on ice for 20min until the agrobacterium-infected state is completely melted; 3) pipetting 5 mul of plasmid with a pipette gun, adding into 30-50 mul of competent cells, gently blowing and mixing with the pipette gun, and standing on ice for 30 min; 4) putting the mixture into liquid nitrogen at medium speed for 5 min; 5) taking out from liquid nitrogen, immediately placing into a 37 deg.C water bath to melt competent cells for 5 min;

6) sucking 900 μ l YEB liquid culture medium with pipette in super clean bench, adding into the mixture, gently blowing, mixing, placing in 28 deg.C shaking table for 200r/min, and 3 hr; 7) melting the solid YEB culture medium in a microwave oven, cooling to about 60, adding 50 mu g/mL kanamycin (Kana) and rifampicin (Rif), and uniformly mixing and pouring the mixture into a plate; 8) putting the shaken bacterial liquid into a centrifuge at 5000rpm, centrifuging for 1min, and enriching thalli; 9) discarding the supernatant, slightly blowing the residual liquid culture medium with a pipette to resuspend the thallus, sucking 20 μ l of the resuspended thallus into the solid culture medium (the residual thallus can be temporarily stored in a refrigerator at 4 ℃), and slightly and uniformly coating on a flat plate with a sterile coating rod (the coating rod rotates in one direction in the coating process); 10) culturing in 28 deg.C incubator, screening positive clone, selecting single clone colony (if sterile colony grows out, coating with the rest bacterial liquid for the second time), and streaking for propagation; 11) and (5) carrying out colony PCR, selecting a colony with bright and correct bands, and shaking and storing the colony.

4 preservation of Strain

1) Agrobacterium colonies containing the correct target fragment were picked with a sterilized pipette tip and placed in 50mL YEB broth, Kana 50. mu.g/mL and Rif 50. mu.g/mL were added to the broth, and the mixture was placed on a shaker at 28 ℃ and cultured overnight at 180 r/min. 2) Preparing glycerol sterilized in an autoclave, sucking 300 mu l of glycerol and 700 mu l of bacterial liquid, adding into a 1.5mL Ep tube, fully shaking and uniformly mixing by using a vortex instrument, and quickly freezing in liquid nitrogen for 5 min. 3) The Ep tube was clipped out with tweezers and stored in a refrigerator at-80 ℃.

Infection of 5-dichromatic Limonium Gmelinii leaves

5.1 shaking bacteria

(1) The agrobacterium liquid containing pHEC401-2gR-LbRSG expression vector and pCAMBIA1300-LbRSG expression vector is taken out in a refrigerator at-80 ℃ and melted in ice bath for 30 min. (2) The suspension was transferred to 50mL YEB medium (containing Kan and Rif at a final concentration of 50. mu.g/mL, 20. mu.M AS) using a pipette gun and shake-cultured at 28 ℃ and 200rpm for 24 hours. (3) And (4) activating the bacterium solution which is shaken for the first time again to enable the activity of the agrobacterium to reach the highest. The first shaken broth was pipetted into a new 50mL YEB medium (containing Kan and Rif with a final solubility of 50. mu.g/mL, 0.2. mu.M AS) and shaken at 200rpm at 28 ℃ for 6 h.

5.2 infection

(1) The shaken bacterial solution was poured into a 50ml centrifuge tube and centrifuged at 6000rpm at 4 ℃ for 10 min. (2) The supernatant was discarded, suspended in MS liquid, and then transferred to 50ml of liquid medium, to which 0.2. mu.M of AS was added. The OD value of the bacterial suspension was about 0.7 (λ 600 nm). (3) And culturing the sterile seedlings for 30 days. (4) The culture medium attached to the lateral roots and the main roots was removed with forceps, and the roots were cut into 1cm long. (5) Placing in prepared bacterial liquid for infection for 17min, continuously shaking during the infection period, and accelerating the infection. (6) The dry bacteria solution is sucked by filter paper, evenly placed on an MS solid culture medium containing glucose and gibberellin, and cultured in dark for 4 d. (7) Transferring to MS solid culture medium containing piperacillin, hygromycin and gibberellin, and culturing under light. (8) After cluster buds grow, the cluster buds are transferred to a rooting culture medium, and meanwhile, leaves are taken to observe the development condition of the saline glands.

Three, subcellular localization

The overexpression vector constructed above is used to transform agrobacterium GV3101, and a method for infecting onion epidermis by agrobacterium is adopted (Sun et al 2007), which comprises the following steps:

1) taking fresh onion bulb, peeling three to four layers of scales on the outer surface, disinfecting with 75% ethanol for 10min, then washing with deionized water for 3 times, cutting the onion epidermis on the inner layer into 1cm multiplied by 1cm, tearing off the cut inner epidermis with a pair of tweezers, washing with sterile water for 3 times, sucking surface water with filter paper, then placing on 1/2MS culture medium, and culturing at 24 ℃ in dark for 24 h.

2) The unloaded GV3101 Agrobacterium carrying the pCAMBIA 1300-35S-sGFP-LbRLG expression vector and pCAMBIA1300-35S-sGFP was removed from a-80 ℃ refrigerator, thawed in an ice bath, added to YEB medium containing Kan and Rif at 50. mu.g/mL, shaken at 28 ℃ and 180rpm overnight.

3) Centrifuging the bacterial solution at 4 deg.C and 6,000rpm for 1min, suspending the bacterial solution with MS liquid medium, adding AS (final concentration 200 μ M), and measuring OD of bacterial solution with ultraviolet spectrophotometer₆₀₀And may be about 1.0.

4) Placing the pre-cultured onion epidermal cells in prepared bacterial liquid, fully submerging, infecting for 30min, then taking out the epidermis by using tweezers, sucking dry the surface bacterial liquid by using filter paper, and paving on 1/2MS culture medium (the final concentration is 20 mu M), and co-culturing for 2d at 24 ℃.

5) The onion skins were removed with forceps and rinsed clean with sterile water and placed directly on a glass slide.

6) DAPI staining: DAPI (4',6-diamidino-2-phenylindole), namely 4',6-diamidino-2-phenylindole, is a fluorescent dye capable of strongly binding to DNA and is commonly used for fluorescent microscope observation. Because DAPI is permeable to intact cell membranes, it can be used for staining of both living and fixed cells, here for staining of the nucleus. The incident light was 358nm and the emergent light was 461nm, at a concentration of 10. mu.g/. mu.l. The dyeing time is 5-10 min.

7) FM4-64 staining: FM4-64 is a non-toxic water-soluble substance capable of specifically staining membranes, and is commonly used for staining cell membranes by utilizing the characteristic that the FM4-64 emits high-intensity fluorescence after being specifically combined with plasma membranes. FM4-64 is dissolved in DMSO, the concentration of mother liquid is 800 μ M, the working concentration is 4 μ M, the incident light of FM4-64 is 559nm, and the emergent light is 648-734 nm. The dyeing time is 1-10 min.

8) Observed with a confocal laser microscope.

Fourth, experimental results and analysis

Construction of 1CRISPR-Cas9 knockout vector

Since the full-length gene cloning is performed according to the third-generation transcriptome sequencing, but many genes contain introns, before designing a gene knockout primer, the genome sequence of the gene needs to be found, and a primer needs to be designed according to a continuous exon sequence so as not to knock out the site on the intron. Through DNMAN mapping (FIG. 14), the template (cDNA) sequence in FIG. 13 was finally determined to design knock-out primers. Taking a proper amount of an intermediate vector pCBC-DT1T2 product containing two target site sequences obtained by PCR, and carrying out 1% agarose gel electrophoresis to obtain a 626bp correct target band; cutting and recovering the gel, connecting a target band to an expression vector pHEC401, transforming the constructed pHEC401-2gR-LbRSG expression vector into escherichia coli, performing colony PCR amplification by using a U6-26F forward primer and a U6-29R reverse primer, amplifying a correct target fragment of 726bp, selecting a monoclonal colony with a correct band, sequencing by using 626F and 629F primers, comparing, and then, containing an editing sequence, indicating that the vector construction is successful, identifying the colony PCR after transforming the agrobacterium as a positive strain, and storing the strain for transforming the wild type limonium bicolor.

2 infecting bicolor limonium leaf explant to obtain transgenic regeneration plant

After agrobacterium is transformed by the LbRSG knockout vector, Limonium bicolor leaves are infected to obtain callus, and a transgenic regeneration strain is screened on a culture medium by utilizing hygromycin. The taproots have now been infected and screened on hygromycin selection medium (FIG. 15), now adventitious buds have grown, and after rooting selection, the LbRSG knockout line 3 strain is obtained (FIG. 16).

3 Limonium bicolor knockout mutant and salt gland observation of epigenetic line

Under the excitation of 330-380nm ultraviolet light, the wild-type bicolor limonium salt gland has blue autofluorescence, and four obvious salt gland luminescence points can be seen, which are formed by thickening intercellular stratum corneum. When the Limonium bicolor explants were infected, the uninfected wild Limonium bicolor was used as a control group, cultured under the same conditions, and then the true leaves formed by redifferentiation of the roots were picked with tweezers, and the saline gland development was observed under DIC and fluorescence microscope (FIG. 17).

Limonium bicolor is infected by agrobacterium containing a gene overexpression vector, and is screened by hygromycin, a cluster bud grows from callus near roots, when the bud grows to the size equal to the true leaf size in the period of D, E, a part of leaves are taken to respectively manufacture temporary mounting pieces, and the salt gland phenotype of the Limonium bicolor Lbrsg1 knockout and overexpression strain is observed under a microscope, as shown in figure 18. The wild type has obvious salt gland with 4 luminous points through ultraviolet autofluorescence and GFP observation. The LbRSG knockout mutant has three-luminous-point salt gland, and the number of constitutional cells of the salt gland is obviously reduced; the phenotypic strain shows that the salt gland component cells are obviously increased, and the salt gland phenotype of an eight-luminous point or a nine-luminous point is shown. Indicating that LbRSG is indeed involved in the regulation of the differentiation of saline glands.

4 onion subcellular localization results

The expression of the GFP reporter gene was detected by observation using a two-photon confocal microscope, and it was revealed that the protein encoded by the LbRSG gene was localized to the cell membrane (fig. 19).

To investigate whether the LbRSG gene plays a key role in the development of limonium bicolor salt glands, this example silenced the gene in limonium bicolor using CRISPR/Cas9 gene knockout technology (Feng et al, 2013). In the experiment, a gene knockout vector is constructed, and then the limonium bicolor is transformed to obtain the regenerated buds. Hygromycin was selected for screening positive regenerated shoots. And after the leaves are mature, observing the development condition of the positive seedling salt gland.

The development of the salt glands of the different lines was observed under DIC and fluorescence microscopy. The saline glands of the normal wild type limonium bicolor have autofluorescence, have four luminous points due to thickening of cell wall cuticle, and the shapes of the saline glands are more regular. When the LbRSG gene in Limonium bicolor is knocked out, the salt gland with three luminous points appears in the leaf blade at the moment, and the diameter of the salt gland is reduced integrally at the moment, which indicates that the number of cells forming the salt gland is reduced at the moment. The above results indicate that the LbRSG gene positively regulates the development of Limonium bicolor salt gland.

In situ hybridization of the invention showed that LbRSG was localized to the saline glands, subcellular localization to the cytoplasmic membrane, and histochemical staining of the promoter showed a possible association with ion transport.

According to the three-generation full-length transcription group information, the full length 669bp of mRNA of LbRSG is obtained by cloning, 222 amino acids are coded, high expression is shown in the salt gland development period, and onion subcellular cells show that the LbRSG is positioned on a cytoplasmic membrane. Bioinformatics analysis shows that the gene encodes a hydrophilic protein, a double-transmembrane domain exists, the outer part of the membrane occupies 64 percent (141 amino acids) to form 1 large extracellular loop, the continuous negatively charged domain possibly participates in ion response, and the C end in the membrane has phosphorylation sites of threonine and serine, possibly leads to enzyme-linked reaction in cells through phosphorylation. The gene is directly positioned on the salt gland through in situ hybridization, and the strong correlation with the development of the salt gland is suggested.

The LbRSG preplanning 3030bp promoter sequence is obtained by a chromosome walking technology, and has the functions of responding abscisic acid, gibberellin, auxin elements (ABRE, GARE-motif and AAAC-motif), low-temperature, drought and light action elements (LTR, MBS, G-Box) and the like besides necessary cis-acting elements (TATA-Box and CAAT-Box). After the arabidopsis thaliana is transformed after a gene-specific promoter is constructed and connected with a GUS vector, the LbRSG is mainly positioned in a conduction tissue, such as veins, a root-tuber junction and root hair, and the possibility that the gene is related to the absorption and response of ions is possibly suggested.

In situ hybridization shows that LbRSG can be directly related to the development of saline glands, and the function of LbRSG genes is further verified by using knockout and overexpression technologies. An LbRSG knockout carrier is constructed by using a CRISPR/Cas9 gene editing technology, LbRSG silent mutant LbRSG is obtained through a Limonium bicolor genetic transformation system, and the development condition of the LbRSG saline gland is determined by adopting a method that the saline gland has blue autofluorescence under ultraviolet excitation light. Compared with wild four-light-point salt gland, the LbRSG silent line LbRSG has a large number of three-light-point salt glands, which indicates that the salt gland dysplasia is effectively caused by the LbRSG knockout. Through the phenotype observation of LbRSG gene knockout strains, the LbRSG is determined to play a positive regulation role in the salt gland development process.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of Shandong Master

<120> Limonium bicolor gene LbRSG and application thereof

<130> PI202110023

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 222

<212> PRT

<213> Limonium bicolor (Limonium bicolor)

<400> 1

Met Ala Ser Arg Gln Ser Pro Leu Leu Ile His Arg Leu Phe Phe Val

1 5 10 15

Asn Ser Leu Leu Ala Pro Ala Ile His Leu Val Leu Ile Leu Ser Leu

20 25 30

Phe Ala Arg His Ser Thr Ala Asn Tyr Ile Gln Gly Gln Ala Thr Gly

35 40 45

Leu Arg His Asn Arg Ala Leu Leu Gly Phe Lys Glu Thr Pro Ser Gly

50 55 60

Gly Asn Thr Thr Phe Glu Cys Ser Pro Ser Gly Pro Cys Ile Ala Cys

65 70 75 80

Gln Tyr Ser Glu Lys Asn Glu Glu Lys Tyr Arg Cys Ser Glu Thr Gly

85 90 95

Tyr Arg Ile Pro Leu Lys Cys Val Lys Ile Val Asn Val Ala Asn Glu

100 105 110

Glu Asn Asp Glu Lys Glu Glu Thr Arg Ser Thr Leu Glu Val Leu Asn

115 120 125

Ser Glu Ser His Thr Asp Ser Gly Leu Arg Gly Ser Glu Asn Val Lys

130 135 140

His Arg Thr Leu Ala Glu Ser Lys Thr Asn Lys Ala Gly Glu Ser Glu

145 150 155 160

Val Tyr Val Thr Tyr Arg Ser Cys Ile Pro Ala Val Asn Glu Glu Lys

165 170 175

Ile Ser Val Val Gly Phe Glu Val Phe Thr Leu Ala Leu Leu Leu Ala

180 185 190

Ser Gly Ser Phe Val Phe Leu Arg Arg Arg Arg Thr Ser Val Ala Ser

195 200 205

Thr Ala Gly Gly Gly Ile Arg Met Gln Thr Asn Pro Arg Phe

210 215 220

<210> 2

<211> 3030

<212> DNA

<213> Limonium bicolor (Limonium bicolor)

<400> 2

aatgcaagga cctgaaggcg agcacacaaa agtctaatct caaccatcat cactagctaa 60

ggttgaagtc ctgaccatat attttgacaa agtggcttcc aacttgtctc caatcaacga 120

tatccacgcc taaatagaga taaataaata gtgtaaccaa caaggtcact tcaaaacaat 180

aacaagggat tgatcgacac acaaagatag agaaagttgt tagatgctta gtaactatct 240

gtttttggat aaagaaaatc aaaacctggt ctgccgaatt gaaccctcga atggtgttac 300

ctcctacaat ggcaatccct ctgttcccca gaggctgaga tatcactgcc tgccgaaatc 360

cacgttgcta gatcaagcag agcaatagac atataggagc agatgctaaa cagcatgatc 420

agcagtattt acctgtgtat ttgcaggaag aaaaggcagc tctgatctcg cagggtaaag 480

agaaaggttt gcaaaatagg tttctacaga cacaacattg aacaaacaac taaattaacg 540

tgaactgaag caattactcg ctgcttaaat gatcaagaca atttaacctc agcgccggac 600

acgagtttaa ctaaagctta cgtgccccag atttcataag cccttgatac aacgtgcctc 660

gaatcagttt tgcagaatct acggtcacag cttcaccacc tcctttgagt gattcagctg 720

ctgttcctat ctgaaacaca caatcgccat cataaacaac gaccaaagac ttgcaacaag 780

tagccgtcga caaagattcc caaaacctgg aaacaaagaa gtacgaattg aaactacttg 840

atccggacgg aatgtgtatg gatatgaatg gtttaagtta caaagtaaga cgtttcatca 900

taccatttca actcagaaac aacatggtca ggaaggtatg gagacattct ttcacactcg 960

acaccttctg gatccaccta acataaaaga aaaaagagcg atcaaaggaa ttcacattca 1020

cactattaga cacagccaga caaagataat tgagggaaaa cagttctaac cttagatatg 1080

gatttcggtc taattgaaac ccatacaaga ccagttagga tacacgaaac tgataaacca 1140

agagctaaca aatccgccct ggattgcgaa ctactcacaa aagatcaact tagatcgtac 1200

gagttgcatg aggcacagac ttgccacatt catagacact agttgttcag aaagataaac 1260

atgaaatttc gtcgacgccg cggtgatctc aattctcaaa ttgaggtgtt attctcatga 1320

tagagatcgc aaagaacgag tgaaggatag aagataaaat cttttacctt ccggcatcgg 1380

cgacgggtgc aatgccggaa accgcgcgat taaagagaac cgcgagcagg gaaaatccgc 1440

caatgtaaat tggtagactt cgaacgacat cgtcgttttc agaaacccaa tccgcaactg 1500

aattcctttt aggcttcggc cctgtatatg cacctcctga atttccctaa gggagagcaa 1560

aattcttaca gaaaggttga agtacatgta aaggcgagca gtttgaaaag gagaagctat 1620

aagtgcgacc tggaagtttc gaggggacga tttagccgtt atgtgacgtc gaaaggtaag 1680

agggtggaga tggagaggaa gggcgctagt atgtttggag cagagatttc gggggaaaaa 1740

atcgccattg cagttaggct gtactgtttg atgccgaagt gtgattgccg tttccatatc 1800

gttacgaagg tctcaattcg ttccccagat tttgtaggca attaacaact agggatgagg 1860

gatttggatt tatccaaatt aactgaaatt tatttttgga tttttaactg atttttaaaa 1920

ctgaatttaa aattttggtt attcgaatat ccaaattata attcggtttg gatttcggtg 1980

ttaaccctat aaaattcggt taaccgaatc atccaaactt ttttttaaag aaaatctcta 2040

aatctttcct taatggttaa tactctttaa acatgacaaa cagaacgtca aaattcataa 2100

ccctatctcg cattgctcca atgcttcaat tatataatgt catactattc ttttttcgtc 2160

aaattaatta tatataactg gcttttttgg aacattgttc atcgctgctc acccctcgtt 2220

tgatgtaact agtcaaatgt ttgagtgagt tctttctctt tctgttgtga tgaattggca 2280

actagggaat tcacaatttt cttgcagctt atggttatta ctgctgctga tcatgaacgt 2340

gaaacaaaaa aaagtgtttg tttttttttt tagttcggtt aactaaaatc cgattttaaa 2400

aatgggtttt tggttttttt tttaaatttc agttaattga aatccgaacc aaagcaaatt 2460

ttggatttca attcggtttt cagttttcaa ctgaaaaaag tttcagattt tggataattt 2520

ggatttcggt taattcagtt ttaatcaaag tttagttaat tcggtttttt aaccaaatta 2580

accaaaatcc aatttttttt tacaatttag attttggtta atcaaattat ccaaatgcta 2640

acctctgtaa caaccgtgag caaaagaaaa ctacccgcag attatgcaat tggattaacc 2700

gaaaaataac ctatggttgg gcctatagtt gggctggata caatagccca ataattagct 2760

aacaaccaaa gttgtcttaa tagtttgtcc ttccgtccct ctctcctcgg ccttaatgcc 2820

tccggttagc gtaactcgga tcggaacccc aaaaatttcg ttggttcgcc cgattattgt 2880

cgtgagattc tgtcttcttt gatcggacat cgacaattct gcatcttccc gacggtgaag 2940

gtggagtaag ccttgcaagt cgaaatacta gcagaactga ttgtgagctc tggtcgactg 3000

tagtatcaga ggacataacc agccattgga 3030

Claims (10)

1. Limonium bicolor gene LbRSG, characterized in that it is a gene encoding the following protein (a) or (b):

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO; or

(b) 1, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.

2. The Limonium bicolor LbRSG promoter is characterized by having the sequence as follows:

i) a nucleotide sequence shown as SEQ ID NO. 2; or

ii) nucleotide sequences with the same functions, wherein one or more nucleotides are substituted, deleted and/or added in the nucleotide sequence shown in SEQ ID NO. 2; or

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO. 2 under stringent conditions, in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS at 65 ℃ and washing the membrane with the solution; or

iv) a nucleotide sequence having more than 90% homology with the nucleotide sequence of i), ii) or iii) and having the same function.

3. A biomaterial containing the gene LbRSG according to claim 1 or the promoter according to claim 2, wherein the biomaterial is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

4. The use of the promoter according to claim 2 for regulating the expression of a downstream gene, wherein the downstream gene comprises the gene LbRSG according to claim 1, and reporter genes GUS, GFP.

5. Use of the gene LbRSG according to claim 1, the promoter according to claim 2 or the biological material according to claim 3 for the preparation of transgenic plants.

6. Use of the gene LbRSG or of the biomaterial containing the gene LbRSG according to claim 1, in any one of the following applications:

(1) used for regulating and controlling the growth and development of salt glands of salt secreting halophytes;

(2) the method is used for plant variety improvement;

(1) the regulation and control in (1) is positive regulation and control;

preferably, the plant is Limonium bicolor.

7. A method for reducing the diameter of the limonium bicolor saline gland, reducing the number of saline gland cells and abnormal saline gland development is characterized in that a genetic engineering means is utilized to weaken the limonium bicolor gene LbRSG to obtain a gene weakening strain; said attenuation comprises knocking out or reducing expression of the gene;

the Limonium bicolor gene Lbrsg is as defined in claim 1;

preferably, the genetic engineering means is selected from one of mutagenesis, site-directed mutagenesis, and homologous recombination.

8. The method of claim 7, wherein a CRISPR/Cas 9-based sgRNA sequence is designed for a target gene LbRSG in Limonium bicolor, a DNA fragment containing the sequence encoding the sgRNA is connected to a vector carrying the CRISPR/Cas to transform the Limonium bicolor, and a transgenic plant with the gene function being deleted is obtained;

preferably, the nucleotide sequence of the sgRNA action site is 5'-AGTATCGATGCAGTGAAAC-3'.

9. The method for increasing the number of the bicolor limonium salt gland cells and promoting the salt gland development is characterized in that the method is selected from the following (i) or (ii):

expressing a protein encoded by the gene LbRSG of claim 1 in a plant;

over-expressing the gene LbRSG of claim 1 in a plant;

the mode of overexpression is selected from the following 1) to 5), or an optional combination:

1) by introducing a plasmid having the gene;

2) by increasing the copy number of the gene on the plant chromosome;

3) by altering the promoter sequence of said gene on the plant chromosome;

4) by operably linking a strong promoter to the gene;

5) by introducing an enhancer.

10. Use of a transgenic plant obtained according to the method of claim 9 for any one of the following applications:

i. for plant breeding;

and ii, planting in saline-alkali soil.

Images