Chinese cabbage bolting-resistant gene BraDREB1D and application thereof

Abstract

The invention belongs to the technical field of plant heredity and genetic engineering, and particularly relates to a Chinese cabbage bolting-resistant gene BraDREB1D and application thereof. Specifically, a low-temperature-resistant key transcription factor BraDREB1D (BraA10g010540.3 C) induced by low temperature is found through combination of a yeast stress library screening technology and transcriptome analysis, belongs to an AP2 type transcription factor, and has a relatively close genetic relationship with arabidopsis thaliana CBF4 / DREB1D in evolution, so that the key transcription factor is named as BraDREB1D. Experiments prove that the key transcription factor can effectively improve the cold resistance of plants, and the application of the key transcription factor BraDREB1D in the field of plant growth. Therefore, the method can be used for improving the quality and yield of plants (such as Chinese cabbages). Meanwhile, the BraDREB1D is used as the marker, so that the performance of plant germplasm, especially the germplasm performance of Chinese cabbage, can be identified, and therefore, the BraDREB1D has a good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetics and genetic engineering, and particularly relates to a Chinese cabbage bolting-resistant gene BraDREB1D and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Chinese cabbage (Brassica rapa L.ssp. Pekinensis) is a semi-cold-tolerant vegetable crop, with an optimal growing temperature of 12-22°C. However, growth and development are inhibited in early winter for autumn-sown cabbage, or early spring for spring-sown cabbage. The plant transitions from a vegetative state to a dormant state, where nutrients are transported from the outer leaves to the bulbs. Flower buds differentiate slowly at the reproductive apex, and bolting begins after the plant returns to green when temperatures are favorable. This impacts the plant's growth and yield. Breeding bolting-tolerant Chinese cabbage varieties in my country began relatively late, and most bolting-tolerant varieties currently on the market come from South Korea and Japan. Therefore, identifying key genes for cold tolerance in Chinese cabbage is crucial for developing new bolting-tolerant varieties through biobreeding. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the present invention aims to provide a Chinese cabbage bolting-tolerance gene, BraDREB1D, and its applications. Specifically, a yeast stress library screening technique combined with transcriptome analysis identified a key low-temperature-tolerant transcription factor, BraDREB1D (BraA10g010540.3C), induced by low temperatures. BraDREB1D is an AP2-type transcription factor and evolutionarily closely related to Arabidopsis thaliana CBF4 / DREB1D, hence its designation as BraDREB1D. Experiments have demonstrated that BraDREB1D can effectively improve plant cold tolerance and inhibit the growth of plant stalks, thus demonstrating its potential as a bolting-tolerance gene. Based on these research findings, the present invention was completed.

[0005] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0006] The first aspect of the present invention provides the use of at least one of the following a1) to a5) in regulating plant bolting resistance;

[0007] a1) BraDREB1D protein;

[0008] a2) proteins in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the BraDREB1D protein and which have the same or similar functions as BraDREB1D;

[0009] a3) a nucleic acid molecule encoding the protein described in a1) or a2);

[0010] a4) a nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of the nucleic acid molecule in a3) and which can encode a protein with the same or similar functions;

[0011] a5) A substance capable of regulating the level or activity of at least one of a1) to a5).

[0012] In the present invention, the amino acid sequence of the BraDREB1D protein is shown in SEQ ID NO.1;

[0013] The sequence of the nucleic acid molecule encoding the protein shown in SEQ ID NO.1 is shown in SEQ ID NO.2.

[0014] In the present invention, the regulation includes both positive and negative regulation. Experiments have shown that overexpressing the BraDREB1D gene, promoting BraDREB1D gene transcription, increasing BraDREB1D protein levels, or promoting BraDREB1D protein activity are beneficial for improving plant cold tolerance and bolting resistance (i.e., inhibiting plant stalk growth).

[0015] A second aspect of the present invention provides a formulation for improving plant bolting resistance, comprising at least one of the following b1) to b6):

[0016] b1) BraDREB1D protein or a nucleic acid molecule encoding BraDREB1D protein;

[0017] b2) an expression vector comprising a nucleic acid encoding a BraDREB1D protein;

[0018] b3) a recombinant host containing b2);

[0019] b4) a promoter or enhancer that increases BraDREB1D gene expression;

[0020] b5) inducers that promote BraDREB1D gene expression;

[0021] b6) A preparation that enhances the activity of BraDREB1D protein.

[0022] In the preparation of the present invention, the amino acid sequence of the BraDREB1D protein is shown in SEQ ID NO. 1. The nucleotide sequence of the nucleic acid molecule encoding the BraDREB1D protein is shown in SEQ ID NO. 2.

[0023] The third aspect of the present invention provides a method for improving plant bolting resistance, which comprises using the preparation of the present invention to increase the level and / or activity of endogenous BraDREB1D protein in plants, or causing plants that do not contain BraDREB1D or have inactivated BraDREB1D to express BraDREB1D protein.

[0024] In some embodiments, the method of causing a plant that does not contain BraDREB1D or has inactivated BraDREB1D to express a BraDREB1D protein comprises:

[0025] A vector containing a nucleic acid encoding BraDREB1D protein is constructed and transformed into Agrobacterium; and the seeds or explants of crops are infected with the Agrobacterium.

[0026] In addition, BraDREB1D can also be used as a marker for screening bolting-resistant plants in plant breeding.

[0027] A fourth aspect of the present invention provides a preparation for screening plants resistant to bolting, comprising:

[0028] Preparations for detecting BraDREB1D gene transcription levels;

[0029] and / or detecting the expression level or active preparation of BraDREB1D protein.

[0030] A fifth aspect of the present invention is the use of the preparation of the present invention in screening plants resistant to bolting.

[0031] A sixth aspect of the present invention provides a method for screening plants resistant to bolting, comprising detecting the BraDREB1D gene transcription level or the BraDREB1D protein expression level or activity in the plant using the preparation of the present invention.

[0032] The detection described herein includes detection of expression or activity levels. For example, Western blot analysis can be used to detect BraDREB1D protein expression levels, while PCR analysis can be used to detect BraDREB1D gene transcription levels. Using the identification method described herein, bolting resistance can be predicted by simply testing young plant tissues, shortening the breeding cycle.

[0033] In the present invention, the plant may be a Cruciferae plant; the plants used to verify the function of BraDREB1D in the present invention include Cruciferae plants, such as Chinese cabbage, Arabidopsis thaliana, etc.

[0034] Beneficial technical effects of one or more of the above technical solutions:

[0035] Research in this study demonstrates that the BraDREB1D gene or protein, an AP2 transcription factor derived from Chinese cabbage, can enhance plant cold tolerance and regulate plant bolting resistance, thus potentially improving plant quality and yield, such as in Chinese cabbage. Using BraDREB1D as a marker enables the identification of plant germplasm, particularly Chinese cabbage germplasm, and therefore has significant practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0037] Figure 1 This is the phylogenetic tree of DREB genes.

[0038] Figure 2 This is the fluorescence quantitative detection of the Arabidopsis strain overexpressing BraDREB1D in the examples of the present invention.

[0039] Figure 3 This is the effect of heterologous overexpression of BraDREB1D on the bolting resistance of Arabidopsis thaliana in the examples of the present invention.

[0040] Figure 4 The effect of heterologous overexpression of BraDREB1D on the cold tolerance of Arabidopsis thaliana in the embodiment of the present invention is shown in FIG.

[0041] A: Electrolyte leakage rate; B: Maximum photochemical efficiency of PSⅡ under dark adaptation; C: Relative expression levels of cold stress-related genes. DETAILED DESCRIPTION

[0042] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0043] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this manual, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. The present invention has used conventional techniques and methods in the field of genetic engineering and molecular biology. Those skilled in the art can adopt other conventional techniques, methods and reagents in this area on the basis of the embodiments provided by the present invention, without being limited to the limitation of the specific embodiments of the present invention.

[0044] In the present invention, the nucleic acid molecule encoding the BraDREB1D protein includes genomic DNA, cDNA, recombinant DNA, mRNA, or hnRNA encoding the BraDREB1D protein; or a nucleic acid molecule that is reverse complementary to the above DNA, cDNA, recombinant DNA, or mRNA.

[0045] The above nucleic acid molecules can be modified or optimized according to actual needs to make gene expression more efficient; for example, (1) the codons of the BraDREB1D gene of the present invention can be changed to conform to the preferences of the recipient plant while maintaining the amino acid sequence of the gene according to the present invention. (2) It can be linked to various plant-expressed promoters to facilitate its expression in plants; the promoters can include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression and also depends on the target species; (3) enhancer sequences, such as intron sequences and viral leader sequences, can be introduced.

[0046] In the present invention, the vector may be a plasmid, cosmid, phage or viral vector. The host may be a fungus, bacterium, algae or cell. The bacterium is preferably Agrobacterium.

[0047] For plants that do not contain BraDREB1D, BraDREB1D gene fragments can be introduced into plant cells by chemical methods, shotgun methods, microinjection, electroporation and other methods. BraDREB1D gene fragments can also be introduced into plant cells by homologous recombination, zinc finger nuclease, TALEN, CRISPR and other methods, which are not specifically limited here.

[0048] The present invention is described in detail below with reference to the accompanying drawings and specific examples. In the following examples, the materials and reagents used were obtained from commercial sources unless otherwise specified. The experimental methods described are conventional methods in the art unless otherwise specified.

[0049] Example

[0050] 1. Test methods

[0051] 1. Gene cloning and expression vector construction

[0052] 1) Using genomic cDNA as a template, specific primers are designed according to the sequence of the target gene for PCR amplification to obtain the target gene fragment.

[0053] 2) Digest the vector plasmid according to the restriction endonuclease reaction temperature.

[0054] 3) Use homologous recombinase to connect the linearized vector with the PCR product of the target fragment.

[0055] 4) The ligation product was transformed into DH5α competent cells and plated on LB medium for resistance screening to obtain positive clones containing the recombinant plasmid.

[0056] 5) The positive clones were identified by PCR and sequenced, and then the plasmids of the positive clones with correct sequencing were extracted.

[0057] 2. Arabidopsis transformation using the inflorescence infiltration method

[0058] 1) Select a suitable Agrobacterium strain (LBA4404), introduce the recombinant plasmid containing the target gene into the Agrobacterium, and screen positive clones with antibiotics. Cultivate the Agrobacterium until the logarithmic growth phase, collect the cells and resuspend them in infection medium (MS liquid medium containing surfactant Silwet L-77 and a certain concentration of sucrose), and adjust the bacterial concentration to OD 600 About 0.8.

[0059] 2) When the Arabidopsis thaliana is in full bloom, immerse the entire inflorescence in the Agrobacterium solution and gently shake it to ensure even contact with the solution. Soak for 5 minutes.

[0060] 3) The dipped Arabidopsis plants were placed in the dark for 16-24 hours for co-cultivation, and then the plants were transferred to a growth chamber for cultivation under normal temperature and light.

[0061] 3. Identification of Transformed Arabidopsis Plants

[0062] 1) qRT-PCR

[0063] Total RNA was extracted from transgenic Arabidopsis thaliana and reverse-transcribed into cDNA according to the kit instructions. qRT-PCR reactions were performed using the cDNA as a template, using specific quantitative primers and a fluorescent dye. Expression of the target gene in transgenic plants was compared with that in non-transgenic plants to confirm expression.

[0064] 2) Phenotypic level identification

[0065] With WT as the control, the positive transgenic plants were transplanted into nutrient soil and cultured normally in the growth chamber for about 30 days to observe the bolting morphological characteristics of the transgenic Arabidopsis plants; then the plants were transferred to a 4°C low-temperature incubator. After 3 days of low-temperature treatment, the relevant physiological and biochemical indicators of the transgenic Arabidopsis were measured.

[0066] 2. Test results

[0067] To verify the biological function of BraDREB1D, we constructed an overexpression vector for BraDREB1D and heterologously expressed BraDREB1D in Arabidopsis thaliana by floral transformation, and obtained three OE-BraDREB1D lines ( Figure 2).

[0068] To verify the biological function of BraDREB1D, we constructed an overexpression vector for BraDREB1D and heterologously expressed BraDREB1D in Arabidopsis thaliana by floral transformation, and obtained three OE-BraDREB1D lines ( Figure 2 ).

[0069] In addition, the cold tolerance of WT and OE-BraDREB1D plants was analyzed at room temperature (22℃) and low temperature (4℃, 3d). Figure 4 The results showed that there was no significant difference between WT and OE-BraDREB1D at room temperature. However, at low temperatures, the OE-BraDREB1D phenotype was less damaged than the WT. The Fv / Fm ratio of OE-BraDREB1D was significantly higher than that of WT, and the EL was significantly lower than that of WT. qPCR revealed that OE-BraDREB1D significantly increased the expression of cold-stress-responsive genes. This suggests that BraDREB1D can enhance plant cold tolerance and further suggests that it may play a role in regulating the bolting resistance of Chinese cabbage.

[0070] The nucleotide / amino acid sequence information of the present invention

[0071] >BraDREB1D protein

[0072] MDPFYTSFSDSFLSIPDHRSPVSDSSECSPKLASSCPKKRAGRKKFRETRHPIYRGVRQRNSGKWVCEVREPNKKSRIWLGTFPTVEMAARAHDVAALALRGRSACLNFADS AWRLRIPESTCPKEIQRAAAEAAMAFQKETATTETTMVEAVKPAEETVGQTGGETTEENGVFYMDEEAGFGMPRFLENMAEEMLLPPPELGWNHNDLTGDADVSLWSF*(SEQ ID NO.1)

[0073] >BraDREB1D gene

[0074] (SEQ ID NO.2)

[0075] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of at least one of a1) to a5) in regulating plant bolting resistance; a1) BraDREB1D protein; a2) proteins in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the BraDREB1D protein and which have the same or similar functions as BraDREB1D; a3) a nucleic acid molecule encoding the protein described in a1) or a2); a4) a nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of the nucleic acid molecule in a3) and which can encode a protein with the same or similar functions; a5) A substance capable of regulating the level or activity of at least one of a1) to a5). The amino acid sequence of the BraDREB1D protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The sequence of the nucleic acid molecule encoding the protein shown in SEQ ID NO.1 is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The plant is a cruciferous plant, preferably Chinese cabbage.

4. A preparation for improving plant resistance to bolting, characterized in that Including at least one of the following b1) to b6): b1) BraDREB1D protein or a nucleic acid molecule encoding BraDREB1D protein; b2) an expression vector comprising a nucleic acid encoding a BraDREB1D protein; b3) a recombinant host containing b2); b4) a promoter or enhancer that increases BraDREB1D gene expression; b5) inducers that promote BraDREB1D gene expression; b6) Preparations that increase the activity of BraDREB1D protein; The amino acid sequence of BraDREB1D protein is shown in SEQ ID NO.

1.

5. The preparation according to claim 4, wherein The nucleotide sequence of the nucleic acid molecule encoding the BraDREB1D protein is shown in SEQ ID NO.

2.

6. A method for improving plant bolting resistance, characterized in that: The preparation according to any one of claims 4 to 5 is used to increase the level and / or activity of endogenous BraDREB1D protein in a plant, or to make a plant that does not contain BraDREB1D or has inactivated BraDREB1D express BraDREB1D protein.

7. The method according to claim 6, wherein The method of causing a plant that does not contain BraDREB1D or has inactivated BraDREB1D to express BraDREB1D protein comprises: A vector containing a nucleic acid encoding BraDREB1D protein is constructed and transformed into Agrobacterium; and the seeds or explants of crops are infected with the Agrobacterium.

8. A preparation for screening plants resistant to bolting, characterized in that: The preparation comprises: Preparations for detecting BraDREB1D gene transcription levels; and / or detecting the expression level or active preparation of BraDREB1D protein.

9. Use of the preparation according to claim 8 in screening plants resistant to bolting.

10. A method for screening plants resistant to bolting, characterized in that: The preparation according to claim 8 is used to detect the transcription level of the BraDREB1D gene or the expression level or activity of the BraDREB1D protein in the plant.

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