CN112625103B - Alfalfa WRKY transcription factor and application thereof in aluminum toxicity and salt stress resistance - Google Patents

Abstract

The invention relates to an application of alfalfa WRKY transcription factor in improving aluminum toxicity resistance and salt stress resistance of transgenic plants. In the first aspect, the WRKY transcription factor protein disclosed by the invention is a protein consisting of an amino acid sequence shown as SEQID N0.2; in a second aspect, the invention also provides a nucleic acid sequence encoding the protein, as shown in SEQ ID N0.1; in a third aspect, the invention provides alfalfa WRKY transcription factor protein and an application of a coding gene thereof in improving aluminum virus resistance and salt stress resistance of transgenic plants; in a fourth aspect, the present invention provides a genetically engineered host cell comprising the MsWRKY22 transcription factor gene; in a fifth aspect, the invention provides an application of the MsWRKY22 nucleotide sequence or amino acid sequence or cloning vector or expression vector or host cell in genetic engineering. The invention provides a theoretical basis for molecular breeding for improving the aluminum toxicity resistance and salt tolerance of the alfalfa by utilizing a genetic engineering technology, and has very important application value.

Description

An alfalfa WRKY transcription factor and its application in tolerance to aluminum toxicity and salt stress

technical field

The invention relates to the field of plant genetic engineering, in particular to an alfalfa WRKY transcription factor and its application in tolerance to aluminum toxicity and salt stress.

Background technique

Alfalfa (Medicago sativa L.) is an important leguminous forage, which enjoys the reputation of "King of Pastures" due to its high yield, excellent grass quality and good palatability. In acidic soil (pH<5.0), aluminum mostly exists in ionic form (Al ³⁺ or Al[OH] ^4- ), which can be toxic to plants. Soil salinization will seriously affect the yield and quality of alfalfa and cause serious economic losses.

WRKY transcription factors are widely involved in plant growth, development, signal transduction, biotic and abiotic stresses, and their N-terminus contains a highly conserved WRKYGQK core motif and a CX4-5CX22-23HXH zinc finger structure. AtWRKY46 in Arabidopsis thaliana can directly and specifically bind to the W-box in the promoter of malate transporter ALMT1. Deletion of this gene can increase the secretion of malate and reduce the accumulation of Al in root cells, thereby improving the aluminum tolerance of plants. toxicity. OsWRKY22 in rice can further regulate the secretion of citric acid in roots by activating the expression of OsFRDL4 to improve the tolerance of plants to aluminum toxicity. Overexpression of OsWRKY45 and OsWRKY72 in rice improves rice tolerance to drought and salt stress. The overexpression of DgWRKY1 and DgWRKY3 in chrysanthemum can improve the salt tolerance of transgenic plants by regulating the activities of antioxidant enzymes. GhWRKY68 can mediate plant responses under salt and drought stress by regulating ABA content and enhancing the transcriptional levels of ABA-related genes.

Since alfalfa is a perennial, cross-pollinated autotetraploid with a complex genetic background, traditional breeding methods still face great challenges in the selection of aluminum-tolerant and salt-tolerant varieties. By means of reverse genetics, genes that can improve aluminum toxicity and salt tolerance were identified and genetically transformed, and their functions in abiotic stress resistance (tolerance) were analyzed. The breeding of alfalfa varieties provides theoretical reference and practical guidance significance.

SUMMARY OF THE INVENTION

How to provide an alfalfa WRKY transcription factor, identify genes that can improve aluminum toxicity and salt tolerance by means of reverse genetics, carry out genetic transformation, and analyze their abiotic stress resistance (tolerance) aspects. It can provide theoretical reference and practical guiding significance for the breeding of Alfalfa-tolerant and salt-tolerant alfalfa varieties.

The first aspect of the present invention provides an alfalfa WRKY transcription factor protein, and the alfalfa WRKY transcription factor protein includes the following (a) or (b) proteins:

(a) a protein consisting of the amino acid sequence shown in SEQ ID No.2;

(b) A protein derived from (a) having the amino acid sequence shown in SEQ ID No. 2 by substitution, deletion or addition of one or several amino acids and having the characteristics of a WRKY transcription factor.

(c) an amino acid sequence having at least 70% homology to the amino acid sequence shown in SEQ ID No. 2.

Further, the alfalfa WRKY transcription factor protein is a protein obtained by deletion, insertion and/or substitution of 1-50 amino acids in the amino acid sequence shown in SEQ ID No. 2.

Further, the alfalfa WRKY transcription factor protein is a protein obtained by adding within 1 to 20 amino acids to the C-terminal and/or N-terminal of the amino acid sequence shown in SEQ ID No. 2.

Further, the alfalfa WRKY transcription factor protein is an artificially modified amino acid sequence with an identity of ≥70% compared with the nucleic acid sequence of SEQ ID No. 2.

The second aspect of the present invention provides a nucleic acid sequence encoding the WRKY transcription factor protein of alfalfa.

Further, the nucleic acid sequence contains a typical WRKY domain and a C2H2 type zinc finger structure, and belongs to the second subfamily of the WRKY transcription factor family.

Further, the nucleic acid sequence is obtained by cloning and/or artificial synthesis in alfalfa.

Further, the nucleic acid sequence of the alfalfa WRKY gene is derived from the variety of alfalfa 'WL525', named MsWRKY22.

Further, this nucleic acid sequence is:

(a) The base sequence is shown in positions 1 to 987 of SEQ ID NO.1;

or (b) a sequence having at least 70% homology with the nucleic acid shown in positions 1 to 987 of SEQ ID NO.1;

or (c) a sequence capable of hybridizing with the nucleic acid shown in positions 1 to 987 of SEQ ID NO. 1.

Further, the nucleic acid sequence is a nucleic acid sequence complementary to the base sequence (a).

Further, the nucleic acid sequence is an artificially modified DNA sequence that has ≥70% identity with the nucleic acid sequence of SEQ ID No. 1.

Further, the nucleic acid sequence is specifically the deletion, insertion and/or substitution of 1 to 90 nucleotides in the nucleic acid sequence shown in positions 1 to 987 of SEQ ID NO. 1, or at the 5' and/or 3' end Add sequences formed within 60 nucleotides.

The third aspect of the present invention provides the application of alfalfa WRKY transcription factor protein and its encoding gene in improving the tolerance of transgenic plants to aluminum poisoning and salt stress.

The fourth aspect of the present invention provides a genetically engineered host cell containing the above-mentioned MsWRKY22 transcription factor gene, or the above-constructed recombinant cloning vector and expression vector.

Further, the host cells are Escherichia coli cells or Agrobacterium cells.

The fifth aspect of the present invention provides the application of MsWRKY22 nucleotide sequence or amino acid sequence or cloning vector or expression vector or host cell in genetic engineering.

The sixth aspect of the present invention provides a plant expression vector comprising the MsWRKY22 gene.

Further, when the gene of the present invention constructs a plant expression vector, any enhanced promoter or inducible promoter can be added before its transcription initiation nucleotide.

The seventh aspect of the present invention provides the application of the above-mentioned MsWRKY22 nucleotide sequence or amino acid sequence or expression vector or host cell in genetic engineering for improving plant tolerance to aluminum toxicity and salt tolerance.

Further, the expression vector carrying the MsWRKY22 of the present invention can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conductivity, Agrobacterium-mediated Transformed plant tissue is grown into plants.

Further, the transformed host can be either a monocotyledonous plant or a dicotyledonous plant.

Invention effect:

1. The present invention clones a MsWRKY22 transcription factor gene related to aluminum toxicity and salt tolerance in alfalfa, the gene can respond to different adversity stresses and hormone treatments, and is located in the nucleus, has transcriptional self-activation activity, and can improve transgenic purple flowers. Aluminium toxicity and salt tolerance of alfalfa.

2. The present invention provides a basis for its effective application, and is of great significance for improving the stress resistance of plants, especially for cultivating alfalfa varieties that are resistant to aluminum toxicity and salt.

Description of drawings

Fig. 1 Schematic diagram of PCR amplification results of MsWRKY22 gene;

Figure 2 Schematic diagram of the expression pattern of MsWRKY22 gene under aluminum and salt treatments;

Figure 3 Schematic diagram of the expression pattern of MsWRKY22 gene under different hormone treatments;

Figure 4 Schematic diagram of the subcellular localization of MsWRKY22 in tobacco epidermal cells;

Figure 5 Schematic diagram of the analysis of MsWRKY22 transcriptional self-activation activity;

Figure 6 Schematic diagram of PCR identification results of MsWRKY22 gene in transgenic alfalfa;

Fig. 7 Schematic diagram of aluminum treatment phenotype and ion content determination of wild-type and MsWRKY22 transgenic alfalfa;

Fig. 8 Schematic diagram of salt treatment phenotype and ion content determination of wild-type and MsWRKY22 transgenic alfalfa.

Detailed ways

The present invention is further described in detail below in conjunction with specific embodiments and with reference to data. It should be understood that these examples are intended to illustrate the invention only, and not to limit the scope of the invention in any way.

Example 1: Cloning and sequence analysis of MsWRKY22 gene

Extraction of total RNA from alfalfa and synthesis of cDNA: Extract the total RNA of alfalfa WL525 leaves with EasyPure Plant RNA Kit (purchased from Quanzhijin), and use TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (purchased from Quanshijin) for reverse reaction. Transcribe, synthesize cDNA.

Design and synthesis of primers: According to the gene chip data of Medicago sativa L. WL525 under Al and control treatments, the differentially expressed WRKY gene fragments were selected and compared in NCBI, and Medicago truncatula (Medicago truncatula) was found. ) in the WRKY gene with higher homology to the gene, design primers according to the gene sequence, carry out homologous cloning in alfalfa WL525, the upstream primer sequence and the downstream primer sequence are as shown in SEQ ID No.3 and SEQ ID No.4 Show.

(F: 5'-TACAGACTTTGTCCCTTTACCTATG-3', R: 5'-TAGTTCCTCCCTACTATACTACACTTCC-3'.)

Using the above cDNA as a template, PCR amplification was performed according to the following reaction system and conditions: 20 μL system, containing 10 μL of 2×EXTaq super PCR Mix (purchased from TaKaRa), 0.8 μL of 10 μmol/L primer F and primer R, 1 μL of cDNA , make up to 20 μL of deionized water. Reaction conditions: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 30s, 57°C for 30s, 72°C for 1 min; extension at 72°C for 7 min. The above amplified fragment was recovered and ligated with the cloning vector pMD18-T (purchased from TaKaRa), identified and then sent to Sangon Bio-sequencing. The sequence result is shown in SEQ ID No. 1.

The full-length MsWRKY22 gene sequence of 987bp was obtained by PCR amplification, encoding a protein composed of 329 amino acid residues, including a conserved WRKY binding domain and a C2H2-type zinc finger structure. The PCR amplification results of MsWRKY22 gene sequence are shown in Figure 1.

Example 2: Expression pattern of MsWRKY22 gene induced by Al, NaCl and hormones

Cultivation and treatment of alfalfa WL525 plants: after removing the coating, the seeds are washed and evenly distributed in a growth plate padded with filter paper, kept moist, and when the first true leaf is grown from germination, the seedlings with consistent growth are selected and grown. Transplant in 1/2 Hoagland nutrient solution for hydroponics. The formula of Hoagland culture medium is as follows: Ca(NO ₃ ) ₂ ·4H ₂ O 0.62g/L, KNO ₃ 0.34g/L, KH ₂ PO ₄ 0.06g/L, NH ₄ NO ₃ 0.053g/L, MgSO ₄ 0.24g /L, MgCl ₂ 0.67mg/L, H ₃ BO ₃ 0.38mg/L, MnSO ₄ 0.2mg/L, ZnSO ₄ 7H ₂ O 0.29mg/L, CuSO ₄ 0.01mg/L, FeSO ₄ 7H ₂ O0 .02785g/L, EDTA-Na ₂ 0.0373g/L, PH 5.7-5.8), 28°C, 16h light/8h dark conditions, and after one week, the following treatments were performed: 0, 100μM AlCl ₃ , 200mM were added to the nutrient solution. NaCl and 100 μM ABA, MeJA, SA, respectively, at 0, 0.5, 1, 3, 6, 9, 12, and 24 hr, take the underground and aerial parts of alfalfa, wrap them in tin foil, and quickly place them in liquid nitrogen, then store them in -80°C ultra-low temperature freezer.

The extraction of total RNA and the synthesis of cDNA were the same as those in Example 1. Real-time fluorescent quantitative PCR primers were designed according to the cDNA sequence of MsWRKY22, and the upstream primer sequences and downstream primer sequences are shown in SEQ ID No.5 and SEQ ID No.6.

(F: 5'-CAAGTACCAGTTGAGAGTCT-3'; R: 5'-CATTGTTGGATCTGTTCTGT-3').

The alfalfa constitutively expressed gene EF-α was used as the internal reference gene, and the upstream and downstream primer sequences were shown in SEQ ID No.7 and SEQ ID No.8.

(F: 5'-GCACCAGTGCTCGATTGC-3'; R: 5'-TCGCCTGTCAATCTTGGTAACAA-3').

Using the Bio-rad real-time quantitative PCR instrument, real-time fluorescent quantitative PCR was carried out with the cDNA of the sample taken above as a template. The reaction system contained 10 μL of 2×SYBR qPCR SuperMix (purchased from Quanzhou Gold), 0.4 μL of Primer F/R, 2 μL of cDNA, and water to a total volume of 20 μL. The reaction program was 94°C for 30s; 95°C for 5s, 57°C for 15s, 72°C for 15s, 40 cycles. Each treatment had 3 biological replicates and 3 technical replicates. 2 ^-ΔΔCT method was used to analyze data, spss14.0 was used for statistical analysis, and EXCEL was used for graphing. The expression patterns are shown in Figures 2 and 3.

Example 3: Subcellular localization of MsWRKY22 in tobacco epidermal cells.

Construction of plant expression vector: The cloned MsWRKY22 gene was introduced into the restriction site and linked to the pMD18-T vector, and then double restriction digestion was performed. After the gel was recovered, it was linked with the linearized pHB-YFP vector with the same restriction enzyme site, transformed into Escherichia coli, and the plasmid was extracted after PCR verification of the bacterial liquid, and transformed into Agrobacterium-competent GV3101.

Tobacco Seedling Cultivation: Spread tobacco seeds evenly in a 1:1 mixture of substrate and vermiculite. After germination, transplant them into flowerpots and water them with 1/2 Hoagland nutrient solution every two days.

Agrobacterium preparation: streak and activate Agrobacterium strains containing pHB-MsWRKY22-YFP and glycerol containing empty load, pick single clones into YEB liquid medium supplemented with antibiotics Kan50 and Rif100, and cultivate to OD600=1.2 at 28°C and 200rpm. Then, centrifuge at 6000 rpm for 10 min, collect the cells, and resuspend to OD600=0.6 with MS liquid medium for use.

Instantaneous transformation of tobacco leaves: Use a 1ml disposable syringe to absorb the above resuspended bacterial solution, select tobacco leaves with the same growth state for injection, and mark the injection area in the leaves with a marker. Observation was performed after culturing in the dark for 48 hours.

Observation under a fluorescence confocal microscope: Take the leaves of the above injection area, place them on a glass slide, and observe and take pictures under a fluorescence confocal microscope. The subcellular localization of MsWRKY22 in tobacco epidermal cells is shown in Figure 4.

Example 4: Analysis of MsWRKY22 Transcriptional Activation Activity

The MsWRKY22 was linked to the pGBKT7 vector by homologous recombination, and transformed into yeast competent Y2H (purchased from Vidida), and the transformation method was referred to the instruction manual. After transformation, it was spread on the solid medium of SD/-Trp. . Pick a single clone in SD/-Trp liquid medium, shake it at 30°C for culture, centrifuge, resuspend it with ddH ₂ O, dilute the stock solution, 10, 100 and 1000 times of the resuspended bacteria, respectively, and pipette 10 μL of the resuspended cells into SD. /-Trp/x-α-gal and SD/-Trp/x-α-gal/AbA plates. Yeast culture medium was purchased from Takara, and the preparation of culture medium refers to the instructions of this series of products. Yeast cells with self-activating activity can grow plaques on the above two media and show blue color. According to the color development results, as shown in Figure 5, MsWRKY22 has transcriptional autoactivation activity.

Example 5: MsWRKY22 gene transformed alfalfa and identification of transgenic lines

Agrobacterium preparation: The pHB-MsWRKY22-Flag Agrobacterium strain stored in the -80°C refrigerator was streaked and activated on YEB solid medium containing antibiotics as Kan50 and Rif100, and after culturing at 28°C for 48 hours, single clones were picked to contain In YEB liquid medium with the same antibiotics, placed on a shaker at 28° C. at a speed of 200 rpm and cultured to OD600=0.8 for infection.

Preparation of explants: The explants were made of leaves of alfalfa WL525 wild-type line (WL525-7) with differentiation ability. Add 0.1% Tween20 in 30% sodium hypochlorite solution to the culture flask with leaves, and place it on a shaker at 100 rpm for 8-10 min for surface sterilization. Then rinse three times with sterile distilled water.

Agrobacterium infection: Centrifuge the Agrobacterium solution prepared in the first step at 6000 rpm for 10 min, discard the supernatant, and resuspend the solution to OD600=0.2-0.4. Pour it into a sterile culture bottle, put in sterilized sterile leaves, tighten the bottle cap and place it in a dry bottle for 10 minutes with a vacuum pump. After that, the culture flask was placed in an ultrasonic cleaner at 40 kHz and 20 °C for 3 min, and then vacuumed for 10 min after taking it out.

Callus induction and differentiation: After vacuuming, use tweezers to pick out the leaves and cover them in the middle of multi-layer sterile paper. After about 30 minutes in the ultra-clean workbench, blot the residual Agrobacterium on the surface of the leaves and transfer them to the co-cultivation medium. in the dark for 5 days. Afterwards, the leaves were cut into 1*1cm ² leaf discs with sterile scissors or scalpel, placed on the screening medium and placed under light to induce callus and screened for 4 weeks, during which subculture was performed every 2 weeks. When more callus was differentiated, the positive callus was transferred to regeneration medium, and after 4 weeks, the callus with differentiated bud points was transferred to stem elongation medium. Transfer to rooting medium after differentiation into leaves. Two weeks after the roots emerged, they were transplanted into sterile substrates and propagated in a growth chamber.

References for the culture medium formula of this genetic transformation method: Chunxiang Fu, Timothy Hernandez, Chuanen Zhou et al. Agrobacterium Protocols, Springer New York, 2015.

Screening and identification of transgenic alfalfa:

About 0.2 g of leaves of wild-type alfalfa and transgenic lines were taken, fully ground in liquid nitrogen, and gDNA was extracted with Plantgenomic DNA extraction kit (purchased from Quanjingold) for hygromycin verification. The hygromycin upstream and downstream primer sequences are shown in SEQ ID No.9 and SEQ ID No.10.

F: 5'-GGATATGTCCTGCGGGTAAA-3'; R: 5'-ATTTGTGTACGCCCGACAGT-3'.

The PCR reaction system was the same as that in Example 1, and the extension time was 1 min. The results of gel electrophoresis are shown in Figure 6, and the obtained strain is a positive strain.

Example 6: Aluminium toxicity analysis of MsWRKY22 transgenic alfalfa

The wild-type and transgenic plants with consistent growth were selected, the stem nodes at the same position were taken, the redundant leaves were removed, and only the top-most fully opened leaves were retained, and the bottom incision was dipped in rooting powder for hydroponics. After 10 days of growth, the control group and the treatment group were respectively set for culture. After 21 days of AlCl ₃ treatment, the growth phenotype was observed, and samples were taken to determine the Al ³⁺ content in the aerial and underground parts.

The results are shown in Figure 7. The growth inhibition of the transgenic lines under AlCl ₃ treatment was significantly lower than that of the wild type, and the ion content of the aerial and underground parts was also significantly lower than that of the wild type.

Example 8: Salt tolerance analysis of MsWRKY22 transgenic alfalfa

Select the wild-type and transgenic plants with consistent growth, take the stem nodes at the same position, remove the redundant leaves, and keep only the leaves that are fully opened at the top. cultured in the material. After 10 days of growth, the control group and the treatment group were respectively set for culture. Pour the nutrient solution every two days. After two weeks of NaCl treatment, the growth phenotype was observed, and samples were taken to determine the Na ⁺ content of the aerial and underground parts.

The results are shown in Figure 8. The growth inhibition of the transgenic lines under NaCl treatment was significantly lower than that of the wild type, and the ion content of the aboveground and underground parts was also significantly lower than that of the wild type.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

sequence listing

<110> Shanghai Jiaotong University

<120> An alfalfa WRKY transcription factor and its application in tolerance to aluminum toxicity and salt stress

<130> 2021

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Claims (5)

1. The alfalfa WRKY transcription factor protein is characterized by being a protein consisting of an amino acid sequence shown in SEQ ID No. 2.

2. The alfalfa WRKY transcription factor protein as claimed in claim 1, wherein the protein is obtained by adding 1-20 amino acids to the C-terminal and/or N-terminal of the amino acid sequence shown in SEQ ID No. 2.

3. A nucleic acid encoding the alfalfa WRKY transcription factor protein of claim 1.

4. The nucleic acid of claim 3, wherein the sequence of said nucleic acid is as shown in positions 1-987 of SEQ ID No. 1.

5. The use of the alfalfa WRKY transcription factor protein and the encoding gene thereof as claimed in any one of claims 1 or 2 for improving tolerance to aluminum toxicity and salt stress of alfalfa.

Images