CN113481210A - Application of cotton GhDof1.7 gene in promotion of salt tolerance of plants - Google Patents

Abstract

The invention discloses cottonGhDof1.7The application of gene in promoting plant salt tolerance belongs to the field of plant gene engineering technology.GhDof1.7The gene has the nucleotide sequence shown in SEQ ID NO. 1 and can encode the amino acid sequence shown in SEQ ID NO. 2. The invention can be used for carrying out technical support on the improvement of stress-resistant molecules of plants, particularly cotton.

Description

Application of cotton GhDof1.7 gene in promotion of salt tolerance of plants

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a cotton GhDof1.7 gene in promotion of salt tolerance of plants.

Background

Soil salinization is a global prime environmental problem, and high salt affects the growth and metabolic processes of plants, thereby causing significant reduction in crop yield and quality. Research shows that after a large amount of salt ions enter plant cells, the intracellular water potential is higher than that of the cells, the water loss of the cells is further caused, and the Na in the cells is also caused⁺/K⁺The proportion is unbalanced, and the osmotic regulation is unbalanced, so that the plants grow slowly or even die due to water shortage.

The molecular cloning technology is used for cloning salt tolerance related genes, and is an important step for researching plant salt tolerance approaches. Compared with the traditional breeding method, the method has the advantages that the biological technology and the genetic technology are utilized to research the functions of the genes, and the characters of target species can be changed more quickly and more efficiently, so that the stress resistance of the plant is enhanced, and the propagation efficiency is improved. In fact, many genes responding to salt stress have been identified as candidate genes for genetic engineering. For example, after the arabidopsis AtMYB44 gene is subjected to stress induced expression, the expression of PP2Cs is further inhibited, so that the tolerance of a plant is improved; under the salt stress, the heterologous expression of the soybean GmbZIP1 can promote stomata to be closed and enhance the stress resistance of the transgenic arabidopsis; after the ricinus communis over-expresses the RcSOS1 gene, the salt resistance function of the plant is improved.

In cotton, there are also many studies on the improvement of plant salt stress resistance by using genetic engineering techniques. For example, the salt resistance of cotton can be improved by transferring genes related to the synthesis of Escherichia coli betaine into the cotton; the cotton GhERF38 gene is over-expressed in Arabidopsis, so that the adaptability of Arabidopsis plants to external stress is poor, and the sensitivity is increased.

When a plant is subjected to salt stress, certain defense measures are firstly taken, such as synthesizing substances through a gene transcription regulation mode and the like, and then the damage is resisted. The plant response mechanism to salt stress mainly comprises osmotic regulation, salt discharge and ion regionalization, active oxygen elimination, gene transcription regulation and the like.

Cotton is an important fiber crop and is widely used in the global textile industry. In regions of the world affected by abiotic stress, one of the major methods to maintain the growing cotton yield is to mine key genes to improve stress tolerance. However, the current research on stress resistance of cotton, especially on salt tolerance genes, is still insufficient.

Disclosure of Invention

The inventor shows that the GhDof1.7 gene plays an important role in salt tolerance of cotton by identifying and analyzing characteristics of a group A member GhDof1.7 gene of MAPK in cotton and combining results of fluorescence quantification, Arabidopsis transformation, VIGS test and the like, and can be used for improving stress-resistant molecules of cotton. Thus, the present invention has been completed.

The invention provides application of a GhDof1.7 gene in promoting salt tolerance of plants, wherein the GhDof1.7 gene has a nucleotide sequence shown in SEQ ID NO. 1.

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

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 protein comprising the amino acid sequence has a relative molecular weight of 27.62kDa and an isoelectric point of 8.64.

The Dof (DNA-binding with one finger) transcription factor consists of 200-400 amino acids, and belongs to the zinc finger protein super-family (zinc finger-family). The N-terminal domain of the Dof family protein consists of 50-52 conserved amino acid residues. The single zinc finger structure consists of 4 cysteine residues and Zn²⁺Covalent binding occurs, and this mode of binding is also unique to the Dof protein structure.

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

In some embodiments of the present invention, the increasing of the expression level of the ghdof1.7 gene in the plant is achieved by: improving the expression of the endogenous GhDof1.7 gene of the plant, or over-expressing the exogenous GhDof1.7 gene in the plant.

In a specific required embodiment of the invention, the overexpression of the exogenous GhDof1.7 gene refers to that the GhDof1.7 gene is transformed into a plant for expression by agrobacterium-mediated transformation by using a plant expression vector.

Further, the GhDof1.7 gene is introduced into a plant cell, tissue or organ 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 a 35S promoter.

In the present invention, the promotion of flowering refers to promotion of the flowering phase of plants to be advanced.

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

The invention has the advantages of

According to the invention, by silencing the GhDof1.7 gene in cotton, the result shows that the GhDof1.7 gene possibly has a key effect on promoting salt tolerance of cotton. The invention can be used for carrying out technical support on the improvement of stress-resistant molecules of plants, particularly cotton.

Drawings

FIG. 1 shows the gene structure, protein sequence and phylogenetic analysis of GhDof1.7. A: (ii) a gene structure; b: the genetic relationship between the Arabidopsis Dof protein and the GhDof1.7 protein; c: and (4) protein sequence alignment.

Figure 2 shows the analysis of the tissue specific expression pattern of ghdof 1.7. A: log (FPKM) values of the GhDof1.7 gene in different tissues in the TM-1 transcriptome database; b: relative expression of GhDof1.7 gene in different tissues.

FIG. 3 shows the salt tolerance of GhDof1.7 transgenic Arabidopsis thaliana. A: WT Arabidopsis and overexpression GhDof1.7 Arabidopsis phenotypes after salt treatment; b: detecting the expression level of the GhDof1.7 gene; c: the expression level of the GhDof1.7 gene is changed; d: detecting the proline content in WT arabidopsis thaliana and transgenic arabidopsis thaliana before and after salt treatment; e: SOD and CAT activities in WT Arabidopsis thaliana and transgenic Arabidopsis thaliana were measured before and after salt treatment.

FIG. 4 shows VIGS cotton phenotype identification and analysis. A: before salt treatment; b: after salt treatment; c: detecting the expression amount; d: detecting the content of chlorophyll and soluble sugar; e: detecting the content of proline; f: and (5) detecting the SOD activity.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, 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 and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The cotton material selected in the embodiment of the invention is upland cotton standard line TM-1, and the cotton material is cultured in a culture room at 25 ℃ in a period of 16h light/8 h dark. Plants for vegetative tissue were planted in a dark 25 ℃ growth chamber with 16h light and 8 h. Plants for obtaining reproductive tissues were planted in the cotton research institute field (Anyang City, Henan, China). In the house, tissues like young leaves were sampled at the early stage of planting, and stalks, true leaves and roots were sampled at two weeks after sowing. Flowers were harvested from the field 10 days after flowering.

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

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

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

1 enzyme and kit: restriction enzyme, modified enzyme, PCR reaction system related enzyme, homologous recombinase, gel recovery kit, cloning kit and plasmid small-scale kit are purchased from Novozam biotech Inc. RNA extraction, reverse transcription reaction, plasmid DNA extraction and PCR fragment purification were purchased from Beijing Tiangen Biochemical technologies. The fluorescence quantitative kit is purchased from health as a century Biotechnology Co., Ltd. GUS staining kit was purchased from Beijing & Vietnam. Proline content, soluble sugar content detection, CAT and SOD activity detection kits and Hoagland nutrient solution were purchased from Beijing Solibao. The medicines required in the experimental process such as sucrose, yeast powder, sodium chloride, antibiotics and the like are purchased from Sigma company.

2, culture medium: LB liquid medium: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), and 10g/L sodium chloride (NaCl); LB solid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract), 10g/L of sodium chloride (NaCl) and 15g/L of Agar powder (Agar), and the volume is fixed to 1L; LB selective medium: before LB plate, adding antibiotic with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 deg.C, shaking up and plating. The reagent solutions mentioned but not listed here were prepared according to the method of the third edition of the molecular cloning instructions, and the biochemical reagents were analytically pure or of higher grade. Preparing culture medium according to the above formula, and autoclaving. If a resistant culture medium is required to be prepared, filter-sterilized antibiotics are added to the sterilized culture medium as required.

3, main instruments: PCR amplification apparatus (BIO-RAD), high speed centrifuge (Hettich MIKRO 200R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR apparatus (ABI7500), electric heating constant temperature incubator (Shanghai Sensin), constant temperature culture oscillator (Shanghai Zhicheng), artificial climate test chamber (Saifu), and artificial climate chamber.

Example 1 bioinformatics analysis and cloning of Cotton GhDof1.7 Gene

The GhDof1.7(Gh _ A10G0541) gene is cloned by using upland cotton TM-1 as a material. The gene is located on A10 chromosome, the length of genome DNA is 4917bp, the length of CDS is 759bp, 252 amino acids are coded, 1 exon is contained, and no intron is contained (figure 1, A). The GhDof1.7 gene is a homologous gene of Arabidopsis AtDof1(AT1G51700), and the protein sequence homology is 52.17%. Phylogenetic trees showed that the arabidopsis Dof transcription factor family is divided into 4 groups (A, B, C and D) or 9 subfamilies, and that the ghdof1.7 protein is most closely related to the members of group a (fig. 1, B). Furthermore, protein sequence analysis showed that homologous genes of different species share the C2C2 domain (fig. 1, C).

The online website predicts the secondary structure of the ghdof1.7 protein, which is found to contain 6.35% alpha helix, 3.97% beta turn, 11.90% extended backbone and 77.78% random coil. Analysis of the physicochemical properties of the GhDof1.7 protein revealed 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 negative and 27 positive 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 on-line predicted protein half-life was about 30h, the instability index was 53.46, the fat index was 40.63, and the average number of protein hydrophobicity (GRAVY) was-0.997. Subcellular localization prediction showed that the protein was localized in the nucleus, and that no transmembrane structure, nor signal peptide, existed. Thus, the protein is an unstable hydrophilic lipid-soluble, non-secreted nuclear protein.

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

ATGCAAGACCCAACGGGCTTTCACCAAATGAAAGCGCCGGCTTTTCAAGAGCAAGAGCAGCAGCAGCTGAAATGCCCCCGCTGTGACTCAACCAACACCAAATTCTGTTACTACAACAACTATAACTTGTCTCAGCCCCGCCATTTCTGCAAGAACTGCCGCCGTTACTGGACTAAAGGCGGCGCCCTCCGTAACATACCCGTCGGTGGCGGCACCCGTAAGGGCACCAAACGCTCCTCCTCCTCCACCAACAAACCTAAGCGCCAACCCAACCCCTCTCCAGACCCCACCCCAAACCAAAAAATCCCTGATCCCTCTCCGCCGCCGCCGAAATCATCATCATCATCGATGTTTCCCCAGCAGATTGTTTTGAACTCGGGGGCTCAGAATTCGGACTCGGATATCGACTCGACCCGGATGTATCTGTTGCCGGTTGATCATCAAGATGGGAAGATGATGGATATCGGCGGGAGCTTCAGCTCGCTGTTGGCTTCGACTGGGCAGTTTGGAAACCTCCTAGAAGGGTTTAATTCAAATGGGTCGGGTTTAAAAACGCTGAATCATTTTGGAGGGAATTTCGATTCGGGTTGTGAAATGGATCAGAATTCGGGTCGGGACCCGCTATTCGGAGAGAGCAGTAAAAACGGAGAGAGTTATTTGGATGTACAGGGCGGTAGGGATACAAGTTGTTGGAGTGGCGATAGCAATGGCTGGCCAGATCTTTCTATTTACACTCCAGGTTCAAGTTTACGGAGATAG

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

MQDPTGFHQMKAPAFQEQEQQQLKCPRCDSTNTKFCYYNNYNLSQPRHFCKNCRRYWTKGGALRNIPVGGGTRKGTKRSSSSTNKPKRQPNPSPDPTPNQKIPDPSPPPPKSSSSSMFPQQIVLNSGAQNSDSDIDSTRMYLLPVDHQDGKMMDIGGSFSSLLASTGQFGNLLEGFNSNGSGLKTLNHFGGNFDSGCEMDQNSGRDPLFGESSKNGESYLDVQGGRDTSCWSGDSNGWPDLSIYTPGSSLRR

example 2 analysis of expression Pattern of GhDof1.7

To determine the specific expression of the GhDof1.7 gene in various tissues of Gossypium hirsutum, the inventors analyzed the expression changes of the gene in various tissues (stamen, pistil, petal, root, stem and leaf) using the TM-1 transcriptome database. As shown in FIG. 2(A), the expression level of GhDof1.7 in different tissues was greatly different. The gene is highly expressed in petals of reproductive organs; high expression in leaves of vegetative organs. The results were further verified by subsequent fluorescent quantitation experiments. It was found that, in accordance 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 the vegetative organ, the gene was highly expressed in the root, and the leaf was inferior (FIG. 3-2B).

1 grinding sample

Different tissues of TM-1 material were placed in liquid nitrogen, ground to powder using a mortar and pestle, and approximately 100mg of sample was placed in a 1.5mL centrifuge tube.

2 extraction of RNA

All following centrifugation steps were carried out at room temperature

(1) And (3) homogenizing treatment: to the milled sample was added 700. mu.L SL (beta-mercaptoethanol added before use) and the sample was mixed by shaking vigorously immediately.

Note that 1: for plant samples with an expected RNA yield of less than 10. mu.g, please use a starting sample size of 100 mg; for starch-rich samples or mature leaves, please increase the amount of lysate SL to 700. mu.L.

Note that 2: because of the rich diversity of plants and the different RNA contents of different growth stages and tissues of the same plant, please select the appropriate amount of plant material according to the specific experimental conditions.

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

(3) The supernatant was transferred to the filtration column CS and centrifuged at 12,000rpm for 2min, and the supernatant from the collection tube was carefully pipetted into a new RNase-Free centrifuge tube, the tip being kept from touching the cell debris in the collection tube.

(4) Adding 0.4 times volume of anhydrous ethanol, mixing, transferring the mixture into adsorption column CR3, centrifuging at 12,000rpm for 15sec, discarding the waste liquid in the collection tube, and returning the adsorption column CR3 to the collection tube.

(5) 350. mu.L of deproteinizing solution RW1 was added to the adsorption column CR3, and centrifuged at 12,000rpm for 15sec, thereby discarding the waste liquid in the collection tube and returning the adsorption column CR3 to the collection tube.

(6) DNaseI working solution: mix 10. mu.L DNaseI stock and 70. mu.L RDD gently.

(7) 80. mu.L of DNaseI working solution was added to CR3 and allowed to stand at room temperature for 15 min.

(8) After standing, 350. mu.L of deproteinizing solution RW1 was added to CR3, centrifuged at 12,000rpm for 15sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

(9) To the adsorption column CR3, 500. mu.L of rinsing solution RW (ethanol was added before use), centrifuged at 12,000rpm for 15sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

(10) Step 9 is repeated.

(11) Centrifuge 2 at 12,000rpm (. about.13,400 Xg)min, placing the adsorption column CR3 into a new RNase-Free centrifuge tube, and dripping 30-50 μ L RNase-Free ddH into the middle part of the adsorption membrane₂O, left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution. Note that: the volume of elution buffer should not be less than 30. mu.L, and too small a volume affects the recovery efficiency. The RNA samples were stored at-70 ℃. If the expected RNA yield is more than 30. mu.g, the RNA solution obtained by centrifugation in step 11 may be added to an adsorption column CR3, and left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution.

To prevent RNase contamination, precautions:

(1) the gloves are often replaced with new ones. Because the skin is often bacteria-bearing, RNase contamination may result;

(2) the RNase-free plastic product and the gun head are used to avoid cross contamination;

(3) RNA is not degraded by RNase while in lysate SL. However, after extraction, plastics and glassware without RNase should be used in the further processing.

(4) The preparation solution should use RNase-Free ddH₂O。

3 Synthesis of reverse transcription cDNA

Sample cDNA was synthesized using PrimeScript of TaKaRa^TMRT reagent Kit was performed with gDNA Eraser Kit (Takara Bio, Dalian). The test process mainly comprises two steps:

(1) removal of genomic dna (gdna) that may remain in the RNA sample;

(2) and (2) carrying out reverse transcription on the RNA obtained in the step (1) into single-stranded cDNA, wherein all system configuration processes need to be carried out on ice.

The specific operation is as follows:

(1) removal of gDNA in RNA samples:

1) configuration of reaction System

2) And standing the prepared system at room temperature for 5-10min, and then transferring the system onto ice for later use.

(2) cDNA Single Strand Synthesis

The preparation of a reaction system:

placing 20 μ L of the prepared and mixed system at 37 deg.C for 30 min; 5sec at 85 ℃; storing at 4 ℃. The reverse transcription cDNA is placed at-20 deg.C for long-term storage.

4 fluorescent quantitative PCR

(1) Primers specific to the GhDof1.7 gene were designed using Primer5.0 software, and the GhACTin7 gene was used as an internal reference gene.

(2) Fluorescent quantitative PCR

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

1) diluting the cDNA stock solution by 5 times;

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

the prepared system was mixed well, centrifuged until no air bubbles were present, and then subjected to fluorescent quantitative PCR using Applied Biosystems 7500: the PCR program was set according to a two-step procedure: pre-denaturation: 2min at 95 ℃; 95 ℃ for 5 sec; 60 ℃, 34sec (this step collects the fluorescence signal), these two steps set 40 cycles; final dissolution curve analysis: 95 ℃ for 15 sec; 60 ℃, 20 sec; 95 ℃ for 15 sec. After the reaction was completed, the data was derived and the expression level of the gene was calculated.

Example 3 cloning of GhDof1.7 Gene and construction of overexpression vector

1 design of Gene primers

In order to amplify the whole length of the gene coding region and add specific enzyme cutting sites, primers containing suitable enzyme cutting sites are designed at the initiation codon ATG and the termination codon respectively according to the CDS sequence of GhDof1.7. The cleavage sites used were XbaI and SacI.

The GhDof1.7 enzyme cutting site primer sequence is as follows:

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

5’-CACGGGGGACTCTAGAATGCAAGACCCAACGGGCTTT-3’

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

5’-GATCGGGGAAATTCGAGCTCCTATCTCCGTAAACTTGAACCT-3’

2 PCR reaction System, procedure and product detection for Gene cloning

(1) PCR reaction system

(2) PCR reaction procedure

(3) Detection of PCR products

And (3) adding 3 mu L of 6 XLoading Buffer into 2 mu L of PCR product, uniformly mixing, spotting in 1% agarose gel, and detecting whether the size of a band is 1416bp or not by electrophoresis.

(4) Gel recovery of PCR products

The Vazyme product purification kit was used, the procedure was as follows:

1) after the DNA electrophoresis is finished, the gel containing the target DNA fragment is cut off rapidly by an ultraviolet lamp, and it is recommended to suck up the liquid on the surface of the gel with a paper towel and cut up, and to remove the excess gel as much as possible. Weighing the grain in the gel (removing the weight of the empty tube), wherein 100mg of the gel is equal to 100 mu L of the gel, and the volume of the gel is taken as the volume of one gel;

2) an equal volume of Buffer GDP was added. Carrying out water bath at 50-55 ℃ for 7-10min, and properly adjusting time according to the size of the gel to ensure that the gel block is completely dissolved. Mixing the sol evenly by reversing for 2 times during the water bath;

3) the droplets on the walls of the tubes were collected by brief centrifugation. The FastPure DNA Mini Columns-G adsorption column is placed in a Collection tube of 2mL Collection Tubes, less than or equal to 700 μ L of sol solution is transferred to the adsorption column, and centrifugation is carried out for 30-60sec at 12,000 Xg. If the sol volume is larger than 700. mu.L, the adsorption column is placed in a collection tube, the remaining sol solution is transferred to the adsorption column, and centrifugation is carried out at 12,000Xg for 30-60 sec.

4) The filtrate was discarded and the adsorption column was placed in the collection tube. Add 300. mu.L Buffer GDP to the adsorption column. Standing for 1 min. Centrifuge at 12,000Xg for 30-60 sec.

5) The filtrate was discarded and the adsorption column was placed in the collection tube. Add 700. mu.L of Buffer GW (to which absolute ethanol had been added) to the adsorption column. Centrifuge at 12,000Xg for 30-60 sec.

6) And (5) repeating the step.

7) The filtrate was discarded and the adsorption column was placed in the collection tube. Centrifuge at 12,000Xg for 2 min.

8) Placing the adsorption column in 1.5mL sterilized centrifuge tube, adding 20-30 μ L sterilized water to the center of the adsorption column, and standing for 2 min. Centrifuge at 12,000Xg for 1 min. The adsorption column was discarded and the DNA was stored at-20 ℃.

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

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

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

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

PCR product with joint and double-enzyme-digested PBI121 plasmid are subjected to Novozam homologous recombinase kit

The One Step Cloning Kit was used for ligation as follows:

the system was placed on ice.

After the system is finished, the components are blown and uniformly mixed, the reaction is carried out for 30min at 37 ℃, the ice-water bath is immediately carried out for 5min, and the components are converted or stored at-20 ℃.

(3) Transformation of E.coli by ligation products

1) Adding 100 mu L of escherichia coli DH5a competence into the ligation reaction system, and carrying out ice bath for 30 min;

2) carrying out water bath heat shock at 42 ℃ for 45-90 sec;

3) ice-bath for 2 min; adding 900 mu L of non-resistant LB liquid culture medium, incubating at 37 ℃ and 190rpm for 1 h;

4) centrifuging to collect bacteria, rotating at 4000rpm for 3min, discarding the supernatant, leaving about 100 μ L, mixing, and coating on an LB plate containing kanamycin resistance;

5) incubated at 37 ℃ overnight.

(4) Detection and sequencing of Positive clones

1) Picking white colonies from the transformation plate, putting the white colonies into a liquid LB culture medium containing Kan, and carrying out shake culture at the constant temperature of 37 ℃ for 8 hours;

2) positive clones were verified by colony PCR, and the correctly verified monoclonals were sent to Shanghai Biotech, Inc. for sequencing, 3 replicates per sequence.

(5) Preservation of positive bacteria liquid

And adding a certain amount of glycerol into the bacterial liquid which is subjected to PCR verification and sequencing to ensure that the final concentration of the glycerol is about 20 percent and storing the glycerol at-80 ℃. The correctly sequenced plasmid was returned for Agrobacterium transfer.

(6) Transformation of Agrobacterium

The agrobacterium tumefaciens LBA4404 competent cells are transformed by a freeze-thaw method, and the specific transformation process is as follows:

1) the Agrobacterium was thawed at-80 ℃ and the ice-water mixture was inserted into ice.

2) Adding 0.01-1 mu g of plasmid DNA into 100 mu L of competence, manually dialing the tube bottom, uniformly mixing, standing on ice for 5min, using liquid nitrogen for 5min, performing ice bath for 5min at 37 ℃ for 5 min.

3) Adding 700. mu.L of nonresistant LB liquid medium, and culturing at 28 ℃ for 2-3h with shaking.

4) 100-150 μ L of the bacterial solution was placed on an LB plate containing kanamycin, rifampicin, and streptomycin, and placed in an incubator at 28 ℃ for 2-3 days while inverted.

5) Selecting positive clones, culturing for 48h at 28 ℃ on an LB liquid culture medium with resistance, and storing the bacterium liquid glycerol with correct bands at the final concentration of about 20% by PCR verification of the bacterium liquid at-80 ℃ for later use.

Example 5 Agrobacterium-mediated transformation of Arabidopsis thaliana

(1) Cultivation of Arabidopsis thaliana

Columbia wild type arabidopsis thaliana transplanted from an 1/2MS flat plate is planted in an artificial climate chamber, and grows to the full-bloom stage, the fruit pods are cut off, and the humidity of nutrient soil at the root of the arabidopsis thaliana is ensured.

(2) Arabidopsis inflorescence infection transformation

For the transformation of Arabidopsis with overexpression vector of 35S, GhDof1.7, the inflorescence infection method is adopted, and the specific operation is as follows:

1) bacterial liquid activation: taking 20 mu L of agrobacterium liquid of a corresponding recombinant vector stored at minus 80 ℃, inoculating the agrobacterium liquid into 1mL of LB liquid culture medium (corresponding antibiotics are added, namely kanamycin, rifampicin and streptomycin), and culturing for 14-18h at 28 ℃ and 180 rpm;

2) expanding and shaking: adding 200 mu L of the activated corresponding bacterial liquid into 50mL of LB liquid culture medium (adding corresponding antibiotics); culturing at 28 deg.C and 180rpm until OD600 of bacterial liquid is about 1.2-1.6 (about 18-20 hr), centrifuging for 8min at 5000g, removing supernatant, and collecting thallus;

3) preparing a medium for infection transformation: 1/2MS halving, 6% sucrose, 0.02% Silwet L-77, pH adjusted to 5.6-5.7 with NaOH;

4) suspending the above thallus with transformation medium to adjust OD600 to 0.6-0.8;

5) dip dyeing: placing arabidopsis inflorescences (mainly unopened buds) in a transformation medium for 30-50sec, and after dip dyeing, keeping arabidopsis flat for 24h under the condition of weak light or dark;

6) culturing the treated arabidopsis thaliana under normal conditions, and spraying water to arabidopsis thaliana leaves every day within one week after infection; to increase transformation efficiency, the infection may be repeated about one week later;

7) after the seeds are matured, the arabidopsis seeds are harvested, namely the transgenic T0 generation seeds.

Example 6 phenotypic characterization of transgenic Arabidopsis plants

(1) The harvested seeds are sterilized and planted on 1/2MS containing kanamycin, then vernalization is carried out for 2 days at 4 ℃, the seeds are transferred to a climatic test box, positive plants grow normally in about 10 days, and negative plants turn yellow in leaves and do not grow any more.

(2) And transplanting the positive arabidopsis thaliana plant into a small flowerpot for planting, extracting DNA after the plant grows for one month, and detecting the DNA by using PCR. The plants of each generation were tested for positive lines until propagation to T3 generations to obtain homozygous transgenic Arabidopsis lines. And (5) carrying out qRT-PCR detection on the T3 generation strain.

Primer for fluorescence quantification of Arabidopsis thaliana internal reference gene UBQ 10:

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

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

And (3) preparing a qRT-PCR reaction system on ice, and carrying out fluorescent quantitative PCR reaction.

The fluorescent quantitative result shows that the expression level of the GhDof1.7 gene in transgenic Arabidopsis is extremely obviously increased (FIG. 3, B). When the inventor wants to bolt arabidopsis, WT arabidopsis and transgenic arabidopsis are watered with 400mM NaCl solution at the same time. After 5 days of treatment, the yellowing of WT leaves was found to be significantly higher than that of Arabidopsis leaves of OE-GhDof1.7 (FIG. 3, A). In addition, the inventors sampled samples at different time periods of transgenic arabidopsis salt treatment, and detected the expression level of the ghdof1.7 gene. As a result, as shown in the graph C in FIG. 3, the expression level of GhDof1.7 decreased first and then continued to increase as the salt treatment time progressed.

In order to observe the influence of salt treatment on the physiological changes in plants, the inventors carried out proline content detection and SOD and CAT activity detection on Arabidopsis thaliana before and after salt treatment. The results show that the proline content, SOD and CAT enzyme activities in GhDof1.7 transgenic arabidopsis after salt treatment are all higher than that of WT after salt treatment; in the case of transgenic arabidopsis, the proline content, SOD and CAT enzyme activities after salt treatment were also significantly higher than those of transgenic plants before salt treatment (fig. 3, D, E). This indicates that overexpression of the GhDof1.7 gene improves the tolerance of Arabidopsis to salt stress.

Example 7 Cotton infection with Virus-induced GhDof1.7 Gene silencing

1 Cotton Material planting

Selecting and planting full TM-1 seeds in a phytotron, wherein the photoperiod and temperature conditions are as follows: irradiating for 16h at 28 ℃; dark 8h, 22 ℃. After the cotyledon of the seedling is flattened and the first true leaf is exposed (about 10 days), the VIGS bacterial liquid injection test is carried out.

2 construction of silencing vectors

Construction of the silencing vector pYL156 for VIGS the vector pYL was double digested with XbaI and BamHI, the plasmid was first double digested and the digested product was recovered from the gel. The primers used were:

upstream primer F

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

Downstream primer R

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

After recovery of the VIGS silent fragment gel, recombinant ligation of the gene fragment and the vector was performed according to the Clonexpress II One Step Cloning Kit (Novozam, Nanjing). The reaction system was transferred into E.coli competent Trans 5. alpha. by the same procedure as described above. The bacterial solution was spread on LB plates supplemented with kanamycin (Kan, 50. mu.g/mL), placed at 37 ℃ and cultured overnight for 12-16h under inversion, and positive single clones were selected and sequenced. And (3) transferring the plasmid with correct sequencing into agrobacterium, performing colony PCR, and storing the bacterial liquid glycerol with correct strips to-80 ℃.

3 injection of bacterial liquid

The specific process of cotton VIGS is as follows:

(1) activating a bacterial liquid: 20 μ L of a strain containing pYL156 plasmid, pYL192 plasmid, pYL156-GhDof1.7 plasmid and pYL156-CAL1 (positive control) plasmid frozen at-80 ℃ was added to a liquid LB medium containing three antibiotics (kanamycin, rifampicin and streptomycin) and cultured at 28 ℃ and 180rpm for 14-16 hours;

(2) expanding and shaking: adding 50-100 μ L of activated bacteria solution into 50mL of liquid LB culture medium containing the above three antibiotics, and culturing at 28 deg.C and 180rpm for 16-20h to make OD600 value of bacteria solution between 1.5-2.0 (the bacteria solution becomes orange yellow). 5000g, centrifuging for 10min, and recovering thalli;

(3) and (3) preparing a transformation medium:

formulation of the transformation medium: MgCl ₂10 mM; MES (2- (4-Morpholino) ethanesulfonic acid), 10mM, pH 5.6 with NaOH; as (acetosyringone), 200 μ M.

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

(4) Uniformly mixing the pCLCrVB and pCLCrVA (no-load), the pCLCrVA of the positive control and the pCLCrVA of the target gene according to the ratio of 1:1 respectively;

(5) cutting the epidermis on the back of the cotton cotyledon by using a 1mL sterile syringe needle, removing the needle, and injecting the uniformly mixed bacterial liquid into the cotyledon until the cotyledon is completely soaked;

(6) and (3) culturing the injected cotton seedlings overnight in the dark, and then placing the cotton seedlings under the light and temperature conditions as follows: 23 ℃; the artificial climate chamber under 16h (light)/8 h (dark) condition is normally managed.

4 identification of silencing lines

After the positive control is whitened, extracting RNA of the sample, carrying out PCR and fluorescent quantitative PCR detection, and planting the seedling with the detected target fragment in a large flowerpot for normal management until the cotton blooms. The expression level of GhDof1.7 in the silent plant is detected by fluorescent quantitative PCR. The results showed that the expression level of ghdof1.7 was reduced by about 3-fold in VIGS plants compared to control plants (fig. 4, C). And then selecting plants with higher silencing efficiency and control plants to carry out salt treatment, and observing after 1 day of treatment. In FIG. 4, panel A shows the null plants and VIGS plants before salt treatment, and panel B shows the control plants and VIGS plants after salt treatment. It is obvious that after salt treatment, VIGS plants have wilting degree more seriously than unloaded plants, which indicates that the gene is possibly related to the salt tolerance of the plants.

To further observe the effect of salt treatment on physiological changes in cotton plants, the inventors measured chlorophyll content, soluble sugar content, proline content and SOD activity in salt-treated VIGS plants and no-load plants, respectively. The results show that the content of all three indicators in VIGS plants is significantly reduced (fig. 4, D and E). In addition, SOD activity in the empty-load control plants was also significantly higher than that in the gene-silenced plants (FIG. 4, F). These results demonstrate that the salt tolerance of cotton plants decreases after silencing the ghdof1.7 gene.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Cotton research institute of Chinese academy of agricultural sciences

Application of cotton GhDof1.7 gene in promotion of salt tolerance of plants

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Artificial Sequence (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

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

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

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

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

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

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

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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

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<213> Artificial Sequence (Artificial Sequence)

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atcctccgtc ttgaccttg 19

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgtccgtcag gcaactcat 19

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tggatcagaa ttcgggtcgg ga 22

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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acttgtatcc ctaccgccct gt 22

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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cacgggggac tctagaatgc aagacccaac gggcttt 37

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<213> Artificial Sequence (Artificial Sequence)

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gatcggggaa attcgagctc ctatctccgt aaacttgaac ct 42

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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agatccagga caaggaaggt attc 24

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgcaggacca agtgaagagt ag 22

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

taccgaattc tctagaatgc aagacccaac gggcttt 37

<210> 12

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctcggtacc ggatccttgg tttggggtgg ggtctgga 38

Claims (9)

1.GhDof1.7Use of a gene for enhancing salt tolerance in plants, wherein said gene is characterized in thatGhDof1.7The gene has a nucleotide sequence shown as SEQ ID NO. 1.

2. The application of the polypeptide coded by the gene of claim 1 in promoting salt tolerance of plants, wherein the polypeptide has an amino acid sequence shown in SEQ ID NO. 2.

3. Use according to claim 1, characterized in that: increase in plantsGhDof1.7The expression level of the gene is used for promoting the salt tolerance of the plant.

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

5. The use of claim 4, wherein the 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.

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

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

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

9. Use according to any one of claims 5 to 8, wherein the plant is cotton, maize, rice, wheat or Arabidopsis thaliana.

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