Cold-resistant and bolting-resistant related gene BraTZF3 of Chinese cabbage and application thereof

Abstract

The invention belongs to the technical field of plant heredity and genetic engineering, and particularly relates to a cold-resistant and bolting-resistant related gene BraTZF3 of Chinese cabbage and application of the cold-resistant and bolting-resistant related gene BraTZF3. A stress-related transcription factor BraA01g008400.3 C is screened by combining a yeast stress library screening technology with transcriptome analysis, the stress-related transcription factor BraA01g008400.3 C belongs to a co-tandem C3H type zinc finger protein and comprises a C-x8-C-x5-C-x3-H zinc finger (ZNF) structural domain, and tests prove that the BraTZF3 is positively correlated to low-temperature stress response and cold resistance, and the BraA01g008400.3 C can be used for preparing the stress-related transcription factor BraA01g008400.3 C. According to the present invention, the BraTZF3 is subjected to heterologous overexpression to positively regulate the cold resistance and the bolting resistance of arabidopsis thaliana, such that the gene provides the positive regulation effect on the cold resistance and the bolting resistance of the plant, especially the Chinese cabbage, and the new tool and the new method are provided for improving the cold resistance and the bolting resistance of the crop by using the gene engineering means so as to provide the 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 cold-resistance and bolting-resistance related gene BraTZF3 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] Low temperature stress is an important environmental factor that restricts plant growth and significantly affects the physiological metabolism and yield formation of crops. As a cool-loving vegetable, Chinese cabbage (Brassica rapa L.ssp.Pekinensis) has basic cold resistance, but when the ambient temperature is below the critical value of 5°C, its nutritional growth process is hindered, which is manifested as delayed heading and stagnant biomass accumulation. Under low temperature adversity, the cell membrane system of Chinese cabbage is damaged, and the content of malondialdehyde (MDA), a membrane lipid peroxidation product, increases sharply; the activity of key antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD) is inhibited, resulting in a weakened ability to scavenge reactive oxygen species (ROS) and an imbalance in the redox balance in the cell. At the same time, low temperature stress destroys the integrity of the photosynthetic membrane system, hinders light energy capture and electron transfer, and significantly reduces photosynthetic efficiency, which directly affects the carbon assimilation capacity of the plant.

[0004] Currently, research on the molecular mechanisms for improving the cold tolerance of Chinese cabbage is relatively weak, and the key regulatory genes have not yet been systematically analyzed. It is worth noting that encountering low temperatures during the heading period will trigger the physiological dormancy of Chinese cabbage, forcing it to start the reproductive growth program early, and consume the organic nutrients stored in the head to differentiate and form flower buds. This abnormal developmental pattern seriously damages the yield and quality of commercial head. Existing studies have shown that growth and development-related genes such as photoperiod response and flowering regulation may be involved in regulating the low-temperature bolting process of Chinese cabbage. Therefore, the discovery of Chinese cabbage's cold-tolerance and bolting-tolerance genes is of great significance for the next step of breeding new bolting-resistant Chinese cabbage varieties through biological breeding methods. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide BraTZF3, a gene related to cold tolerance and bolting resistance in Chinese cabbage, and its application. Specifically, the present invention screened a stress-related transcription factor BraA01g008400.3C through yeast stress library screening technology combined with transcriptome analysis. It belongs to a co-tandem C3H type zinc finger protein, containing a C-x8-C-x5-C-x3-H zinc finger (ZNF) domain, and has a close genetic relationship with Arabidopsis OXIDATION-RELATED ZINC FINGER 2 (AtTZF3, At4g29190), so it is named BraTZF3. Experiments have shown that it plays a (positive) regulatory role in regulating cold tolerance and bolting resistance in Chinese cabbage. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0007] In a first aspect of the present invention, a Chinese cabbage zinc finger protein gene is provided, which is named BraTZF3. The Chinese cabbage zinc finger protein gene is selected from:

[0008] (a1) the nucleotide sequence shown in SEQ ID NO. 1;

[0009] (a2) a nucleotide sequence that encodes a protein having the same amino acid sequence as the nucleotide sequence of (a1) but differs in sequence due to the degeneracy of the genetic code;

[0010] (a3) a nucleotide sequence having ≥90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) with the nucleotide sequence shown in (a1) or (a2) and encoding a nucleotide sequence having the same or similar functional protein;

[0011] (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

[0012] The second aspect of the present invention provides a zinc finger protein, which is encoded by the above-mentioned Chinese cabbage zinc finger protein gene.

[0013] Furthermore, the zinc finger protein has:

[0014] (b1) the amino acid sequence shown in SEQ ID NO. 2;

[0015] (b2) a protein derived from (b1) which has the same function as (b1) but has one or more amino acid residues substituted and / or deleted and / or added;

[0016] (b3) Proteins encoded by other genes that have an amino acid sequence identity of at least 90% with the amino acid sequence shown in (b1) and have the same or similar activity as the zinc finger protein shown in (b1).

[0017] Furthermore, the proteins in (b1)-(b3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0018] The third aspect of the present invention provides a recombinant expression vector, a transgenic cell line, a host bacteria or a transgenic plant containing the above-mentioned Chinese cabbage zinc finger protein gene.

[0019] In a specific embodiment of the present invention, the recombinant expression vector is obtained by connecting the Chinese cabbage zinc finger protein gene to an expression vector, and the expression vector can be any one or more of a viral vector, a plasmid, a phagemid, a cosmid, or an artificial chromosome.

[0020] The transgenic cell line can be isolated, in vitro, cultured, or preferably a part of a plant; wherein the plant cell can be a crop cell, the crop is a dicotyledonous crop, further a cruciferous crop, and Chinese cabbage is preferred.

[0021] The host bacteria can be eukaryotic or prokaryotic, such as bacteria, fungi, actinomycetes, etc.

[0022] Furthermore, the bacteria may be from the genera Escherichia, Flavobacterium, Agrobacterium, Pseudomonas, Bacillus, etc., and further may be, for example, Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis, or Bacillus pumilus. The fungus may be yeast. The fungus may be from the genera Fusarium, Verticillium, Aspergillus, Cephalosporium, etc. The actinomycetes may be from the genera Streptomyces, Nocardia, Neurospora, etc.

[0023] The transgenic plant may be a transgenic crop, more preferably a dicotyledonous crop, further preferably a cruciferous crop, with Chinese cabbage being preferred.

[0024] A fourth aspect of the present invention provides the use of the Chinese cabbage zinc finger protein gene, zinc finger protein, recombinant expression vector containing the Chinese cabbage zinc finger protein gene, transgenic cell line, host bacteria or transgenic plant in any one or more of the following:

[0025] (c1) regulating plant cold tolerance and / or bolting resistance;

[0026] (c2) Improving and breeding plants.

[0027] Among them, in said (c1), the regulation of plant cold tolerance and / or bolting resistance: promoting (increasing) the expression of the Chinese cabbage zinc finger protein gene BraTZF3 in the plant, thereby promoting / increasing the plant cold tolerance and bolting resistance (manifested as heterologous overexpression of BraTZF3 positively regulating the cold tolerance and bolting resistance of Arabidopsis thaliana).

[0028] In said (c2), improving and cultivating plants specifically refers to improving and cultivating plant varieties with cold tolerance and / or bolting tolerance; said plants may be crops, more preferably dicotyledonous crops, further preferably cruciferous crops, among which cabbage is preferred.

[0029] A fifth aspect of the present invention provides a method for improving and cultivating cold-tolerant and / or bolting-tolerant plants, the method comprising increasing the expression level and / or activity of the BraTZF3 gene in the target plant.

[0030] In the above method, the expression level and / or activity of the BraTZF3 gene in the target plant can be increased by, for example, introducing a recombinant expression vector containing the BraTZF3 gene, operably linking a strong promoter to the BraTZF3 gene, and introducing an enhancer, etc., which are not specifically limited here.

[0031] The target plant can be any plant at any developmental stage, and in particular, the plant is a crop, more preferably a dicotyledonous crop, with Chinese cabbage being preferred. The above method can achieve the cultivation and improvement of crops, especially Chinese cabbage with cold tolerance and / or bolting tolerance.

[0032] Beneficial technical effects of one or more of the above technical solutions:

[0033] The above technical scheme uses yeast stress library screening technology combined with transcriptome analysis to screen out a stress-related transcription factor BraA01g008400.3C, which belongs to a co-tandem C3H type zinc finger protein and contains a C-x8-C-x5-C-x3-H zinc finger (ZNF) domain. Experimental verification shows that BraTZF3's response to low temperature stress is positively correlated with cold tolerance, and heterologous overexpression of BraTZF3 positively regulates the cold tolerance and bolting resistance of Arabidopsis, indicating that this gene plays a positive regulatory role in the cold tolerance and bolting resistance of plants, especially Chinese cabbage, providing new tools and methods for improving the cold tolerance and bolting resistance of crops using genetic engineering means, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 This is a diagram showing the phylogenetic relationship between TZF genes in Chinese cabbage and Arabidopsis thaliana.

[0036] Figure 2 This is the effect of low temperature on the expression of BraTZF3 gene in Chinese cabbage in the examples of the present invention.

[0037] Figure 3 This is the fluorescence quantitative detection of the Arabidopsis strain overexpressing BraTZF3 in the examples of the present invention.

[0038] Figure 4 The effect of heterologous overexpression of BraTZF3 on the cold tolerance of Arabidopsis thaliana in the embodiment of the present invention is shown in FIG. 1 ; wherein A is the maximum photochemical efficiency (F) of PSⅡ under dark adaptation in different treatment groups under different temperature conditions. v / F m ); B is the electrolyte leakage rate of different treatment groups under different temperature conditions; C is the effect of different treatment groups on the expression of cold stress response genes.

[0039] Figure 5 This is the effect of heterologous overexpression of BraTZF3 on the bolting resistance of Arabidopsis thaliana in the examples of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Example

[0044] 1. Test methods

[0045] 1. RNA Extraction and Fluorescence Quantification

[0046] Total RNA was extracted from the leaves or roots of the seedlings using the Trizol method, and the concentration and quality of mRNA were determined using a micro-UV spectrophotometer (KAIAO, China).

[0047] 1) Take 0.1-0.2g of plant material in a pre-cooled mortar and add liquid nitrogen to grind thoroughly.

[0048] 2) Transfer the ground powder to an RNase-free centrifuge tube, add 1 mL of Trizol, and shake thoroughly.

[0049] 3) Let stand at room temperature for 5 minutes.

[0050] 4) For every 1 mL of Trizol used, add 0.2 mL of chloroform and shake vigorously for 15 seconds.

[0051] 5) Centrifuge at 10,000 × g at 2-8°C for 15 min and transfer the upper aqueous phase to a new centrifuge tube.

[0052] 6) Add 0.5 mL of isopropanol, mix thoroughly by inversion, and incubate at -20°C for 20 min.

[0053] 7) Centrifuge at 10,000 × g at 2-8°C for 10 min and discard the supernatant.

[0054] 8) Add 1 mL of 75% anhydrous ethanol (prepared with DEPC water), vortex vigorously, and centrifuge at 7500×g at 2-8°C for 5 min.

[0055] 9) Remove the supernatant, air-dry the pellet at room temperature, and dissolve the pellet in 50-100 μL RNase-free water.

[0056] Perform mRNA reverse transcription and gene expression analysis according to the instructions of the reverse transcription kit and real-time fluorescence quantitative PCR kit. The specific procedures are as follows:

[0057] 1) Reverse transcription

[0058]

[0059] Mix gently, incubate at 37°C for 2 min, 50°C for 15 min, and 85°C for 5 sec.

[0060] 2) Prepare qRT-PCR reaction system

[0061]

[0062] 3) qRT-PCR reaction conditions

[0063]

[0064] The fluorescence signal of the gene was monitored using a Light Cycler 96 real-time fluorescence quantitative PCR instrument (Roche, Switzerland). -ΔΔCT The relative expression level of each target gene was calculated using the formula.

[0065] 2. Gene cloning and expression vector construction

[0066] 1) The PCR reaction system for gene cloning is as follows:

[0067]

[0068]

[0069] The reaction conditions are:

[0070]

[0071] 2) The vector plasmid is digested according to the restriction endonuclease reaction temperature. The reaction system is as follows:

[0072]

[0073] 3) Detect the target band by 1.0% agarose gel electrophoresis and recover the fragment by gel excision.

[0074] 4) Use homologous recombinase to connect the linearized vector and the target fragment:

[0075]

[0076] 5) The ligation product was transformed into DH5α competent cells and plated on LB medium for resistance screening at 37°C.

[0077] After 12 h of culture, positive clones were screened by PCR and sequenced, and plasmids were extracted from positive clones with correct sequencing results.

[0078] 3. Arabidopsis Transformation and Identification

[0079] 1) Thaw competent Agrobacterium on ice and add 0.1-0.5 μg plasmid DNA per 20 μL competent culture.

[0080] 2) Gently shake to mix. Incubate on ice, in liquid nitrogen, in a 37°C water bath, and on ice for 5 min each. Add LB medium and incubate at 28°C with shaking for 2-3 h.

[0081] 3) After centrifugation, retain 100 μL of resuspended cells and apply to 25 μg mL -1 Kan and 50 μg mL -1 The cells were cultured on Rif's YEP solid medium at 28°C for 48 h, and positive clones were screened by PCR.

[0082] 4) Inflorescence Dip Transformation of Arabidopsis: After the Arabidopsis inflorescence has formed, add 2 mL of activated Agrobacterium containing the expression vector plasmid to 250 mL of YEP medium containing resistance selection medium and incubate with shaking overnight at 28°C. Collect the cells and resuspend them in transformation medium to an OD600 of 0.8. Immerse the inflorescence in the dipping solution for 5 minutes, incubate in the dark for one day, and then incubate in a constant temperature incubator at 20°C.

[0083] 5) Screening and identification of transgenic lines: Transgenic Arabidopsis seeds were screened using a resistance culture medium, and seedlings that grew normally on the resistance culture medium were selected for planting until the seeds were homozygous (approximately T4 generation).

[0084] 2. Test results

[0085] (1) BraTZF3 response to low temperature stress is positively correlated with cold tolerance

[0086] Two high-generation Chinese cabbage inbred lines, 'Y2' (cold-sensitive) and 'Y7' (cold-tolerant), with significant differences in cold tolerance, were used as experimental materials to determine the response of BraTZF3 to low temperature stress. The results showed that low temperature could significantly induce the expression of BraTZF3, and the expression level of BraTZF3 in 'Y7' was always significantly higher than that in 'Y2' (cold-sensitive). Figure 2 ).

[0087] (2) Heterologous overexpression of BraTZF3 positively regulates cold tolerance and bolting resistance in Arabidopsis

[0088] To verify the biological function of BraTZF3, we constructed a BraTZF3-pCAMBIA3300 overexpression vector and heterologously expressed it in Arabidopsis thaliana by genetic transformation, and obtained three OE-BraTZF3 lines ( Figure 3 ).

[0089] Using WT and OE-BraTZF3 as experimental materials, samples were taken from plants treated at room temperature (22°C) and low temperature (4°C, 3 days) to identify their cold tolerance and analyze the expression of cold stress response genes. Figure 4 The results showed that there was no significant difference between WT and OE-BraTZF3 under normal temperature conditions. Under low temperature conditions, the plants showed slow growth, wilting and water loss of leaves. Compared with WT, the OE-BraTZF3 phenotype was less damaged. The maximum photochemical efficiency of PSⅡ (F v / F m) was significantly higher than that of WT, and the electrolyte leakage rate of OE-BraTZF3 was significantly lower than that of WT. qPCR results showed that OE-BraTZF3 significantly increased the expression levels of cold stress-responsive genes (AtCBF1, AtCBF2, AtCBF3, AtICE1, AtKIN1, AtCOR47, AtRD29A). These results indicate that BraTZF3 can positively regulate plant cold tolerance.

[0090] Furthermore, heterologous overexpression of BraTZF3 in Arabidopsis significantly inhibited the growth of Arabidopsis inflorescences ( Figure 5 ), indicating that BraTZF3 is a bolting-resistant gene in Chinese cabbage.

[0091] The nucleotide / amino acid sequence information of the present invention

[0092] >BraTZF3

[0093]

[0094] >BraTZF3

[0095] MMIGDTRRTYPTVEIPTWPVSEDFTAGDVYSPVMNSPDCSMLEALAALQRYLPSNEPDPDSDPDLFGPDSPIDAYSCDHFRMYDFKVRRCARGRSHDWTECPYAHPGEKARRRDPRKYSYSGTACPDFRKGGCVKGDSCEFAHGVFECWLHPARYRTQPCKDGGNCRRRVCFFAHSPDQLRYL PNRSPDRVDSFDVSSPMRHSCARAFQLSISPVSGSPPVSPRADSGSQSLSRSLGSNSINDVVSSFRNLQFEKVKSFPPSYNNPLRCYQSGFGSPRGSILGPGFQSLPTTPTRPGNMDIWENGIEEEPAMERVVESGRELRAKMFEKLSKENCMDRVEPDPDHNSGEGPDVEWVSELVM*(SEQ IDNO.2)

[0096] 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. A Chinese cabbage zinc finger protein gene, named BraTZF3, characterized in that: The Chinese cabbage zinc finger protein gene is selected from: (a1) the nucleotide sequence shown in SEQ ID NO. 1; (a2) a nucleotide sequence that encodes a protein having the same amino acid sequence as the nucleotide sequence of (a1) but differs in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes a nucleotide sequence having the same or similar functional protein; (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

2. A zinc finger protein, characterized in that The zinc finger protein is encoded by the Chinese cabbage zinc finger protein gene according to claim 1; Furthermore, the zinc finger protein has: (b1) the amino acid sequence shown in SEQ ID NO. 2; (b2) a protein derived from (b1) which has the same function as (b1) but has one or more amino acid residues substituted and / or deleted and / or added; (b3) Proteins encoded by other genes that have an amino acid sequence identity of at least 90% with the amino acid sequence shown in (b1) and have the same or similar activity as the zinc finger protein shown in (b1).

3. A recombinant expression vector, transgenic cell line, host bacteria or transgenic plant containing the Chinese cabbage zinc finger protein gene according to claim 1.

4. The recombinant expression vector, transgenic cell line, host bacteria or transgenic plant according to claim 3, wherein: The recombinant expression vector is obtained by connecting the Chinese cabbage zinc finger protein gene according to claim 1 to an expression vector; The transgenic cell line is isolated, in vitro, cultured, or preferably a part of a plant; wherein the plant cell may be a crop cell, and the crop is a dicotyledonous crop, further Chinese cabbage; The host bacteria are eukaryotic or prokaryotic bacteria, such as bacteria, fungi, actinomycetes, etc. The transgenic plant is a transgenic crop, and the crop is a dicotyledonous crop, further comprising Chinese cabbage.

5. Use of the recombinant expression vector, transgenic cell line, host bacteria or transgenic plant of the Chinese cabbage zinc finger protein gene according to claim 1, the Chinese cabbage zinc finger protein according to claim 2 or the Chinese cabbage zinc finger protein gene according to claim 3 in any one or more of the following: (c1) regulating plant cold tolerance and / or bolting resistance; (c2) Improving and breeding plants.

6. The use according to claim 5, characterized in that In (c1), the regulation of plant cold tolerance and / or bolting resistance is specifically manifested as: promoting (increasing) the expression of the Chinese cabbage zinc finger protein gene BraTZF3 in the plant, thereby promoting / increasing the plant cold tolerance and bolting resistance.

7. The use according to claim 5, characterized in that In the above-mentioned (c2), improving and cultivating plants specifically refers to improving and cultivating plant varieties with cold tolerance and / or bolting tolerance.

8. A method for improving and cultivating cold-tolerant and / or bolting-tolerant plants, characterized in that: The method comprises increasing the expression level and / or activity of the Chinese cabbage zinc finger protein gene BraTZF3 in the target plant.

9. The method according to claim 8, wherein: The method for increasing the expression level and / or activity of the BraTZF3 gene in the target plant comprises introducing a recombinant expression vector containing the BraTZF3 gene, operably linking a strong promoter to the BraTZF3 gene, and introducing an enhancer.

10. The method according to claim 8, wherein The plant is a crop, further a dicotyledonous crop, and further Chinese cabbage.

Images