Application of CDK8 gene in improving plant salt tolerance
By constructing CDK8 overexpression vector and transferring it to Arabidopsis, its biological function under salt stress was analyzed, and the problem of insufficient salt tolerance in plants under salt stress was solved, achieving a significant improvement in plant salt tolerance.
Patent Information
- Application Number
- CN202211146908.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-19
- Publication Date
- 2025-04-25
- Estimated Expiration
- 2042-09-19
AI Technical Summary
Plants exhibit low germination rate, growth delay and yellowing of leaves under salt stress. The biological function and mechanism of action of CDK8 gene in plant salt tolerance have not been clarified in the prior art.
By obtaining the nucleotide sequence information of the Arabidopsis mediator CDK8, the CDK8 overexpression vector was constructed, and transferred to Arabidopsis through Agrobacterium-mediated method, the CDK8 overexpression transgenic line was obtained, and its biological function under salt stress was analyzed.
Under salt stress, the seed germination rate, cotyledon greening rate, main root length and survival rate of CDK8 overexpressing transgenic lines were higher than those of wild-type and cdk8 mutants, significantly improving the salt tolerance of plants.
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Figure CN115807027B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of CDK8 gene in improving plant salt tolerance. Background Art
[0002] Salt stress is one of the important factors that limit plant growth and crop yield. Usually, under salt stress, plants show low seed germination rate and delayed growth in the vegetative and reproductive stages, and in severe cases, the leaves of plants turn yellow and die. Therefore, how plants perceive and regulate salt stress responses, thereby regulating plant growth adaptation, is an important biological issue that plays an important role in improving plant stress resistance and crop yield.
[0003] CDK8 is a subunit of the kinase module of the eukaryotic mediator complex, and plays an important role in plant growth and development and adaptation to environmental stress. In recent years, studies have found that CDK8 is involved in the development of the leaf wax layer and regulates the synthesis of plant defense genes and secondary metabolites, and cdk8 mutants show unique disease resistance. In addition, CDK8 also integrates abscisic acid (ABA) signals with drought stress responses, positively regulating plant drought resistance. However, the biological function and mechanism of CDK8 in plant salt tolerance are still unclear. Therefore, revealing the biological function of the mediator complex CDK8 in plant salt tolerance can provide rich genetic resources and theoretical basis for molecular breeding of stress-resistant crops. Summary of the invention
[0004] The purpose of the present invention is to provide application of CDK8 gene in improving salt tolerance of plants.
[0005] In order to achieve the above object, the technical solution adopted by the present invention is summarized as follows:
[0006] Based on the Arabidopsis genome sequencing published on the TAIR official website (http: / / www.arabidopsis.org / ), the nucleotide sequence information of Arabidopsis mediator CDK8 was obtained. The nucleotide sequence length of the CDK8 gene (AT5G63610) coding frame is 1413 bp, its nucleotide sequence is shown in SEQ ID NO.1, and it consists of 470 amino acids. Its sequence is shown in SEQ ID NO.2, and its molecular weight is about 52.80 kD.
[0007] The present invention also constructs a series of plant expression vectors, and the expression vectors, recombinant vectors or transgenic plant lines containing the above-mentioned genes and host cells containing the vectors also fall within the protection scope of the present invention in terms of improving the salt tolerance of plants.
[0008] The protein encoded by the above CDK8 gene is selected from:
[0009] (1) Its amino acid sequence is shown in SEQ ID NO.2;
[0010] (2) a protein derived from (1) which is formed by replacing, deleting or adding one or more (e.g., 1-30; preferably 1-20; more preferably 1-10; such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO.2 and has the function of the protein of (1);
[0011] (3) A protein derived from (1) that has 80% (preferably more than 90%, such as 95%, 98%, 99% or higher) homology with the protein sequence defined in (1) and has the function of the protein of (1).
[0012] That is to say, the functions of the genes protected by the present invention include not only the above-mentioned CDK8 gene, but also the functions of homologous genes with higher homology to SEQ ID NO.1 (such as the homology is higher than 40%; preferably higher than 50%; preferably higher than 60%; more preferably higher than 70%; more preferably higher than 80%; more preferably higher than 90%; more preferably higher than 95%; more preferably higher than 98%) in salt tolerance.
[0013] The present invention also transfers the recombinant vector with the exogenous gene into wild-type Arabidopsis thaliana by Agrobacterium floral dipping method to obtain Arabidopsis thaliana plants over-expressing CDK8 protein.
[0014] The main purpose of the present invention is to construct a CDK8 overexpression transgenic line and analyze the biological functions of the cdk8 mutant and the CDK8 overexpression transgenic line under salt stress, thereby providing gene resources for molecular breeding of crop salt resistance.
[0015] The biological function of the Arabidopsis mediator CDK8 disclosed in the present invention in plant salt tolerance is specifically manifested in:
[0016] (1) Under salt stress, the seed germination rate of the cdk8 mutant was lower than that of the wild type, while the seed germination rate of the CDK8 overexpression line was higher than that of the wild type;
[0017] (2) Under salt stress, the cotyledon greening rate of the cdk8 mutant was lower than that of the wild type; while the cotyledon greening rate of the CDK8 overexpression line was higher than that of the wild type;
[0018] (3) Under salt stress, the relative taproot length of the cdk8 mutant was shorter than that of the wild type, while the relative taproot length of the CDK8 overexpression line was longer than that of the wild type.
[0019] (4) Under salt stress, the survival rate of the cdk8 mutant was lower than that of the wild type, while the survival rate of the CDK8 overexpression line was higher than that of the wild type.
[0020] The above applications were concluded through experiments simulating salt stress in culture medium and soil.
[0021] According to its function, salt-tolerant plants can be obtained by genetic modification. Specifically, the CDK8 gene can be introduced into the target plant to obtain a transgenic plant, which has higher salt tolerance than the target plant.
[0022] Specifically, the CDK8 gene can be introduced into the target plant through the recombinant expression vector. In the method, the recombinant expression vector can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants.
[0023] Specifically, in order to improve the excellent traits of plants, the present invention also protects a new plant breeding method, comprising the following steps (1) or (2):
[0024] (1) By increasing the activity of CDK8 protein in the target plant, a plant with stronger cold resistance than the target plant is obtained;
[0025] (2) Promoting the expression of the CDK8 gene in the target plant to obtain a plant with stronger cold resistance than the target plant;
[0026] The implementation method of "promoting the expression of CDK8 gene in target plants" can be as follows (1) or (2) or (3):
[0027] (1) Introducing the CDK8 gene into the target plant;
[0028] (2) introduction of strong promoters and / or enhancers;
[0029] (3) Other common methods in this field.
[0030] Among them, the target plant (target plant), the target plant described in the present invention is Arabidopsis thaliana.
[0031] Target gene, also known as target gene, is a gene used for gene recombination, changing the characteristics of receptor cells and obtaining the expected expression product in genetic engineering design and operation. It can be from the organism itself or from a different organism.
[0032] In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, flower plants, or forestry plants, etc. The plants can be, for example (but not limited to): dicotyledons, monocotyledons or gymnosperms.
[0033] As a preferred embodiment, the "plant" includes but is not limited to Arabidopsis thaliana, and any plant having the gene or a gene homologous thereto is applicable.
[0034] The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, and our target gene or nucleic acid is present in these different parts. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, and similarly, each of the aforementioned objects contains the target gene / nucleic acid.
[0035] The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and the progeny obtained using the method of this patent have the same characteristics.
[0036] The invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also further relates to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or proteins. The invention also relates to foods or food additives obtained from the relevant plants.
[0037] Advantages of the present invention:
[0038] (1) The present invention obtained the Arabidopsis CDK8 gene by cloning the plant genome, constructed a CDK8 overexpression vector, and transferred it into Columbia Arabidopsis (Col-0) using an Agrobacterium-mediated method to obtain a CDK8 overexpression transgenic line. The inventors revealed the role of CDK8 in plant salt tolerance through simulation experiments of salt stress in culture medium and soil, and provided gene resources for molecular breeding of crop salt tolerance.
[0039] (2) Salt-tolerant plants can be obtained through transgenic methods. Specifically, the CDK8 gene can be introduced into the target plant to obtain a transgenic plant. The salt tolerance of the plant is higher than that of the target plant, which provides a new approach for plant salt tolerance breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1 This is the electrophoresis diagram of the CDK8 gene amplification product;
[0041] In the figure, Figure 1 A is the electrophoresis diagram of CDK8 gene amplification. Figure 1 B is the PCR electrophoresis of Escherichia coli culture medium containing the CDK8-YFP overexpressing recombinant vector.
[0042] Figure 2 is CDK8 overexpression transgenic and cdk8 mutant identification;
[0043] In the figure, Figure 2 A is the immunoblot of WT, cdk8 mutant and CDK8 transgenic. GFP antibody was from SIGMA, USA, with the catalog number 11814460001. Ponceau red was from Beijing Solebow Technology Co., Ltd., used for total protein staining. Figure 2 B is the PCR electrophoresis diagram for identification of cdk8-1 and cdk8-2 mutants.
[0044] Figure 3 is the seed germination phenotype of the wild-type Arabidopsis line (WT), cdk8 mutant, and CDK8 overexpression line;
[0045] In the figure, Figure 3 A is the germination of seeds of wild-type Arabidopsis thaliana (WT), cdk8 mutant, and CDK8 overexpression line on 1 / 2MS medium and 1 / 2MS medium containing 75 mM or 100 mM NaCl. Figure 3 B is the statistics of cotyledon greening rate after germination of seeds of wild-type Arabidopsis thaliana line (WT), cdk8 mutant and CDK8 overexpression line on 1 / 2MS medium and 1 / 2MS medium containing 75mM or 100mM NaCl.
[0046] Figure 4 It is the taproot length phenotype of the wild-type Arabidopsis line (WT), cdk8 mutant, and CDK8 overexpression line;
[0047] In the figure, Figure 4 A is the main root growth of wild-type Arabidopsis strain (WT), cdk8 mutant and CDK8 overexpression line in 1 / 2MS medium and 1 / 2MS medium containing 75mM or 100mM NaCl; Figure 4 B is the main root length statistics of wild-type Arabidopsis lines (WT), cdk8 mutants, and CDK8 overexpression lines on 1 / 2MS medium and 1 / 2MS medium containing 75 mM or 100 mM NaCl.
[0048] Figure 5 is the survival phenotype of wild-type Arabidopsis strain (WT), cdk8 mutant, and CDK8 overexpression strain before and after salt stress treatment;
[0049] In the figure, Figure 5 A is the growth of wild-type Arabidopsis strain (WT), cdk8 mutant and CDK8 overexpression strain before and after salt stress treatment. Figure 5B is the survival rate statistics of wild-type Arabidopsis strain (WT), cdk8 mutant and CDK8 overexpression line after salt treatment. DETAILED DESCRIPTION
[0050] The present invention will be described in detail below through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.
[0051] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.
[0052] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.
[0053] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.
[0054] The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to these. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include a coding sequence in a cDNA, and / or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA.
[0055] In addition, in order to have a more intuitive understanding of the technical solution of the present invention, some professional terms involved in the present invention are explained as follows:
[0056] "Mutant" refers to an individual with a mutation that has phenotypic characteristics different from the wild type.
[0057] "Expression vectors" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene.
[0058] The Arabidopsis thaliana used for CDK8 gene amplification in the present invention is the Columbia ecotype (Col-0), and its T-DNA insertion mutant was donated by others.
[0059] The inventors cloned the coding frame sequence of the CDK8 gene using molecular cloning technology, constructed a CDK8 overexpression vector, and obtained a CDK8 overexpression transgenic line by Agrobacterium tumefaciens flower dipping method. The present invention also verified the function of the overexpression transgenic plant under salt stress.
[0060] 1. Gene source and isolation:
[0061] Based on the whole genome sequencing of Arabidopsis published on the official website of TAIR (http: / / www.arabidopsis.org / ), the nucleotide sequence information of Arabidopsis mediator CDK8 was obtained. The nucleotide sequence length of the coding frame of the CDK8 gene (AT5G63610) is 1413bp, consisting of 470 amino acids, and the molecular weight is approximately 52.80kD. Sequence-specific primers (F: 5′-ATGGGAGATGGGAGTTCC-3′; R: 5′-TTAGAGGCGTCTGGATTTGTT-3′) were designed using Primer Premier 5 software. The cDNA of Arabidopsis seedlings of the Col-0 ecotype was used as a template, and Primer STAR Max high-fidelity enzyme amplification (Takara) was used, with an annealing temperature of 55°C. The gene cloning results are shown in the figure. Figure 1 In Figure 1 As shown in A.
[0062] 2. Functional identification of CDK8 gene
[0063] In order to study the role of CDK8 gene in plant salt tolerance, its function was compared and analyzed by Arabidopsis cdk8 mutant and CDK8 overexpression transgenic.
[0064] 2.1 Construction of overexpression materials
[0065] The target fragment obtained by PCR was cloned by Gateway recombination cloning technology BP reaction (Gateway TM BPClonase TMEnzyme Mix, Thermo Fisher) was connected to pDONR-207. Then, the LR reaction (Gateway TM LRClonase TM Enzyme Mix, Thermo Fisher) was cloned into the p35S::YFP plant overexpression vector. Named CDK8-YFP. Then transformed into Escherichia coli. Then, the bacterial solution containing the recombinant vector was identified by PCR ( Figure 1 B) Select the correct bacterial culture to extract plasmid and send it for sequencing.
[0066] 2.2 Screening of CDK8 overexpression transgenic positive strains
[0067] The constructed recombinant plasmid was transferred into Agrobacterium GV3101 by liquid nitrogen freeze-thaw method, shaken overnight, transferred and shaken to OD = 0.8, resuspended with transgenic buffer, infected Col-0 Arabidopsis inflorescence, protected from light for one day, cultured in a long-day light incubator, and harvested seeds were screened on 1 / 2MS solid medium containing herbicide (Basta). The screening process is as follows: surface disinfection with 10% sodium hypochlorite solution for 15 minutes, rinsed with sterile water 5 times, and vernalized at 4℃ for three days. Seeds were sown in 1 / 2MS medium with pH = 5.8 and containing a final concentration of 50μg / mL Basta. Cultured in a light incubator with 16h / 8h light and dark, 25℃, and 70% relative humidity. Using wild-type Arabidopsis as a control, the seedlings resistant to Basta were screened and moved to + medium (nutrient soil: vermiculite = 1:1) after 7 days and cultured under the above conditions. After harvesting T0 generation seeds, T1 positive plants were screened on 1 / 2MS medium containing herbicides. Proteins were extracted after true leaves were expanded, and positive plants CDK8-YFP#6 and CDK8-YFP#11 ( Figure 2 A). Harvest seeds and multiply them until the T3 generation is homozygous.
[0068] 2.3 Mutant homozygosity detection
[0069] Based on the sequence number of Arabidopsis T-DNA insertion mutants, the sequencing sequences were downloaded and synthesized from the mutant query website (http: / / signal.salk.edu / tdnaprimers.2.html). cdk8-1 (SALK_138675) is LP: 5'-TTGGTCTTGGCATCGATCTAC-3', RP: 5'-TTGGTGAAGGCACTTATGGTC-3'; cdk8-2 (SALK_016169) is LP: 5'-CACTATTCCGTGCTCTTCTGC-3', RP: 5'-TTTCCTGATCGTCGATTTTTG-3'. Homozygous identification was performed by PCR ( Figure 2 B).
[0070] 2.4 Phenotypic analysis of mutants and transgenic lines
[0071] In order to analyze the sensitivity of seed germination to salt in wild-type Arabidopsis thaliana strains (WT), cdk8 mutants and CDK8 overexpression transgenic lines. The following experimental method was used: freshly harvested seeds were surface disinfected with 10% sodium hypochlorite solution for 15 minutes, rinsed with sterile water 5 times, and vernalized at 4°C for three days. After that, germination experiments were carried out in 1 / 2MS medium and 1 / 2MS medium containing 75mM or 100mMNaCl, and the cotyledon greening rate was counted 7 days after seed germination. The results showed that on normal 1 / 2MS medium, the cotyledon greening rate of wild-type WT, cdk8 mutant and CDK8 overexpression line was roughly the same, almost 100%; under 75mM or 100mMNaCl treatment, compared with wild-type WT, the cotyledon greening rate of cdk8 mutant seeds was much lower than that of wild-type WT; and under 100mM NaCl treatment, the cotyledon greening rate of CDK8 overexpression line was significantly higher than that of wild-type WT ( Figure 3 A and Figure 3 B).
[0072] To analyze the sensitivity of taproot growth to salt in wild-type Arabidopsis thaliana (WT), cdk8 mutants, and CDK8 overexpressing transgenic lines, the following experimental method was used: freshly harvested seeds were surface sterilized with 10% sodium hypochlorite solution for 15 min, rinsed 5 times with sterile water, and germinated on 1 / 2MS medium for 3 days after vernalization at 4°C. After that, the taproot length was counted after being transferred to 1 / 2MS medium and 1 / 2MS medium containing 75mM or 100mM NaCl for 7 days. The results showed that on normal 1 / 2MS medium, the taproot length of the cdk8 mutant was shorter than that of the WT and CDK8 overexpression lines, and there was no significant difference in taproot length between the WT and CDK8 overexpression lines; under 75mM NaCl treatment, the taproot length of the cdk8 mutant was much shorter than that of the WT and CDK8 overexpression lines, and the taproot length of the CDK8 overexpression line was slightly longer than that of the WT but with no significant difference; under 100mM NaCl treatment, the taproot length of the cdk8 mutant was much shorter than that of the WT and CDK8 overexpression lines, and the taproot length of the CDK8 overexpression line was significantly longer than that of the WT ( Figure 4 A and B). To further analyze the sensitivity of the cdk8 mutant taproot to salt, we used the ratio of taproot length after stress to taproot length of normal growth to evaluate. The results showed that under 75mM or 100mM NaCl treatment, the relative taproot length ratio of the cdk8 mutant was lower than that of the WT and CDK8 overexpression lines, while the relative taproot length ratio of the CDK8 overexpression line was higher than that of the WT and cdk8 mutant ( Figure 4 B).
[0073] In order to analyze the sensitivity of wild-type Arabidopsis strains (WT), cdk8 mutants and CDK8 overexpression transgenic lines to salt in soil growth, the following experimental method was used: the sterilized vernalized seeds were transferred to a mixed soil containing nutrient soil: vermiculite = 1:1 after growing for 7 days in 1 / 2MS medium. Each pot had the same weight and contained 9 seedlings. They were cultured in a light incubator with a 16h / 8h light-dark cycle, 25℃, and 70% relative humidity until the water content reached 40%, and then treated with 200mM NaCl for 2 weeks. The survival rate was calculated after 1 week of recovery. The results showed that after salt treatment, the leaves of the cdk8 mutant turned yellow and the degree of dehydration was greater than that of the WT and CDK8 overexpression lines. After recovery, the survival rate of the cdk8 mutant was lower than that of the WT and CDK8 overexpression lines, while the survival rate of the CDK8 overexpression line was significantly higher than that of the WT and cdk8 mutants ( Figure 5 A and B).
[0074] The above results indicate that the CDK8 gene positively regulates the plant's salt stress response; overexpression of the CDK8 gene in plants helps improve the plant's salt tolerance.
[0075] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.
Claims
1. The application of CDK8 gene in improving plant salt tolerance is characterized by: The nucleotide sequence of the CDK8 gene is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.
2. The use according to claim 1, characterized in that: By constructing a CDK8 overexpression vector, salt-tolerant transgenic plants were obtained.
3. The use according to claim 1, characterized in that: The salt tolerance is manifested as: (1) Under salt stress, the seed germination rate of the cdk8 mutant was lower than that of the wild type, while the seed germination rate of the CDK8 overexpression line was higher than that of the wild type; (2) Under salt stress, the cotyledon greening rate of the cdk8 mutant was lower than that of the wild type; while the cotyledon greening rate of the CDK8 overexpression line was higher than that of the wild type; (3) Under salt stress, the relative taproot length of the cdk8 mutant was shorter than that of the wild type, while the relative taproot length of the CDK8 overexpression line was longer than that of the wild type. (4) Under salt stress, the survival rate of the cdk8 mutant was lower than that of the wild type, while the survival rate of the CDK8 overexpression line was higher than that of the wild type.
4. A method for improving plant salt tolerance, characterized in that: The method is the following (1) or (2): (1) By increasing the activity of CDK8 protein in the target plant, a plant with stronger salt tolerance than the target plant is obtained; (2) Promoting the expression of the CDK8 gene in the target plant to obtain a plant with stronger salt tolerance than the target plant; The nucleotide sequence of the CDK8 gene is shown in SEQ ID NO.1, the amino acid sequence of the CDK8 protein is shown in SEQ ID NO.2, and the target plant is Arabidopsis thaliana.