Application of TaCML4-D gene and encoded protein thereof in improving salt tolerance of wheat

Abstract

The invention discloses an application of a TaCML4-D gene and an encoding protein thereof in improving the salt tolerance of wheat, and belongs to the technical field of gene engineering. A novel wheat salt tolerance related gene TaCML4-D is found from wheat, the salt tolerance of the wheat can be improved by overexpressing the gene TaCML4-D, and therefore, overexpressing the gene and applying the gene to a backbone germplasm with poor salt tolerance has a great significance in improving the salt tolerance of the wheat and improving the salt tolerance of the wheat. The method has important economic value and social benefit.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular to a kind of TaCML4-D Gene and its encoding protein in the application of improving the salt tolerance of wheat BACKGROUND Soil salinization is one of the main abiotic stress factors restricting wheat production. According to statistics, the global area of salinized soil has exceeded 800 million hectares, and is showing a trend of increasing year by year. Salt stress can cause a series of physiological and metabolic disorders such as imbalance of cell osmotic pressure, ion toxicity and oxidative damage in wheat, thereby inhibiting the growth and development of wheat and leading to reduced yield or even no yield. Therefore, cultivating salt-tolerant wheat varieties is an effective way to alleviate the impact of salinized soil on wheat production.

[0002] Traditional salt-tolerant wheat breeding methods mainly rely on intervarietal crossing and phenotypic screening, but there are problems such as long breeding cycle, complex inheritance of salt tolerance traits, and low screening efficiency. With the development of genetic engineering technology, by cloning salt-tolerant related genes and introducing them into wheat, cultivating transgenic salt-tolerant wheat varieties has become an important direction of wheat salt-tolerant breeding.

[0003] At present, the reported plant salt-tolerant genes include osmoregulation-related genes (such as proline synthetase genes), ion balance-related genes (such as Na + / H + antiporter genes), antioxidant-related genes (such as superoxide dismutase genes), etc., but the application effect of these genes in wheat still needs to be improved, and the salt tolerance mechanisms of different genes are different. Therefore, it is of great significance to mine new wheat salt-tolerant genes, analyze their salt-tolerance molecular mechanisms, and apply them to transgenic breeding to improve the salt tolerance of wheat. SUMMARY

[0004] In view of the above prior art, the purpose of the present application is to provide a kind of TaCML4-D Gene and its encoding protein in the application of improving the salt tolerance of wheat. The present application finds a new wheat salt-tolerant related gene TaCML4-D , overexpression TaCML4-D Gene can improve the salt tolerance of wheat, which is of great significance for the cultivation of salt-tolerant wheat.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows: The first aspect of the present application provides TaCML4-D The application of the gene in (1) or (2) as follows: (1) improving the salt tolerance of wheat; (2) cultivating salt-tolerant wheat varieties; The TaCML4-D Gene is a DNA molecule as shown in i) or ii) or iii) as follows: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO. 2; iii) A DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.2.

[0006] The term "identity" used here refers to sequence similarity to native nucleic acid sequences. Identity can be evaluated using computer software, such as the BLAST algorithm (Altschul). et al. 1990. Journal of Molecular Biology 215:403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90:5873-5877).

[0007] In the aforementioned nucleic acid molecules, the 90% or more identity can be at least 90%, 92%, 93%, 95%, 96%, 98%, or 99% identity.

[0008] This invention has found that: through overexpression TaCML4-D The gene can improve the survival rate of wheat under salt stress and reduce the ion leakage rate; thus improving the salt tolerance of wheat, which is of great significance for breeding salt-tolerant wheat varieties.

[0009] A second aspect of the present invention provides the use of TaCML4-D protein in improving the salt tolerance of wheat; said TaCML4-D is a protein as shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0010] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, MYC tag, GST tag, and / or SUMO tag, etc.

[0011] In the above applications, improving wheat's salt tolerance specifically means: increasing the survival rate of wheat under salt stress conditions and reducing ion leakage rate.

[0012] A third aspect of the invention provides a way to promote TaCML4-D The application of substances that enhance gene expression or increase the activity and / or content of TaCML4-D in the following (1) or (2): (1) Improve the salt tolerance of wheat; (2) Breed salt-tolerant wheat varieties.

[0013] In some preferred embodiments of the present invention, promoting TaCML4-D The substance used for gene expression can be any of the following: C1) contains TaCML4-D Gene expression cassettes; C2) contains TaCML4-D Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TaCML4-D Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TaCML4-D Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TaCML4-D Transgenic plant tissue containing genes, or transgenic plant tissue containing the expression cassette described in C1).

[0014] Existing plant expression vectors can be used to construct structures containing... TaCML4-D Recombinant gene vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors.

[0015] use TaCML4-D When constructing recombinant vectors using genes, any type of enhancing or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation initiation region can originate from the transcription initiation region or a structural gene.

[0016] A fourth aspect of the present invention provides a method for improving the salt tolerance of wheat, comprising: inducing salt into the wheat plant... TaCML4-D The steps of gene overexpression.

[0017] In the above method, the wheat plant is made to... TaCML4-D Gene overexpression is achieved through the following methods: Exogenous transfer TaCML4-D Genes; or the introduction of genes that can activate or enhance wheat TaCML4-D DNA fragments at the transcriptional or translational level of a gene.

[0018] A fifth aspect of the present invention provides a method for breeding a salt-tolerant wheat variety, comprising the following steps: Transition to wild-type wheat TaCML4-D Genes were used to obtain transgenic wheat, which has a higher salt tolerance than wild-type wheat; Salt-tolerant wheat varieties can be bred by self-pollinating genetically modified wheat with enhanced salt tolerance or by crossing it with other salt-tolerant wheat varieties.

[0019] The beneficial effects of this invention are: This invention has discovered a new wheat salt tolerance-related gene. TaCML4-D overexpression TaCML4-D The gene can enhance the salt tolerance of wheat. Therefore, overexpressing this gene and applying it to backbone germplasm with poor salt tolerance has important economic and social value for improving the salt tolerance of wheat, thereby increasing wheat yield and quality. Attached Figure Description

[0020] Figure 1 Phylogenetic analysis of TaCML4-D homologous proteins.

[0021] Figure 2 Wheat overexpression vector pUbi::TaCML4-D Schematic diagram of the carrier structure.

[0022] Figure 3 : TaCML4-D Identification results of gene overexpression lines Figure 4 Gene knockout strains Tacml4-D Comparison of phenotype, survival rate, and ion leakage rate with the control Fielder phenotype.

[0023] Figure 5 Gene overexpression lines TaCML4-D Comparison chart with the control Fielder phenotype. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, 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 application pertains.

[0025] As described above, it is of great significance to mine new salt-tolerant genes in wheat, analyze their salt-tolerance molecular mechanisms, and apply them to transgenic breeding for improving the salt tolerance of wheat.

[0026] Gene TraesCS5D02G392200 (http: / / wheatomics.sdau.edu.cn / genes / ; version number: genome IWGSC v2.0) is a function-unknown gene in the wheat genome.

[0027] In order to study the function of gene TraesCS5D02G392200, the inventors named the gene as TaCML4-D Gene, the nucleotide sequence of the coding region of which is shown in SEQ ID NO. 1; the amino acid sequence of the encoded TaCML4-D protein is shown in SEQ ID NO. 2. Specifically as follows: TaCML4-D Gene (SEQ ID NO. 1): ATGGAACAGGCGCTGACGGGCGAGCAGATGGTGGCGTTCCAGGAGGCCTTCTCCCTCTTCGACAAGAACGGCGATGGATGCATCAGCTTGGAAGAGCTGGCCGCGGTGACTCGCTCCCTCGGCCTCGACCCGACCAACCAGGAGCTCAATGACATGATGCGTGAAGTCGACATGGATGGGAACGGCACCATTGATTTCCAGGAGTTCTTGAGCCTCATTGCCAGGAAGATGCAGGACGGAGACGCCGACGAAGAGCTCAAGGAAGCTTTCGAGGTCCTGGACAAGGATCGAAATGGTTTCATCTCCCCTGTTGAGCTGAGGACGGTGATGATCAATCTCGGGGAGAAGATGACCGACGAGGAGGTCGAGCAGATGATCAGGGAGGCGGACACCGATGGTGACGGGCAGGTGAACTACGATGAATTCGTGCTCATGATGAAGAATGCTGAGCGCAAGATAACTGGGTGA.

[0028] TaCML4-D protein (SEQ ID NO. 2): MEQALTGEQMVAFQEAFSLFDKNGDGCISLEELAAVTRSLGLDPTNQELNDMMREVDMDGNGTIDFQEFLSLIARKMQDGDADEELKEAFEVLDKDRNGFISPVELRTVMINLGEKMTDEEVEQMIREADTDGDGQVNYDEFVLMMKNAERKITG.

[0029] Phylogenetic analysis of TaCML4-D protein is shown as Figure 1

[0030] The inventors investigated the survival rate and ion leakage rate of the overexpression strain and knockout strain of the gene under salt stress condition by constructing TaCML4-D The results showed that the overexpression of the gene can improve the salt tolerance of plants, and can be used as a new salt-tolerant gene for breeding of salt-tolerant wheat varieties. TaCML4-D

[0031] The test materials used in the embodiments of the application are all conventional test materials in the art and can be purchased through commercial channels. The experimental methods not specified in detail are carried out according to the conventional test methods or according to the operation instructions recommended by the suppliers.

[0032] Example 1 TaCML4-D Cloning of the gene 1. Extraction and purification of total RNA of wheat: The gene was amplified from Chinese spring wheat. TaCML4-D The RNA extraction kit used in this experiment was UltraPure RNA Extraction Kit provided by Beijing Kangwei Reagent Biotechnology Co., Ltd., and the specific experimental steps were carried out according to the instructions of the extraction kit.

[0033] In order to ensure that the quality of RNA meets the requirements, the purity and concentration of the purified RNA sample were detected by spectrophotometer and agarose gel electrophoresis, respectively, wherein the purity and concentration standards are: the purity of RNA is OD 260 / 280 and OD 260 / 230 are both in the range of 1.8-2.0, and the concentration of RNA is in the range of 1.0-2.0 µg / µL.

[0034] 2. Synthesis of the first strand of cDNA: The synthesis kit of the first strand of cDNA used in this experiment was completed by FastQuant RT Kit (with gDNase) of Tiangen Biotech Co., Ltd.

[0035] 3. TaCML4-D Cloning of the gene: ​​PCR amplification was performed using the following primer pairs with reverse-transcribed cDNA as template: Upstream primer: 5'-GAATTCATGGAACAGGCGCTGAC-3'; Downstream primer: 5'-ACTAGTCCCAGTTATCTTGCGCTCA-3'.

[0036] The PCR amplification system was 2 μL of upstream primer (10 pmol / μL), 2 μL of downstream primer (10 pmol / μL), 10 μL of 2x Phanta Max Master Mix, 1 μL of cDNA template, and ddH2O was added to make up the total volume to 20 μL.

[0037] The amplification conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 40 s, 34 cycles; 72°C extension for 5 min.

[0038] Example 2: TaCML4-D Construction of gene overexpression vector and knockout vector 1. Construction of overexpression vector: (1) 4 μL of the PCR product of Example 1 was ligated with pENTR TM / D-TOPO Vector, and the operation steps were performed according to the pENTR TM / D-TOPO Vector product manual.

[0039] (2) The ligation product was transformed into E. coli DH5a, and cultured overnight on LB solid medium containing kanamycin (100 mg / L).

[0040] (3) A single colony was picked and cultured overnight in LB liquid medium containing kanamycin (100 mg / L). Plasmid DNA was extracted by alkaline method, and sequence determination was performed.

[0041] (4) The sequence of the amplified product was analyzed by sequencing, which was shown in SEQ ID NO. 1, indicating that TaCML4-D the gene had been ligated to pENTR TM / D-TOPO Vector, and the construction of the cloning vector was completed.

[0042] (5) The cloning vector was digested with restriction enzymes ApaI and EcoR V The target fragment was detected by electrophoresis and recovered.

[0043] (6) The enzyme-digested cloning vector and pC186 vector were connected by LR reaction, and the operation steps were performed according to the LR instruction of lifetechnologies company product.

[0044] (7) The ligation product was transformed into E. coli DH5α, and cultured on LB solid medium containing kanamycin (50 mg / L) overnight.

[0045] (8) Single colonies were picked and cultured in LB liquid medium containing kanamycin (50 mg / L) overnight. Plasmid DNA was extracted by alkaline method, and sequencing analysis was performed to construct the overexpression vector with correct sequencing analysis pUbi:: TaCML4-D Figure 2

[0046] 2. Construction of knock-out vector: (1) Design sgRNA in E-CRISP (http: / / www.e-crisp.org / E-CRISP / ). By analyzing the results of target points in wheatomics website blast, specific target points targeting only A gene locus (TraesCS5A02G382800.1), B gene locus (TraesCS5B02G387200.1) and D gene locus (TraesCS5D02G392200.1) were screened. TaCML4 Target point one: CGGTGACTCGCTCCCTCGGC NGG;

[0047] Target point two: GTCGTACTACCACCAGCAGG NGG. " N " in the target sequence represents any nucleotide of A, T, C and G.

[0048] (2) Construction of Crispr-Cas9 gene knockout vector.

[0049] pCBC-DT1T2 diluted 100 times was used as a template for four-primer PCR amplification. -BsF / -BsR is the normal primer concentration; -F0 / -R0 is diluted 20 times for PCR amplification:

[0050] -5DMT1T2-F: aataat TaCML4 AAGCgTCCTGGACAAGGATCGAAA; GGTCTC -5DMT1T2-F0: gTCCTGGACAAGGATCGAAA TaCML4 gttttagagctagaaatagc; TaCML4 ​​​-5DMT1T2-R0: CGGTGACTCGCTCCCTCGGCGCTTCTTGGTGCC; TaCML4 -5DMT1T2-R: attatt GGTCTCTAAAC CGGTGACTCGCTCCCTCGG.

[0051] PCR amplification system is 1 μL F primer (50 pmol / μL), 1 μL diluted F0 primer (2.5 pmol / μL), 1 μL R primer (50 pmol / μL), 1 μL diluted R0 primer (2.5 pmol / μL), 5 μL 10×PCR buffer, 2 μL dNTP mixture (10 mmol / L), 0.5 μL EVO DNA polymerase (5 U), 1 μL pCBC-DT1T2 template, and DEPC·H2O to make up the total volume to 25 μL.

[0052] The amplification conditions are: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min, 32 cycles; 72℃ extension for 5 min.

[0053] Recover the PCR amplification product, and establish the enzyme digestion and ligation system, as follows: The ligation product is transformed into E. coli Top10, and cultured on LB solid medium containing kanamycin (100 mg / L) overnight. Single colonies are picked and cultured in LB liquid medium containing kanamycin (100 mg / L) overnight. Plasmid DNA is extracted by alkaline method, and subjected to sequence determination. The sequence of the amplification product is analyzed by sequencing, which is shown in the target point, indicating that the target point sequence has been connected to the pUBE413 Vector vector, TaCML4 The gene knockout vector (pCBC-DT1T2) is constructed. TaCML4-Crispr ).

[0054] Example 3: Creation of transgenic plants (1) Agrobacterium infection: The expression vector pUbi:: TaCML4-D and TaCML4-Crispr knockout vector constructed in Example 1 are transformed into Agrobacterium EHA105, and Agrobacterium infection solution for transformation is obtained. The prepared Agrobacterium infection solution is sucked into a 2 mL centrifuge tube containing Fielder wheat immature embryos, and gently inverted for 45 s to mix, so that the immature embryos are immersed in the bacterial solution. If the immature embryos cannot be immersed in the bacterial solution, they can be centrifuged briefly. After standing for 5 min, the bacterial solution and the immature embryos are poured into a sterile disposable culture dish, and half of the bacterial solution is sucked off.

[0055] (2) Co-culture: The young embryo was picked up with a sterilized scalpel and placed on the co-culture medium (MS powder 2.12 g, sucrose 10 g, 2,4-D 2 mg, glutamine 5 mg, hydrolyzed casein 0.5 g, silver nitrate 4 mg, agar 8 g, distilled water to 1 L; pH = 5.8) during which the scalpel was sterilized several times to avoid contamination affecting the subsequent experiment.

[0056] The culture dish was sealed with sealing film and placed in a 23℃ dark incubator for co-culture.

[0057] (3) Callus induction culture: After 2 days of co-culture, the young embryo axis was cut off and placed on the recovery medium (MS powder 2.12 g, sucrose 10 g, 2,4-D 2 mg, glycine 2 mg, silver nitrate 4 mg, vitamin B5 0.5 mg, agar 8 g, distilled water to 1 L; pH = 5.8) and cultured in the dark at 25℃.

[0058] After 5 days, the young embryo callus began to swell and produce callus. The swollen callus was transferred to screening medium A (MS powder 2.12 g, sucrose 10 g, zeatin 5 mg, IAA 0.5 mg, glycine 2 mg, hygromycin 15 mg, silver nitrate 4 mg, vitamin B5 0.5 mg, agar 8 g, distilled water to 1 L; pH = 5.8), and the browned and dead young embryo tissue was discarded and continued to be cultured in the dark at 25℃.

[0059] After 14 days, larger callus was formed, the callus was cut in half, the cut surface was in contact with the medium, and was transferred to screening medium B (MS powder 2.12 g, sucrose 10 g, 6-BA 5 mg, IAA 0.5 mg, glycine 2 mg, hygromycin 30 mg, silver nitrate 4 mg, vitamin B5 0.5 mg, agar 8 g, distilled water to 1 L; pH = 5.8) and continued to be cultured in the dark at 25℃.

[0060] (4) Differentiation culture: After 21 days, the callus was transferred to differentiation medium (MS powder 2.12 g, sucrose 10 g, zeatin 5 mg, IAA 0.5 mg, glycine 2 mg, hygromycin 15 mg, vitamin B5 0.5 mg, asparagine 5 mg, glutamine 5 mg, silver nitrate 4 mg, agar 8 g, distilled water to 1 L; pH = 5.8), and the callus began to differentiate into buds and was cultured in a light incubator at a temperature of 25℃ and a light intensity of 2000 lx.

[0061] After the callus tissue differentiates into green seedlings, they are transferred to rooting medium (MS powder 2.12g, sucrose 10g, IAA 0.5mg, paclobutrazol 0.5mg, glycine 1mg, hygromycin 15mg, vitamin B5 0.5mg, asparagine 5mg, glutamine 5mg, silver nitrate 4mg, agar 8g, distilled water to a final volume of 1L; pH=5.8) until the seedlings have grown 4-5 leaves, then transplanted to a greenhouse or artificial climate chamber for robust seedling cultivation.

[0062] (5) Identification of transgenic plants 1) Primers for identifying gene knockout lines are as follows: A. Primers for genome identification: F:CGGGATCGGGAATTCAGGAT; R:CAGTACCACAAGCAGCGAATT.

[0063] B genome identification primers: F:GGCTGTGGTGACATTACTTGT; R:TAAAGGTGGCTAGCATAACTTGCA.

[0064] D genome identification primers: F:AAAATCTTTGACCGCACGTGGT; R:CGAGATTGATCATCACCGTCCTCAG.

[0065] 2) Identification of gene expression levels in overexpression lines Gene expression levels were identified by RT-PCR using the following primers: F:ATCCCCAAGCTCGTTTGAGG; R:CCCTGATCATCTGCTCGACC.

[0066] Tublin-F:GATGCAGCCAACAACTTCGCC; Tublin-R:CAGTTCCACCTCCAACAGCGT.

[0067] Upon identification, this invention yielded two wheat [products / items]. TaCML4 Mutant plants ( Tacml 4#1-2 and Tacml 4#2-1). Compared with wild-type Fielder wheat, the mutant plants... Tacml 4#1-2 TaCML4 The gene underwent the following mutation: TaCML4-A The nucleotide at position 107 is deleted;TaCML4-B Nucleotides at positions 106, 107, 288, and 289 are deleted; TaCML4-D A nucleotide G was added after the 120th nucleotide.

[0068] Compared to wild-type Fielder wheat, mutant plants Tacml4#2-1 of TaCML4 The gene underwent the following mutation: TaCML4-A The nucleotide at position 107 is deleted; TaCML4-B Nucleotides 106, 107, and 289-291 are deleted; TaCML4-D The nucleotide at position 107 is deleted.

[0069] The identification results of the overexpression lines are as follows: Figure 3 As shown, select TaCML4 strains with overexpressed genes TaCML4 - #1- 12 and TaCML4 - #4-2 For use in subsequent experiments.

[0070] Example 5: Salt tolerance study of transgenic lines 1. Test method: Select seeds of wild-type Fielder wheat, and those obtained in Example 4. TaCML4 Mutant plants ( Tacml 4#1-2 and Tacml 4#2-1) Seeds of generation T3 TaCML4-D Gene overexpression lines ( TaCML4 - #1-12 and TaCML4 - #4-2 Seeds of generation T1. Plump seeds were hydroponically cultured, sterilized with 70% ethanol for 15 min, then rinsed 5 times with ddH2O, and placed in a 4℃ incubator in the dark for 3 days. After 3 days, they were removed and allowed to germinate at room temperature. Once they reached the one-leaf stage, they were inserted into a hydroponic box containing Hoagland's nutrient solution. When they reached the two-leaf, one-heart stage, they were treated with 250 mM Hoagland's nutrient solution for salt treatment. TaCML4 Mutant plants and wild-type Fielder plants (as controls) were treated with salt for 14 days. TaCML4-D Gene overexpression lines and wild-type Fielder lines as controls were treated with salt for 21 days.

[0071] One week after salt treatment, observe the survival of the plants and calculate the survival rate. Survival rate = (Number of surviving plants / Total number of plants) × 100%; Criteria: no new green leaves produced, all withered after recovery for one week after salt treatment, considered as death.

[0072] Determination of ion leakage rate after recovery for one week after salt treatment: take the aerial part, wash with ddH2O, put into centrifuge tube, add appropriate amount of ddH2O, first measure the conductivity of ddH2O as R0, second measure the conductivity of material as R1, then boil until the leaves are not green, measure the conductivity as R2 when it cools to room temperature. Ion leakage rate is (R1-R0) / (R2-R0) x 100%.

[0073] 2. Test results: The results are shown in Figure 4 and Figure 5 , which show that compared with wild type Fieder, gene knockout lines #1-2 and #2-1 show lower survival rate and higher ion leakage rate, indicating that gene knockout lines greatly reduce the salt tolerance of wheat.

[0074] Compared with wild type Fieder, TaCML4-D the growth of gene overexpression lines under salt stress conditions is significantly improved, and under salt stress conditions, TaCML4-D gene overexpression lines have higher survival rate and lower ion leakage rate, proving that overexpression of TaCML4-D gene can improve the salt tolerance of wheat.

[0075] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. TaCML4-D The application of genes in the following (1) or (2): (1) Improve the salt tolerance of wheat; (2) Breed salt-tolerant wheat varieties; The TaCML4-D A gene is a DNA molecule as shown in i), ii), or iii): i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO. 2; iii) A DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.

2.

2. Application of TaCML4-D protein in improving the salt tolerance of wheat; wherein TaCML4-D is the protein shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. The application according to claim 2, characterized in that, Specifically, improving wheat's salt tolerance involves increasing the survival rate of wheat under salt stress conditions and reducing ion leakage rate.

4. Promote TaCML4-D The application of substances that enhance gene expression or increase the activity and / or content of TaCML4-D in the following (1) or (2): (1) Improve the salt tolerance of wheat; (2) Breed salt-tolerant wheat varieties.

5. The application according to claim 4, characterized in that, Promote TaCML4-D The substance used for gene expression is any one of the following: C1) contains TaCML4-D Gene expression cassettes; C2) contains TaCML4-D Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TaCML4-D Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TaCML4-D Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TaCML4-D Transgenic plant tissue containing genes, or transgenic plant tissue containing the expression cassette described in C1).

6. A method for improving the salt tolerance of wheat, characterized in that, include: In wheat plants TaCML4-D The steps of gene overexpression.

7. The method according to claim 6, characterized in that, In wheat plants TaCML4-D Gene overexpression is achieved through the following methods: Exogenous transfer TaCML4-D Genes; or the introduction of genes that can activate or enhance wheat TaCML4-D DNA fragments at the transcriptional or translational level of a gene.

8. A method for breeding a salt-tolerant wheat variety, characterized in that, Includes the following steps: Transition to wild-type wheat TaCML4-D Genes were used to obtain transgenic wheat, which has a higher salt tolerance than wild-type wheat; Salt-tolerant wheat varieties can be bred by self-pollinating genetically modified wheat with enhanced salt tolerance or by crossing it with other salt-tolerant wheat varieties.

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