CN113481210B - Application of cotton GhDof1.7 gene in promoting plant salt tolerance - Google Patents

Abstract

The invention discloses the application of cotton GhDof1.7 gene in promoting plant salt tolerance, and belongs to the technical field of plant genetic engineering. The GhDof1.7 gene has the nucleotide sequence shown in SEQ ID NO:1 and can encode the amino acid sequence shown in SEQ ID NO:2. Using the present invention, technical support can be provided for the improvement of stress resistance molecules of plants, especially cotton.

Description

Application of cotton GhDof1.7 gene in promoting plant salt tolerance

technical field

The invention belongs to the technical field of plant genetic engineering, in particular to the application of cotton GhDof1.7 gene in promoting plant salt tolerance.

Background technique

Soil salinization is a major global environmental problem. High salinity affects plant growth and metabolic processes, resulting in a significant reduction in crop yield and quality. Studies have shown that a large amount of salt ions will enter plant cells, not only making the intracellular water potential higher than the extracellular water potential, further causing cell water loss, but also causing the intracellular Na ⁺ /K ⁺ ratio imbalance, osmotic adjustment imbalance, and ultimately lead to plant growth. Slow growth or even death due to lack of water.

Cloning of salt-tolerance-related genes by molecular cloning technology is an important step in the study of plant salt-tolerance pathways. Compared with traditional breeding methods, the use of biotechnology and genetic technology to study the function of genes can change the traits of target species faster and more efficiently, thereby enhancing plant stress resistance and improving reproductive efficiency. In fact, many genes responsive to salt stress have been identified as candidate genes for genetic engineering. For example, after the Arabidopsis AtMYB44 gene is induced by stress, the expression of PP2Cs is further inhibited, thereby improving plant tolerance; under salt stress, heterologous expression of soybean GmbZIP1 can promote stomatal closure and enhance the stress resistance of transgenic Arabidopsis ; After overexpression of RcSOS1 gene in castor oil, the function of plant salt tolerance was improved.

In cotton, there are also many studies using genetic engineering technology to improve the ability of plants to resist salt stress. For example, the transfer of genes related to betaine synthesis in Escherichia coli into cotton can improve the salt resistance of cotton; the overexpression of cotton GhERF38 gene in Arabidopsis makes Arabidopsis plants less adaptable to external stress and sensitive at the same time sex increase.

When plants suffer from salt stress, they will first take certain defensive measures, such as synthesizing some substances through gene transcription regulation, etc., to resist damage. The mechanisms by which plants respond to salt stress mainly include osmotic regulation, salt efflux and ion compartmentalization, reactive oxygen species scavenging, and gene transcription regulation.

Cotton is an important fiber crop that is widely used in the global textile industry. In regions of the world affected by abiotic stress, one of the main ways to maintain positive growth in cotton yields is to mine key genes to improve stress resistance. However, the current research on cotton stress resistance, especially salt tolerance genes, is still insufficient.

SUMMARY OF THE INVENTION

The inventors identified and characterized the GhDof1.7 gene, a member of group A of MAPK in cotton, combined with the results of fluorescence quantification, transformation of Arabidopsis thaliana and VIGS experiments, and showed that the GhDof1.7 gene plays an important role in cotton salt tolerance. It can be used for the improvement of cotton anti-stress molecules. Thus, the present invention has been completed.

The present invention provides the application of GhDof1.7 gene in promoting plant salt tolerance, and the GhDof1.7 gene has the nucleotide sequence shown in SEQ ID NO:1.

The open reading frame of GhDof1.7 gene is 759bp.

In some embodiments of the invention, the nucleotide sequence set forth in SEQ ID NO:1 is capable of encoding the amino acid sequence set forth in SEQ ID NO:2. The relative molecular weight of the protein including this amino acid sequence is 27.62 kDa, and the isoelectric point is 8.64.

Dof (DNA-binding with one finger) transcription factor is composed of 200-400 amino acids and belongs to the single zinc finger super-family (zinc finger super-family). The N-terminal domain of Dof family proteins consists of 50-52 conserved amino acid residues. The single zinc finger structure is produced by the covalent binding of four cysteine residues to Zn ²⁺ , which is also unique to the Dof protein structure.

In some embodiments of the present invention, the expression level of the GhDof1.7 gene is increased in a plant to promote salt tolerance in the plant.

In some specific embodiments of the present invention, increasing the expression of GhDof1.7 gene in plants is achieved by the following methods: increasing the expression of endogenous GhDof1.7 gene in plants, or overexpressing exogenous GhDof1 in plants .7 Genes.

In a specific required embodiment of the present invention, the overexpression of the exogenous GhDof1.7 gene means that the GhDof1.7 gene is transformed into a plant through Agrobacterium-mediated transformation using a plant expression vector for expression.

Further, the GhDof1.7 gene is introduced into plant cells, tissues or organs through a plant expression vector.

Further, the plant expression vector drives the expression of the GhDof1.7 gene through a constitutive or inducible promoter.

Still further, the constitutive promoter is the 35S promoter.

In the present invention, the promoting flowering refers to promoting the flowering period of plants to advance.

In the present invention, the plant is cotton, corn, rice, wheat or Arabidopsis.

The beneficial effects of the present invention

The present invention silences the GhDof1.7 gene in cotton, and the results show that the GhDof1.7 gene may play a key role in promoting cotton salt tolerance. Using the present invention, technical support can be provided for the improvement of plants, especially cotton, for the improvement of stress resistance molecules.

Description of drawings

Figure 1 shows the gene structure, protein sequence and phylogenetic analysis of GhDof1.7. A: gene structure; B: relationship between Arabidopsis Dof protein and GhDof1.7 protein; C: protein sequence alignment.

Figure 2 shows GhDof1.7 tissue-specific expression pattern analysis. A: log(FPKM) value of GhDof1.7 gene in different tissues in TM-1 transcriptome database; B: relative expression level of GhDof1.7 gene in different tissues.

Figure 3 shows the salt tolerance of transgenic GhDof1.7 Arabidopsis. A: Phenotype of WT Arabidopsis and GhDof1.7 overexpressed Arabidopsis after salt treatment; B: Detection results of GhDof1.7 gene expression; C: Changes of GhDof1.7 gene expression; D: WT before and after salt treatment Detection of proline content in Arabidopsis and transgenic Arabidopsis; E: Detection of SOD and CAT activities in WT Arabidopsis and transgenic Arabidopsis before and after salt treatment.

Figure 4 shows VIGS cotton phenotypic identification and analysis. A: Before salt treatment; B: After salt treatment; C: Detection of expression level; D: Detection of chlorophyll and soluble sugar content; E: Detection of proline content; F: Detection of SOD activity.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

Example

The following examples are used herein to demonstrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention, and thus can be regarded as preferred solutions for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein and still obtain the same or similar results, without departing from the spirit or scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the public references herein and the materials to which they refer are incorporated by reference .

Those skilled in the art will recognize, or be aware of through routine experimentation, many equivalents to the specific embodiments of the invention described herein.

The cotton material selected in the embodiment of the present invention is that the cotton material is upland cotton standard line TM-1, and the material is cultured in a culturing room at 25°C with a cycle of 16h light/8h dark. Plants for vegetative tissue were grown in a 25°C growth chamber with 16h of light and 8h of darkness. Plants for obtaining reproductive tissue were grown in the experimental field of Cotton Research Institute, Chinese Academy of Agricultural Sciences (Anyang City, Henan Province, China). Indoors, tissues such as young leaves were sampled early in planting, and stalks, true leaves, and roots were sampled two weeks after sowing. Flowers were collected from the field 10 days after flowering.

The Arabidopsis used for transformation in the examples of the present invention is Colombia wild-type Arabidopsis (Col-0 ecotype).

The tobacco material used in the embodiments of the present invention is N. benthamiana.

The reagents and consumables used in the embodiment of the present invention are as follows:

1 Enzymes and kits: restriction endonucleases, modification enzymes, PCR reaction system-related enzymes, homologous recombinases, gel recovery kits, cloning kits, and plasmid mini-prep kits were purchased from Novozymes Biotechnology Co., Ltd. RNA extraction, reverse transcription reaction, plasmid DNA extraction and PCR fragment purification were purchased from Beijing Tiangen Biochemical Technology Company. Fluorescence quantitative kits were purchased from Kangwei Century Biotechnology Co., Ltd. GUS staining kit was purchased from Beijing Huayueyang. Proline content, soluble sugar content detection, CAT and SOD activity detection kits and Hoagland nutrient solution were purchased from Beijing Soleibo. Drugs required in the experimental process, such as sucrose, yeast powder, sodium chloride, and antibiotics, were purchased from Sigma Company.

2 Medium: LB liquid medium: tryptone (Tryptone) 10g/L, yeast extract (Yeastextract) 5g/L, sodium chloride (NaCl) 10g/L; LB solid medium: tryptone (Tryptone) 10g/L L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, agar powder (Agar) 15g/L, dilute to 1L; LB selection medium: before LB plating, to be cultured After autoclaving and cooling to 55 °C, add the corresponding concentration of antibiotics, shake well and spread the plate. The various reagent solutions mentioned but not listed in this article are prepared according to the method on the third edition of "Molecular Cloning Experiment Guide", and the biochemical reagents are of analytical grade or above. The medium was prepared according to the above formula and autoclaved. If a resistance medium is required, filter sterilized antibiotics are added to the sterilized medium as needed.

3 Main instruments: PCR amplifier (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (ABI7500), electric heating Constant temperature incubator (Shanghai Senxin), constant temperature culture oscillator (Shanghai Zhicheng), artificial climate test box (Saifu), artificial climate chamber.

Example 1 Bioinformatics analysis and cloning of cotton GhDof1.7 gene

The GhDof1.7 (Gh_A10G0541) gene was cloned using upland cotton TM-1 as material. The gene is located on the A10 chromosome with a genomic DNA length of 4917 bp and a CDS length of 759 bp, encoding 252 amino acids, including 1 exon and no intron (Fig. 1, A). The GhDof1.7 gene is the homologous gene of Arabidopsis AtDof1 (AT1G51700), and its protein sequence homology is 52.17%. The phylogenetic tree showed that the Arabidopsis Dof transcription factor family was divided into 4 groups (A, B, C, and D) or 9 subfamilies, and the GhDof1.7 protein was most closely related to the members of group A (Fig. 1, B). Furthermore, protein sequence analysis revealed that homologous genes from different species share the C2C2 domain (Fig. 1, C).

The online website predicted the secondary structure of GhDof1.7 protein and found that the protein contained 6.35% α helix, 3.97% β turn, 11.90% extended backbone and 77.78% random coil. The analysis of the physicochemical properties of GhDof1.7 protein found that the molecular weight (Mw) of the protein was 27.62kDa, and the isoelectric point (pI) was 8.64. In addition, the protein contains 23 negatively charged and 27 positively charged amino acid residues, is rich in serine (13.5%), glycine (10.7%) and proline (8.7%), and contains a small amount of tryptophan (1.2%) . The protein half-life predicted online was about 30h, the instability index was 53.46, the fat index was 40.63, and the protein hydrophobicity average number (GRAVY) was -0.997. Subcellular localization prediction showed that the protein was localized in the nucleus, and there was no transmembrane structure and no signal peptide. Therefore, the protein is an unstable hydrophilic lipid-soluble non-secreted nuclear protein.

The GhDof1.7 open reading frame sequence is (SEQ ID NO: 1):

ATGCAAGACCCAACGGGCTTTCACCAAATGAAAGCGCCGGCTTTTCAAGAGCAAGAGCAGCAGCAGCTGAAATGCCCCCGCTGTGACTCAACCAACACCAAATTCTGTTACTACAACAACTATAACTTGTCTCAGCCCCGCCATTTCTGCAAGAACTGCCGCCGTTACTGGACTAAAGGCGGCGCCCTCCGTAACATACCCGTCGGTGGCGGCACCCGTAAGGGCACCAAACGCTCCTCCTCCTCCACCAACAAACCTAAGCGCCAACCCAACCCCTCTCCAGACCCCACCCCAAACCAAAAAATCCCTGATCCCTCTCCGCCGCCGCCGAAATCATCATCATCATCGATGTTTCCCCAGCAGATTGTTTTGAACTCGGGGGCTCAGAATTCGGACTCGGATATCGACTCGACCCGGATGTATCTGTTGCCGGTTGATCATCAAGATGGGAAGATGATGGATATCGGCGGGAGCTTCAGCTCGCTGTTGGCTTCGACTGGGCAGTTTGGAAACCTCCTAGAAGGGTTTAATTCAAATGGGTCGGGTTTAAAAACGCTGAATCATTTTGGAGGGAATTTCGATTCGGGTTGTGAAATGGATCAGAATTCGGGTCGGGACCCGCTATTCGGAGAGAGCAGTAAAAACGGAGAGAGTTATTTGGATGTACAGGGCGGTAGGGATACAAGTTGTTGGAGTGGCGATAGCAATGGCTGGCCAGATCTTTCTATTTACACTCCAGGTTCAAGTTTACGGAGATAG

The amino acid sequence encoded by GhDof1.7 is (SEQ ID NO: 2):

MQDPTGFHQMKAPAFQEQEQQQLKCPRCDSTNTKFCYYNNYNLSQPRHFCKNCRRYWTKGGALRNIPVGGGTRKGTKRSSSSTNKPKRQPNPSPDPTPNQKIPDPSPPPPKSSSSSMFPQQIVLNSGAQNSDSDIDSTRMYLLPVDHQDGKMMDIGGSFSSLLASTGQFGNLLEGFNSNGSGLKTLNHFGGNFDSGCEMDQNSGRDPLFGESSKNGESYLDVSSLGGRDTSCWSGNGESSKNGESYLDVSSLGG

Example 2 Expression pattern analysis of GhDof1.7

In order to determine the specific expression of GhDof1.7 gene in various tissues of Upland cotton, the inventors used the TM-1 transcriptome database to analyze the expression changes of this gene in different tissues (stamen, pistil, petal, root, stem and leaf) analysis. As shown in Figure 2(A), the expression levels of GhDof1.7 in different tissues were quite different. The gene is highly expressed in petals of reproductive organs; highly expressed in leaves of vegetative organs. Subsequently, fluorescence quantitative experiments were carried out to further verify the above results. It was found that consistent with the above results, the GhDof1.7 gene was indeed highly expressed in cotton petals. Therefore, the inventors speculate that the GhDof1.7 gene may also be involved in the flowering process of upland cotton. However, in vegetative organs, the gene was highly expressed in roots, followed by leaves (Fig. 3, B).

1 grinding sample

Different tissues of TM-1 material were placed in liquid nitrogen, ground to powder with a mortar and a grinding rod, and about 100 mg of the sample was placed in a 1.5 mL centrifuge tube.

2 RNA extraction

All centrifugation steps below were performed at room temperature

(1) Homogenization treatment: Add 700 μL of SL (add β-mercaptoethanol before use) to the ground sample, and immediately shake vigorously to mix the sample.

Note 1: For plant samples with expected RNA yields less than 10μg, please use a starting sample volume of 100mg; for starch-rich samples or mature leaves, increase the amount of Lysate SL to 700μL.

Note 2: Since the plant diversity is very rich, and the RNA content of different growth and development stages and different tissues of the same plant is different, please choose the appropriate amount of plant material according to the specific experimental situation.

(2) Centrifuge at 12,000 rpm for 2 min.

(3) Transfer the supernatant to the filter column CS, centrifuge at 12,000 rpm for 2 min, carefully pipette the supernatant in the collection tube into a new RNase-Free centrifuge tube, and avoid touching the cell debris in the collection tube with the pipette tip.

(4) Add 0.4 times the volume of supernatant anhydrous ethanol, mix well, transfer the mixture into adsorption column CR3, centrifuge at 12,000 rpm for 15sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

(5) Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

(6) DNaseI working solution: Take 10 μL of DNaseI stock solution and 70 μL of RDD solution and mix gently.

(7) Add 80 μL of DNaseI working solution to CR3, and stand at room temperature for 15 min.

(8) After standing, add 350 μL of deproteinized solution RW1 to CR3, centrifuge at 12,000 rpm for 15 sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

(9) Add 500 μL of rinsing solution RW to the adsorption column CR3 (add ethanol before use), centrifuge at 12,000 rpm for 15 sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

(10) Repeat step 9.

(11) Centrifuge at 12,000 rpm (~13,400×g) for 2 min, put the adsorption column CR3 into a new RNase-Free centrifuge tube, and add 30-50 μL RNase-Free ddH ₂ O to the middle of the adsorption membrane. Place for 2 min, and centrifuge at 12,000 rpm (~13,400 × g) for 1 min to obtain RNA solution. Note: The volume of elution buffer should not be less than 30 μL, and the recovery efficiency will be affected if the volume is too small. Please store RNA samples at -70°C. If the expected RNA yield is greater than 30 μg, the RNA solution obtained by centrifugation in step 11 can be added to the adsorption column CR3, placed at room temperature for 2 min, and centrifuged at 12,000 rpm (~13,400 × g) for 1 min to obtain the RNA solution.

To prevent RNase contamination, precautions:

(1) Change the gloves frequently. Because the skin often contains bacteria, it may lead to RNase contamination;

(2) Use RNase-free plastic products and pipette tips to avoid cross-contamination;

(3) RNA will not be degraded by RNase in lysis buffer SL. However, RNase-free plastic and glassware should be used for further processing after extraction.

(4) RNase-Free ddH ₂ O should be used to prepare the solution.

3 Synthesis of reverse transcribed cDNA

The synthesis of sample cDNA was performed using TaKaRa's PrimeScript ^™ RT reagent Kit with gDNAEraser (Tao Bio, Dalian). The test process mainly includes two steps:

(1) Removal of possible residual genomic DNA (gDNA) in RNA samples;

(2) Reverse-transcribe the RNA obtained in step (1) into single-stranded cDNA, and all system configuration processes need to be performed on ice.

The specific operations are as follows:

(1) Removal of gDNA in RNA samples:

1) Configuration of the reaction system

2) After placing the prepared system at room temperature for 5-10 minutes, place the system on ice for later use.

(2) cDNA single-strand synthesis

The configuration of the reaction system:

A total of 20 μL of the above prepared and mixed system was placed at 37 °C for 30 min; at 85 °C for 5 sec; and stored at 4 °C. The reverse transcribed cDNA can be stored at -20°C for long-term storage.

4 Fluorescence quantitative PCR

(1) Primer 5.0 software was used to design specific primers for GhDof1.7 gene, and GhActin7 gene was used as internal reference gene.

(2) Fluorescence quantitative PCR

This was done using the UltraSYBR Mixture (Low ROX) kit from Cwbio (China) and an AppliedBiosystems 7500 instrument. The specific process is as follows:

1) Dilute the above-mentioned cDNA stock solution by 5 times;

2) Configuration of the reaction system (operation on ice):

Mix the configured system, centrifuge until there are no bubbles, and then use Applied Biosystems 7500 to perform real-time PCR: set up the PCR program according to the two-step method: pre-denaturation: 95°C for 2min; 95°C, 5sec; 60°C, 34sec (this step Collect fluorescence signal), these two steps are set for 40 cycles; the final dissolution curve analysis: 95°C, 15sec; 60°C, 20sec; 95°C, 15sec. After the above reaction is completed, the data is exported and the expression level of the gene is calculated.

Example 3 Cloning of GhDof1.7 gene and construction of overexpression vector

1 Gene primer design

In order to amplify the full length of the coding region of the gene and add a specific restriction site, according to the CDS sequence of GhDof1.7, primers containing suitable restriction sites were designed at the start codon ATG and the stop codon, respectively. The cleavage sites used were XbaI and SacI.

The primer sequence of GhDof1.7 restriction site is as follows:

GhDof1.7-F (SEQ ID NO: 7)

5’-CACGGGGGACTCTAGAATGCAAGACCCAACGGGGCTTT-3’

GhDof1.7-R (SEQ ID NO: 8)

5’-GATCGGGGAAATTCGAGCTCCTATCTCCGTAAACTTGAACCT-3’

2 PCR reaction system, procedure and product detection of gene cloning

(1) PCR reaction system

(2) PCR reaction program

(3) Detection of PCR products

Take 2 μL of PCR product, add 3 μL of 6×Loading Buffer, mix well, spot on a 1% agarose gel, and check whether the band size is about 1416bp by electrophoresis.

(4) Gel recovery of PCR products

Using Vazyme product purification kit, the steps are as follows:

1) After DNA electrophoresis, quickly cut off the gel containing the target DNA fragments with a UV lamp. It is recommended to use a paper towel to absorb the liquid on the surface of the gel and chop it up, and try to remove the excess gel. Weigh the food in the gel (remove the weight of the empty tube), 100 mg of gel is equivalent to a volume of 100 μL, as a gel volume;

2) Add an equal volume of Buffer GDP. 50～55℃ water bath for 7-10min, adjust the time according to the size of the gel to ensure that the gel block is completely dissolved. During the water bath, invert and mix twice to accelerate the sol;

3) Briefly centrifuge to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G adsorption column in the Collection Tubes 2mL collection tube, transfer ≤700μL of the sol to the adsorption column, and centrifuge at 12,000Xg for 30-60sec. If the sol volume is greater than 700 μL, put the adsorption column in the collection tube, transfer the remaining sol liquid to the adsorption column, and centrifuge at 12,000×g for 30-60sec.

4) Discard the filtrate and place the adsorption column in the collection tube. Add 300 μL of Buffer GDP to the adsorption column. Let stand for 1 min. Centrifuge at 12,000 x g for 30-60 sec.

5) Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL of Buffer GW (anhydrous ethanol has been added) to the adsorption column. Centrifuge at 12,000 x g for 30-60 sec.

6) Repeat step 5.

7) Discard the filtrate and place the adsorption column in the collection tube. Centrifuge at 12,000 × g for 2 min.

8) Place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 20-30 μL of sterilized water to the center of the adsorption column, and place for 2 min. Centrifuge at 12,000×g for 1 min. Discard the adsorption column and store the DNA at -20°C.

Example 4 Construction of PBI121-GhDof1.7 plant expression vector

(1) Double enzyme digestion and gel recovery of PBI121 plasmid

The PBI121 plasmid was double digested with XbaI and SacI, and the large fragment product of the PBI121 vector was recovered by electrophoresis. The enzyme digestion reaction system is as follows:

(2) The ligation of PCR gel recovery product and enzyme-digested PBI121 plasmid

One Step Cloning Kit进行连接，连接反应如下：The PCR product with the adapter and the double-digested PBI121 plasmid were used with the Novel Biogen Recombinase Kit

The One Step Cloning Kit is connected, and the connection reaction is as follows:

The system was configured on ice.

After the system is completed, mix the components by pipetting, react at 37°C for 30 minutes, immediately take an ice-water bath for 5 minutes, and transform or store at -20°C.

(3) Transformation of ligation product into Escherichia coli

1) Add 100 μL of Escherichia coli DH5a competent to the ligation reaction system, ice bath for 30 min;

2) 42℃ water bath heat shock for 45～90sec;

3) Ice bath for 2 min; add 900 μL non-resistant LB liquid medium, incubate for 1 h at 37°C, 190 rpm;

4) Collect bacteria by centrifugation, 4000 rpm, 3 min, discard the supernatant, leave about 100 μL of the mixture, and coat the LB plate containing kana-resistant;

5) Incubate overnight at 37°C at a constant temperature.

(4) Detection and sequencing of positive clones

1) Pick white colonies from the transformation plate, put them into liquid LB medium containing Kan, and cultivate at 37°C in a constant temperature shaker for 8 hours;

2) Colony PCR verifies the positive clones, and sends the verified single clones to Shangya Biotechnology Co., Ltd. for sequencing, with 3 replicates for each sequence.

(5) Preservation of positive bacterial solution

A certain amount of glycerol was added to the bacterial liquid verified by PCR and the sequencing was correct, so that the final concentration of glycerol was about 20%, and it was stored at -80°C. Return the correctly sequenced plasmid for Agrobacterium transformation.

(6) Transformation of Agrobacterium

The freeze-thaw method was used to transform Agrobacterium tumefaciens LBA4404 competent cells, and the specific transformation process was as follows:

1) The Agrobacterium is melted at -80°C, and the mixture of ice and water is inserted into the ice.

2) Add 0.01 to 1 μg of plasmid DNA to 100 μL of competent cells, mix by hand to the bottom of the tube, and then stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C for 5 minutes, and ice bath for 5 minutes.

3) Add 700 μL of non-resistant LB liquid medium, shake and culture at 28°C for 2-3 hours.

4) Take 100-150 μL of bacterial liquid on the LB plate containing kana, rifampicin and streptomycin, and place it upside down in a 28°C incubator for 2-3 days.

5) Select positive clones and culture them on LB liquid medium with resistance for 48 hours at 28°C. The final concentration of bacterial liquid glycerol with the correct bands verified by bacterial liquid PCR is about 20%, and stored at -80 °C for future use.

Example 5 Agrobacterium-mediated transformation of Arabidopsis

(1) Arabidopsis culture

The Colombian wild-type Arabidopsis thaliana transplanted from the 1/2MS plate was planted in an artificial climate room and grew to the full bloom period. The fruit pods that had been set were cut off, and the humidity of the nutrient soil of the Arabidopsis thaliana roots was ensured.

(2) Arabidopsis inflorescence infection and transformation

For the transformation of Arabidopsis thaliana with the overexpression vector of 35S::GhDof1.7, the inflorescence infection method is used, and the specific operations are as follows:

1) Bacterial liquid activation: Take 20 μL of the Agrobacterium bacterial liquid corresponding to the recombinant vector stored at -80°C, inoculate it into 1 mL of LB liquid medium (add corresponding antibiotics: kanamycin, rifampicin and streptomycin), 28 ℃, 180rpm, culture for 14-18h;

2) Shake: Take 200 μL of the activated corresponding bacterial solution and add it to 50 mL of LB liquid medium (add the corresponding antibiotics); at 28°C, 180 rpm, cultivate until the bacterial solution OD600 value is about 1.2-1.6 (about 18-20h). ), 5000g, centrifuged for 8min, discarded the supernatant, and collected the cells;

3) Preparation of the medium for infection and transformation: 1/2 MS halved, 6% sucrose, 0.02% Silwet L-77, pH adjusted to 5.6-5.7 with NaOH;

4) Suspend the above bacterial cells with the transformation medium, and adjust the OD600 to 0.6-0.8;

5) Dip dyeing: place the Arabidopsis inflorescence (mainly unopened buds) in the transformation medium for 30-50 sec, and after the dip dyeing, place the Arabidopsis thaliana flat for 24 hours under low light or dark-light conditions;

6) placing the treated Arabidopsis thaliana under normal conditions for cultivation, and spraying the Arabidopsis thaliana leaves with water every day within one week after infection; in order to improve the transformation efficiency, repeated infection can be carried out after about one week;

7) After maturity, harvest Arabidopsis seeds, which are the transgenic TO generation seeds.

Example 6 Phenotypic identification of transgenic Arabidopsis plants

(1) The harvested seeds are sterilized and planted on 1/2 MS containing kanamycin, then vernalized at 4°C for 2 days, and transferred to an artificial climate test box. The positive plants will grow normally in about 10 days, while the negative plants will grow normally. Plant leaves turn yellow and no longer grow.

(2) The positive Arabidopsis thaliana plants were transplanted into small flower pots for planting, and DNA was extracted and detected by PCR after one month of growth. Each generation of plants should be tested for positive lines until they propagate to the T3 generation to obtain homozygous transgenic Arabidopsis lines. T3 generation lines were tested by qRT-PCR.

Primers for fluorescence quantification of Arabidopsis internal reference gene UBQ10:

Upstream primer: 5'-AGATCCAGGACAAGGAAGGTATTC-3' (SEQ ID NO:9)

Downstream primer: 5'-CGCAGGACCAAGTGAAGAGTAG-3' (SEQ ID NO: 10)

The qRT-PCR reaction system was prepared on ice, and the fluorescence quantitative PCR reaction was carried out.

Fluorescence quantitative results showed that the expression level of GhDof1.7 gene was significantly increased in transgenic Arabidopsis (Fig. 3, B). The inventors simultaneously watered WT Arabidopsis and transgenic Arabidopsis with 400 mM NaCl solution when Arabidopsis was about to bolt. After 5 days of treatment, it was found that the degree of yellowing of WT leaves was significantly higher than that of OE-GhDof1.7 Arabidopsis thaliana leaves (Fig. 3, A). In addition, the inventors sampled the transgenic Arabidopsis at different time periods of salt treatment to detect the expression level of the GhDof1.7 gene. As a result, as shown in panel C in Figure 3, the expression of GhDof1.7 first decreased and then continued to increase with the salt treatment time.

In order to observe the effect of salt treatment on the physiological changes in plants, the inventors detected the proline content, SOD and CAT activities of Arabidopsis before and after salt treatment. The results showed that the proline content, SOD and CAT enzyme activities in GhDof1.7 transgenic Arabidopsis after salt treatment were higher than those in WT after salt treatment; for transgenic Arabidopsis, the proline content after salt treatment , SOD and CAT enzyme activities were also significantly higher than those of transgenic plants before salt treatment (Fig. 3, D, E). This indicated that overexpression of the GhDof1.7 gene improved the tolerance of Arabidopsis to salt stress.

Example 7 Virus-induced GhDof1.7 gene silencing cotton infection

1 Cotton material planting

The plump TM-1 seeds were selected and planted in an artificial climate chamber. The photoperiod and temperature conditions were: light for 16h, 28°C; dark for 8h, 22°C. After the cotyledons of the seedlings were flattened and the first true leaves were exposed (about 10 days), the VIGS bacterial liquid injection test was carried out.

2 Construction of silencing vector

The silencing vector pYL156 of VIGS was constructed by double digestion with XbaI and BamHI. First, double digestion was performed on the plasmid, and the digestion product was recovered by glue. The primers used are:

upstream primer F

5'-TACCGAATTCTCTAGAATGCAAGACCCAACGGGCTTT-3' (SEQ ID NO: 11)

Downstream primer R

5'-GCTCGGTACCGGATCCTTGGTTTGGGGTGGGGTCTGGA-3' (SEQ ID NO: 12)

After the VIGS silencing fragment gel was recovered, the recombinant ligation of the gene fragment and the vector was carried out according to the instructions of the ClonExpress II One Step Cloning Kit (Noviz, Nanjing). The reaction system was transferred into E. coli competent Trans 5α, and the specific steps were the same as described above. The bacterial solution was spread on the LB plate with kanamycin (Kan, 50 μg/mL) added, placed at 37°C, inverted overnight for 12-16 h, and positive single clones were selected for sequencing. The plasmids with the correct sequencing were transferred into Agrobacterium, and colony PCR was performed.

3 Bacterial liquid injection

The specific process of cotton VIGS is as follows:

(1) Activated bacterial solution: add 20 μL of the strains containing pYL156 plasmid, pYL192 plasmid, pYL156-GhDof1.7 plasmid and pYL156-CAL1 (positive control) plasmid frozen at -80°C into the strain containing tertiary antibodies (kanamycin, rifampicin and streptomycin), cultured at 28°C and 180rpm for 14-16h;

(2) Shake: add 50-100 μL of activated bacterial solution to 50 mL of liquid LB medium containing the above three antibiotics, and culture at 28°C and 180 rpm for 16-20 h, so that the OD600 value of the bacterial solution is within 1.5-2.0 time (the bacterial liquid generally turns orange-yellow at this time). 5000g, centrifuged for 10min, and the bacteria were recovered;

(3) The deployment of transformation medium:

Formulation of transformation medium: _MgCl2 , 10 mM; MES (2-(4-Morpholino)ethanesulfonic acid), 10 mM, pH 5.6 with NaOH; AS (acetosyringone), 200 [mu]M.

The cells collected above were suspended with transformation medium, adjusted to an OD600 value of about 1.5, and allowed to stand at room temperature for more than 3 hours (protected from light).

(4) The media of pCLCrVB and pCLCrVA (empty), pCLCrVA of positive control, and pCLCrVA of target gene were mixed at 1:1 respectively;

(5) Cut the epidermis on the back of the cotton cotyledon with a 1mL sterile syringe needle, remove the needle, and inject the mixed bacterial solution into the cotyledon until the cotyledon is completely infiltrated;

(6) The injected cotton seedlings were cultivated overnight in the dark, and then placed in an artificial climate chamber under the conditions of light and temperature: 23°C; 16h (light)/8h (dark) for normal management.

4 Identification of silent strains

After the positive control was albino, the RNA of the samples was extracted, and PCR and real-time quantitative PCR were performed. The expression of GhDof1.7 in silenced plants was detected by real-time PCR. The results showed that the expression level of GhDof1.7 in VIGS plants was about 3 times lower than that in control plants (Fig. 4, C). Then, plants with higher silencing efficiency and control plants were selected for salt treatment, and observed after 1 day of treatment. Figure A in Figure 4 shows empty plants and VIGS plants before salt treatment, and Figure B in Figure 4 shows control plants and VIGS plants after salt treatment. It can be clearly seen that VIGS plants wilt more seriously than empty plants after salt treatment, indicating that this gene may be related to the salt tolerance of plants.

In order to further observe the effect of salt treatment on the physiological changes of cotton plants, the inventors measured chlorophyll content, soluble sugar content, proline content and SOD activity in VIGS plants and empty plants after salt treatment, respectively. The results showed that the contents of all three indicators were significantly reduced in VIGS plants (Fig. 4, D and E). In addition, the SOD activity in the empty control plants was also significantly higher than that in the gene-silenced plants (Fig. 4, F). These results demonstrated that the salinity tolerance of cotton plants decreased after silencing the GhDof1.7 gene.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention.

sequence listing

<110> Cotton Research Institute, Chinese Academy of Agricultural Sciences

<120> Application of cotton GhDof1.7 gene in promoting plant salt tolerance

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 759

<212> DNA

<213> Artificial Sequence

<400> 1

atgcaagacc caacgggctt tcaccaaatg aaagcgccgg cttttcaaga gcaagagcag 60

cagcagctga aatgcccccg ctgtgactca accaacacca aattctgtta ctacaacaac 120

tataacttgt ctcagccccg ccatttctgc aagaactgcc gccgttactg gactaaaggc 180

ggcgccctcc gtaacatacc cgtcggtggc ggcacccgta agggcaccaa acgctcctcc 240

tcctccacca acaaacctaa gcgccaaccc aacccctctc cagaccccac cccaaaccaa 300

aaaatccctg atccctctcc gccgccgccg aaatcatcat catcatcgat gtttccccag 360

cagattgttt tgaactcggg ggctcagaat tcggactcgg atatcgactc gacccggatg 420

tatctgttgc cggttgatca tcaagatggg aagatgatgg atatcggcgg gagcttcagc 480

tcgctgttgg cttcgactgg gcagtttgga aacctcctag aagggtttaa ttcaaatggg 540

tcgggtttaa aaacgctgaa tcattttgga gggaatttcg attcgggttg tgaaatggat 600

cagaattcgg gtcgggaccc gctattcgga gagagcagta aaaacggaga gagttatttg 660

gatgtacagg gcggtaggga tacaagttgt tggagtggcg atagcaatgg ctggccagat 720

ctttctattt acactccagg ttcaagttta cggagatag 759

<210> 2

<211> 252

<212> PRT

<213> Artificial Sequence

<400> 2

Met Gln Asp Pro Thr Gly Phe His Gln Met Lys Ala Pro Ala Phe Gln

1 5 10 15

Glu Gln Glu Gln Gln Gln Leu Lys Cys Pro Arg Cys Asp Ser Thr Asn

20 25 30

Thr Lys Phe Cys Tyr Tyr Asn Asn Tyr Asn Leu Ser Gln Pro Arg His

35 40 45

Phe Cys Lys Asn Cys Arg Arg Tyr Trp Thr Lys Gly Gly Ala Leu Arg

50 55 60

Asn Ile Pro Val Gly Gly Gly Thr Arg Lys Gly Thr Lys Arg Ser Ser

65 70 75 80

Ser Ser Thr Asn Lys Pro Lys Arg Gln Pro Asn Pro Ser Pro Asp Pro

85 90 95

Thr Pro Asn Gln Lys Ile Pro Asp Pro Ser Pro Pro Pro Pro Lys Ser

100 105 110

Ser Ser Ser Ser Met Phe Pro Gln Gln Ile Val Leu Asn Ser Gly Ala

115 120 125

Gln Asn Ser Asp Ser Asp Ile Asp Ser Thr Arg Met Tyr Leu Leu Pro

130 135 140

Val Asp His Gln Asp Gly Lys Met Met Asp Ile Gly Gly Ser Phe Ser

145 150 155 160

Ser Leu Leu Ala Ser Thr Gly Gln Phe Gly Asn Leu Leu Glu Gly Phe

165 170 175

Asn Ser Asn Gly Ser Gly Leu Lys Thr Leu Asn His Phe Gly Gly Asn

180 185 190

Phe Asp Ser Gly Cys Glu Met Asp Gln Asn Ser Gly Arg Asp Pro Leu

195 200 205

Phe Gly Glu Ser Ser Lys Asn Gly Glu Ser Tyr Leu Asp Val Gln Gly

210 215 220

Gly Arg Asp Thr Ser Cys Trp Ser Gly Asp Ser Asn Gly Trp Pro Asp

225 230 235 240

Leu Ser Ile Tyr Thr Pro Gly Ser Ser Leu Arg Arg

245 250

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 3

atcctccgtc ttgaccttg 19

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 4

tgtccgtcag gcaactcat 19

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 5

tggatcagaa ttcgggtcgg ga 22

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 6

acttgtatcc ctaccgccct gt 22

<210> 7

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 7

cacgggggac tctagaatgc aagacccaac gggcttt 37

<210> 8

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 8

gatcgggggaa attcgagctc ctatctccgt aaacttgaac ct 42

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 9

agatccagga caaggaaggt attc 24

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 10

cgcaggacca agtgaagagt ag 22

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 11

taccgaattc tctagaatgc aagacccaac gggcttt 37

<210> 12

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 12

gctcggtacc ggatccttgg tttggggtgg ggtctgga 38

Claims (7)

1.GhDof1.7Use of a gene for enhancing salt tolerance in plants, characterized by increasing the salt tolerance in plantsGhDof1.7The expression level of the gene to promote salt tolerance of plants, theGhDof1.7The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the plant is cotton and arabidopsis thaliana.

2. The use of claim 1, wherein the amino acid sequence of the polypeptide encoded by said gene is as shown in SEQ ID NO. 2.

3. Use according to claim 1, wherein said increase is in plantsGhDof1.7The expression level of the gene is realized by the following method: increasing plant endogenesisGhDof1.7Expression of genes, or overexpression of foreign sources in plantsGhDof1.7A gene.

4. The use of claim 3, wherein said overexpression exogenous sourceGhDof1.7The gene refers toGhDof1.7The gene is transformed into the plant for over-expression by agrobacterium mediation by utilizing the plant expression vector.

5. Use according to claim 4, characterized in that saidGhDof1.7The gene is introduced into a plant cell, tissue or organ by a plant expression vector.

6. Use according to claim 5, wherein said plant expression vector drives said plant expression vector through a constitutive or inducible promoterGhDof1.7Expression of the gene.

7. Use according to claim 6, wherein the constitutive promoter is the 35S promoter.

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