A CrBZR1 gene and its application

Abstract

The present invention belongs to the field of plant genetic engineering technology, and specifically relates to a CrBZR1 gene and its application. The present invention provides a CrBZR1 gene, and the nucleotide sequence of the CrBZR1 gene of the present invention is shown in SEQ ID NO.1. The present invention promotes the expression of the CrBZR1 gene in the plant by overexpressing the CrBZR1 gene in the plant, increases the content of the encoded protein, and thus improves the cold resistance of the plant; at the same time, the present invention significantly reduces the cold resistance of the plant by silencing the expression of the CrBZR1 gene in the plant. This technical effect also verifies the function of the CrBZR1 gene in improving the cold resistance of plants, and can be used for the cultivation of cold-resistant plants, providing new gene resources for plant cold-resistant molecular breeding, and providing new genetic resources for the implementation of green agriculture and sustainable agricultural development.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a CrBZR1 gene and an application thereof. Background Art

[0002] Low temperatures not only severely limit the scope of citrus cultivation but are also the main abiotic factor restricting the development of the citrus industry. Therefore, cultivating new cold-resistant citrus varieties is extremely important for the sustainable, stable and healthy development of the citrus industry.

[0003] The rapid development of biotechnology has provided new avenues for plant breeding. Genetic engineering allows for targeted genetic improvement of crops, demonstrating its significant utility in developing new stress-resistant crop varieties (materials). The discovery and identification of stress-resistant genes is a prerequisite and key to creating plants with resilient genes through genetic engineering. Although screening for cold-resistant genes is ongoing, the resulting cold-resistant genes still fail to effectively achieve cold tolerance, thereby failing to effectively improve plant cold resistance and meeting the needs of scientific research and production. Summary of the Invention

[0004] The object of the present invention is to provide a CrBZR1 gene and its application. The CrBZR1 gene of the present invention can effectively enhance the cold resistance of plants, especially improve the cold resistance of citrus plants.

[0005] The present invention provides a CrBZR1 gene, the nucleotide sequence of the CrBZR1 gene is shown as SEQ ID NO.1.

[0006] The present invention also provides a recombinant expression vector, which includes the CrBZR1 gene described in the above technical solution.

[0007] Preferably, the initial vector of the recombinant expression vector includes a plasmid vector.

[0008] Preferably, the CrBZR1 gene is inserted between the Xba I and Sma I sites of the initial vector.

[0009] The present invention also provides an engineered bacterium, comprising the CrBZR1 gene described in the above technical solution or the recombinant expression vector described in the above technical solution.

[0010] The present invention also provides the use of the CrBZR1 gene, the recombinant expression vector or the engineered bacteria described in the above technical solution in improving plant cold resistance and / or breeding cold-resistant plants.

[0011] Preferably, the plant comprises a citrus plant.

[0012] Preferably, the citrus plants include oranges and / or lemons.

[0013] The present invention also provides a method for cultivating cold-resistant plants, comprising: promoting the expression of the CrBZR1 gene described in the above technical solution in the target plant to obtain the cold-resistant plant.

[0014] Preferably, the expression of the CrBZR1 gene in the target plant is promoted by introducing the CrBZR1 gene or the recombinant expression vector described in the above technical solution into the target plant to obtain the plant.

[0015] Beneficial effects:

[0016] The present invention provides a CrBZR1 gene, which is isolated and cloned from the extremely cold-resistant variety Chongyi wild orange, and belongs to the new gene CrBZR1 of the BZR family. The nucleotide sequence of the CrBZR1 gene is shown in SEQ ID NO. 1. The embodiments of the present invention increase the content of the encoded protein by overexpressing the CrBZR1 gene in plants, thereby improving the cold tolerance of the plants. At the same time, the embodiments of the present invention also silence the expression of the CrBZR1 gene in plants, thereby significantly reducing the cold resistance of the plants. This technical effect also verifies the function of the CrBZR1 gene in improving the cold resistance of plants. The gene can be used for the cultivation of cold-resistant plants, providing new genetic resources for plant cold-resistant molecular breeding, and providing new genetic resources for the implementation of green agriculture and sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 It is a technical flow chart of the present invention;

[0019] Figure 2 This is a fluorescence schematic diagram of the subcellular localization of the CrBZR1 gene in Example 2 of the present invention;

[0020] Figure 3 Schematic diagram of the identification of transcriptional self-activation of CrBZR1 protein in Example 3 of the present invention, wherein A is a schematic diagram of the segmented situation of the CrBZR1 amino acid sequence, and B is a schematic diagram of the identification of transcriptional self-activation of the full-length CrBZR1 amino acid sequence and different fragments;

[0021] Figure 4 Schematic diagram of the identification of the transcriptional inhibitory activity of the CrBZR1 protein in Example 4 of the present invention, wherein A is a schematic diagram of the vector construction and results of the dual luciferase activity assay, and B is a schematic diagram of the vector construction and results of the GUS staining assay;

[0022] Figure 5 Schematic diagram of the construction of the CrBZR1 recombinant expression gene vector in Example 5 of the present invention, wherein A is a schematic diagram of the construction of the overexpression recombinant vector, and B is a schematic diagram of the construction of the silent expression recombinant vector;

[0023] Figure 6 Schematic diagram of PCR identification of the CrBZR1 gene overexpression vector and empty-transformed tobacco and regenerated plants in Example 6 of the present invention, wherein a in A is a co-cultured leaf, b in A is a screening cultured leaf, c in A is a rooted leaf, d in A is a soil-cultured seedling, and B is a gel image of the PCR identification results of transgenic tobacco;

[0024] Figure 7 Figures 6A and 6B show the phenotypic and physiological index measurements of CrBZR1-overexpressing tobacco, wild-type (WT), and empty transgenic tobacco tissue culture seedlings before and after low-temperature treatment, wherein A shows the tobacco phenotype before low-temperature treatment, after 2 hours of treatment at 4°C, and after 2 hours of recovery; B shows the relative conductivity results at 0 hours and 2 hours of low-temperature treatment at 4°C; and C shows the malondialdehyde content results at 0 hours and 2 hours of low-temperature treatment at 4°C.

[0025] Figure 8 Schematic diagram of positive identification of lemon transformed with CrBZR1 gene overexpression vector and regenerated plants in Example 7 of the present invention, wherein A, a are co-cultivated stem segments, A, b are screening cultured stem segments, A, c are elongation growth of resistant lemon buds, A, d are micro-bud grafting of resistant lemon buds onto rootstock trifoliate orange, A, e are soil-cultured plants, B, a are PCR identification schematics, B, b are expression levels of CrBZR1 gene in overexpressed lemons and wild-type lemons, and B, c are semi-quantitative identification schematics;

[0026] Figure 9 The CrBZR1 overexpression lemon and wild type (CK L ) Phenotypes of potted seedlings before -4℃ treatment, after -4℃ treatment for 8 h, and after recovery for 16 h;

[0027] Figure 10 Schematic diagram of the transformation of Chongyi wild orange with the CrBZR1 gene silencing expression vector (VIGS) and PCR identification of regenerated plants in Example 8 of the present invention, wherein A and a represent co-culture of germinating buds, A and b represent soil-cultured plants, and B represents a PCR-identified positive plant;

[0028] Figure 11 Schematic diagram of the comparison of cold resistance between the VIGS-silenced Chongyi wild orange (pTRV2-CrBZR1) and the control (pTRV2) of the CrBZR1 gene in Example 8 of the present invention, wherein A is the phenotypic diagram of the silenced Chongyi wild orange and the control after -5°C low temperature treatment for 12 hours, B is the relative conductivity after -2°C low temperature treatment for 6 hours, and C is the malondialdehyde content after -2°C low temperature treatment for 6 hours. DETAILED DESCRIPTION

[0029] The present invention provides a CrBZR1 gene, the nucleotide sequence of the CrBZR1 gene is shown as SEQ ID NO.1.

[0030] The nucleotide sequence of the CrBZR1 gene described in the present invention is specifically: 5’-ATGACAGCGGGAGGCTCATCAGGGAGGCTGCCGACGTGGAAGGAGAGAGAGAACAACAAGAGGAGGGAGAGAAGGAGAAGAGCCATAGCCGCTAAGATATTTTCTGGGCTTAGAGCTGAGGGCAACTTTAAGCTACCAAAACATTGTGACAATAACGAGGTTTTGAAAGCCCTCTGTGCTGAGGCTGGCTGGATCGTTGAAGAAGATGGTACCACTTATCGCAAGGGAAGCAGGCCACCGCGAACCCCATCTGAAATTACAGGCGGTTCAGCAAACATCAGTACATGTTCCTCTGTTCAACCAAGCCCTCAATCCTCAGCTTTCCCCAGTCCTGTGCCCTCTTATCAGGCCAGCCCAACTTCATCCTCATTTCCTAGTCCCACTCGTTTCGAGGGAAATCCCTCAAATTACATTCTCCCGTTCCTTCAAAACATTGCTTCAGTTCCTACAAATTTACCTCGTCTTAGAATATCCAATAGTGCTCCAGTAACCCCACCTCTTTCATCTCCAACTTCTAGAGGTCCAAAACGAAAGACTGATTGGGAATCCATTTCAAATGGCACCTTGAGCTCATTTCGCCATCCCCTGTTTGCGGTATCTGCTCCTTCAAGCCCTACTCGTCGCCAACATTTTACACCTGCCACAATACCAGAATGTGACGAGTCTGATGCATCCACTGTGGATTCTGGTCGTTGGGTCAGTTTCCAGACAGTAGCAACCTTAGCAGCCCCTCCTTCGCCTACTTTTAACCTTATCAAACCTGTGGTTCAGAAGAATTGTATTCAAAAGGCAGTTGATGGGCATAAAGGCCTAGCTTGGGGAATGGCAGCAGAGAGGGGGCAAGTTTCGGAGTTTGAATTTGAGAGTGAGAGAGTGAAGCCTTGGGAGGGTGAGAGAATTCACGAGGTAGGTGTTGATGATCTTGAGCTTACACTTGGAAGTGGCAAGGCACGTGGATAA-3’。

[0031] The nucleotide sequence shown in SEQ ID NO.1 of the present invention is the open reading frame of the CrBZR1 gene, which is 960 bp in length and is located in the cell nucleus.

[0032] The CrBZR1 gene of the present invention is isolated and cloned from Chongyi wild orange (Citrus reticulata).

[0033] The present invention preferably further provides a method for isolating and cloning the CrBZR1 gene, comprising the following steps: using the cDNA of Chongyi wild orange as a template, performing PCR amplification on the cDNA using amplification primers to obtain the CrBZR1 gene.

[0034] The cDNA template of the present invention is preferably obtained by reverse transcription of RNA extracted from Chongyi wild orange leaves. The present invention does not particularly limit the RNA extraction method and reverse transcription method, and conventional RNA extraction and reverse transcription methods in the art can be used. In the embodiment, the total RNA from Chongyi wild orange leaves was extracted using the RNAiso Plus kit (the kit was purchased from TAKARA, and the operation method was in accordance with the instructions); the extracted Chongyi wild orange RNA was used to synthesize the first chain of cDNA with reference to the operating manual of the TOYOBO reverse transcription kit.

[0035] The nucleotide sequence of the forward primer of the amplification primer pair of the present invention is preferably as shown in SEQ ID NO. 3, specifically: 5'-GGA GAAAGAGAAGAACCTAATGG-3', and the nucleotide sequence of the reverse primer is preferably as shown in SEQ ID NO. 4, specifically: 5'-AAGCTCTTTCCTCTCAAGCCATG-3'. The amplification primer pair of the present invention is preferably designed using Primer Premier 5.0 with the sequence of the sweet orange CrBZR1 gene (Cs1g23700) as the target sequence.

[0036] The present invention has no special limitation on the system and procedure of PCR amplification, and amplification can be performed according to the selected kit or reagent instructions.

[0037] The amino acid sequence of the CrBZR1 protein encoded by the CrBZR1 gene of the present invention is preferably as shown in SEQ ID NO.2, specifically: MTAGGSSGRLPTWKERENNKRRERRRRAIAAKIFSGLRAEGNFKLPKHCDNNEVLKALCAE AGWIVEEDGTTYRKGSRPPRTPSEITGGSANISTCSSVQPSPQSSAFPSPVPSYQASPTSSSFPSPTRFEGNPSNYILPFLQNIASVPTNLPRLRISNSAPVTPPLSSPTSRGPKRKTDWESISNGTLSSFRHPLFAVSAPSSPTRRQHFTPATIPECDESDASTVDSGRWVSFQTVATLAAPPSPTFNLIKPVVQKNCIQKAVDGHKGLAWGMAAERGQVSEFEFESERVKPWEGERIHEVGVDDLELTLGSGKARG, a total of 320 amino acids. The isoelectric point of the CrBZR1 protein of the present invention is 9.48, and the predicted molecular weight is 34.64 kDa.

[0038] The present invention also provides a recombinant expression vector, which includes the CrBZR1 gene described in the above technical solution. The initial vector of the recombinant expression vector of the present invention preferably includes a plasmid, more preferably a 1300-EGFP vector. The recombinant expression vector of the present invention preferably includes 1300-EGFP-CrBZR1; the 1300-EGFP-CrBZR1 of the present invention is preferably obtained by introducing the nucleotide sequence shown in SEQ ID NO.1 into the 1300-EGFP vector between the restriction sites of Xba I and Sma I. The present invention has no special limitation on the construction process of the recombinant expression vector, and the conventional construction process in this field can be used.

[0039] The present invention also provides an engineered bacterium comprising the CrBZR1 gene or the recombinant expression vector described in the above technical solution. The engineered bacterium is preferably obtained by transferring the recombinant expression vector described in the above technical solution into a bacterial strain; the bacterial strain preferably comprises fungi or bacteria, further comprising bacteria, more preferably Agrobacterium tumefaciens, and most preferably Agrobacterium tumefaciens GV3103. The present invention does not specifically limit the construction process of the engineered bacterium; conventional construction processes in the art may be employed.

[0040] The present invention also provides the use of the CrBZR1 gene described in the above technical solution, the recombinant expression vector described in the above technical solution, or the engineered bacteria described in the above technical solution in improving plant cold resistance and / or breeding cold-resistant plants. The plants described in the present invention preferably include citrus plants, more preferably citrus and / or lemons, and more preferably citrus and lemons. The present invention enhances the cold tolerance of plants and improves their cold resistance by increasing the expression of the CrBZR1 gene in plants.

[0041] The present invention also provides a method for cultivating cold-resistant plants, comprising: promoting the expression of the CrBZR1 gene described in the above technical solution in the target plant to obtain the cold-resistant plant. The promoting expression of the CrBZR1 gene in the target plant of the present invention is preferably to introduce the cold-resistant gene or the recombinant expression vector described in the above technical solution into the target plant. The CrBZR1 gene of the present invention is preferably transferred into the target plant in the form of the recombinant expression vector so that the CrBZR1 gene is overexpressed in the target plant; the transfer method preferably includes genetic transformation, and the specific steps of the genetic transformation are not particularly limited, and conventional steps in the art can be used.

[0042] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] The technical process of the following embodiment is as follows Figure 1 shown.

[0044] Example 1

[0045] The isolation and cloning steps of CrBZR1 are as follows:

[0046] 1. First, use the RNAiso Plus kit to extract total RNA from Chongyi wild orange leaves (the kit was purchased from TAKARA, and the operation method was according to the manufacturer's instructions). The extracted Chongyi wild orange RNA was used to synthesize the first strand of cDNA according to the instructions of the TOYOBO reverse transcription kit.

[0047] 2. Based on the sequence of the sweet orange CrBZR1 (Cs123700) gene, the forward primer and reverse primer shown in SEQ ID NO.3 and SEQ ID NO.4 were designed using Primer Premier 5.0 to form an amplification primer pair.

[0048] 3. Using the cDNA of Chongyi wild orange as a template, PCR was used to amplify CrBZR1 of Chongyi wild orange.

[0049] The PCR amplification system is: TaKaRa LATaq (5U / μL) 0.25 μL, 10× LATaq Buffer II (Mg 2+ Plus) 5 μL, dNTP Mixture (2.5 mM each) 4 μL, forward primer and reverse primer (10 μM) 2 μL each, template cDNA 1 μL, sterile water 35.75 μL.

[0050] PCR was performed according to the following program: pre-denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, followed by extension at 72°C for 5 min.

[0051] After PCR amplification, electrophoresis was performed on a 1.2% agarose gel using TAE buffer on a DYY-6C electrophoresis instrument (Liuyi, Beijing) for 30 min at 120 V and 150 mA. The target band was excised under UV light, and the specific target band was recovered using an Axygen gel extraction kit (Corning Life Sciences, Inc.) according to the manufacturer's instructions.

[0052] 5. The purified product was recovered and ligated with the pMD18-T vector (Takara, Japan).

[0053] The ligation system consisted of 0.5 μL of pMD18-T Vector, 5 μL of Solution I, and 4.5 μL of the purified PCR product. Ligation was performed overnight at 16°C. Transformation was then performed into competent E. coli DH5α (Beijing Quanshijin Biotechnology Co., Ltd.) using the heat shock method. Positive clones were selected for PCR verification using the aforementioned amplification primers (using the same PCR protocol as described above) and sequenced (performed by Wuhan Qingke Xinye Biotechnology Co., Ltd.).

[0054] The above operation amplifies a fragment of 1238bp. The ORF Finder on NCBI carries out open reading frame prediction and finds that the sequence contains a coding region sequence of 960bp in length, which encodes a protein of 320 amino acids with a molecular weight of 34.64kDa and a theoretical isoelectric point of 9.48. The gene-encoded protein is compared with the Arabidopsis thaliana AtBZR1 core amino acid sequence using DNAMAN 8.0 and finds that they all contain NLS nuclear localization signal domain, bHLH domain, BIN2 phosphorylation domain, PEST domain and C-terminal conserved domain. Therefore, the gene is named CrBZR1, and the cDNA sequence is shown in SEQ ID NO.1, and the coded protein sequence is shown in SEQ ID NO.2.

[0055] Example 2

[0056] The subcellular localization of the CrBZR1 gene was performed as follows:

[0057] ExPASy online analysis results showed that the CrBZR1 protein sequence has a nuclear localization signal. In this example, Nicotiana benthamiana transient expression and the plant subcellular localization vector pBI-EGFP were used to study the subcellular localization of CrBZR1.

[0058] Subcellular localization primers were designed to amplify the ORF sequence of CrBZR1 gene and inserted between the XbaI and SmaI restriction sites on the pBI-EGFP vector.

[0059] Subcellular localization primers:

[0060] Forward primer (SEQ ID NO. 7): 5′-TCTAGAAAGAGAGACGCATCTGGAGTAAT-3′;

[0061] Reverse primer (SEQ ID NO. 8): 5'-CCCGGGTCCACGTGCCTTGCCACTTCCAAGT-3'.

[0062] Amplification system: 1-5 TM 25 μL of 2× High-Fidelity Master Mix (Qingke Xinye Biotechnology Co., Ltd.), 2 μL of forward primer and reverse primer, 2 μL of Chongyi wild orange cDNA in Example 1, and 19 μL of ddH2O.

[0063] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 5 min after the cycle was completed.

[0064] After PCR amplification, all PCR products were loaded on 1.2% agarose gel for electrophoresis, and the specific target bands were recovered using the Axygen gel recovery kit (Corning Life Sciences Co., Ltd.) according to the instructions. Carrier (Quanshijin, Beijing), connection system is 1 μL of Cloning Vector and 4 μL of PCR purified product.

[0065] After ligation at room temperature for 10 min, the ligation solution was introduced into competent Escherichia coli DH5α (Beijing Quanshijin Biotechnology Co., Ltd.) by heat stimulation. Positive clones were selected using primers for the target gene sequence for PCR verification (consistent with the PCR procedure for constructing the vector above) and sequenced by Wuhan Qingke Xinye Biotechnology Co., Ltd.

[0066] The pEASY-CrBZR1 recombinant vector plasmid and the pBI-EGFP vector plasmid cloned in Example 1 were simultaneously cut with endonucleases Xba I and Sma I. The double enzyme digestion system was as follows: Xba I 1 μL, Sma I 1 μL, 10×T buffer 2 μL, 0.1% BSA 2 μL, and plasmid DNA 14 μL.

[0067] The double enzyme digestion system was incubated overnight at 37°C. Qualified bands were detected by agarose gel electrophoresis and excised. The target bands were recovered using a gel extraction kit. The pEASY-CrBZR1 recombinant vector plasmid, digested with Xba I and Sma I, and the pBI-EGFP vector plasmid were ligated with T4 DNA Ligase to obtain the 35S-CrBZR1-pBI-EGFP recombinant vector. This vector was then transformed into Agrobacterium competent cells GV3101 (Weidi Biotechnology Co., Ltd., Shanghai). Transformation procedures are described in the manufacturer's instructions.

[0068] The transient expression transformation method of Nicotiana benthamiana was used to detect the localization of the CrBZR1 gene. The steps for transient expression in Nicotiana benthamiana are as follows:

[0069] (1) Bacterial liquid activation: Add 10 μL of cryopreserved Agrobacterium (35S-CrBZR1-pBI-EGFP and pBI-EGFP Agrobacterium) and 1 mL of liquid LB (50 mg / L Kan, 25 mg / L Rif) to a 2.0 mL sterile centrifuge tube and culture in a shaking incubator at 28°C and 225 rpm for 16 to 18 h.

[0070] (2) Expansion culture of bacterial solution: Take 300 μL of activated bacterial solution and add it to 30 mL of liquid LB (50 mg / L Kan, 25 mg / L R-Lif), and culture it in a shaking incubator at 28°C and 225 rpm for 12 to 16 hours until the bacterial solution OD reaches 0. 600 The value is around 0.6.

[0071] (3) Collection and washing of bacterial liquid: Use a 50 mL centrifuge tube and centrifuge at 4000 rpm for 5 min to collect the bacterial liquid, discard the supernatant, and then use 5 mL of washing solution (10 mmol / LMgCl2, 10 mmol / LMES, 150 μmol / LAs, pH = 5.6).

[0072] (4) Determination of bacterial solution OD value: discard the supernatant, add an appropriate amount of cleaning solution to resuspend the bacterial solution, and make the bacterial solution OD 600 The value is about 0.6, and it is allowed to stand in the dark at room temperature for 2 to 3 hours.

[0073] (5) Injection: Select tobacco leaves that are approximately 4 weeks old. Use a disposable syringe with the needle removed to inject the bacterial solution from the back of the leaf. 2–3 leaves from both the experimental and control groups were injected and incubated in the greenhouse for two days. Two days later, 10 μg / mL DAPI staining solution (Beijing Lanjie Keke Co., Ltd.) was injected into the tobacco leaves from the back. Fluorescence was then observed using a confocal microscope.

[0074] The results are as follows Figure 2 As shown, in Figure 2 In this study, the pBI-EGFP-CrBZR1 binary vector expresses the 35S-CrBZR1-pBI-EGFP fusion protein. Fluorescence from the empty pBI-EGFP plasmid is distributed throughout the cell, including the cell membrane and nucleus, while fluorescence from the 35S-CrBZR1-pBI-EGFP fusion protein is concentrated only in the nucleus, indicating that the CrBZR1 gene is localized in the nucleus.

[0075] Example 3

[0076] CrBZR1 transcriptional autoactivation activity analysis, the steps are as follows:

[0077] The pGBKT7 vector was used for recombinant construction to verify whether CrBZR1 has transcriptional autoactivation activity. _ The N domain is distributed between 34th and 117th aa and is presumed to be the transcriptional activation region. _ The N domain is divided into three segments: CrBZR1-1 (1th-33th aa), CrBZR1-2 (34th-117th aa), CrBZR1-3 (118th-320th aa), such as Figure 3 As shown in A in . The full-length open reading frame of the CrBZR1 gene and the gene fragments encoding CrBZR1-1, CrBZR1-2 and CrBZR1-3 were respectively connected to the pGBKT7 vector (inserted between the EcoR I and BamH I sites). After sequencing confirmed that the sequence was correct, the four fusion expression vectors and the empty vector (pGBKT7) were transformed into the yeast strain Y2HGold (Shanghai Angyu Biotechnology Co., Ltd.), and then cultured on three different deletion media: SD / -Leu / -Trp, SD / -Leu / -Trp / -Ade / -His and SD / -Leu / -Trp / -Ade / -His+X-α-gal. The results are shown in FIG. Figure 3 As shown in B.

[0078] The results showed that yeast cells transformed with the empty vector and cells transformed with the four fusion vectors could only grow in the deficiency medium SD / -Leu / -Trp, but could not grow in deficiency medium such as SD / -Leu / -Trp / -Ade / -His and SD / -Leu / -Trp / -Ade / -His, indicating that CrBZR1 does not have transcriptional autoactivation activity and may function in the form of a complex.

[0079] Example 4

[0080] The steps for analyzing the transcriptional repression activity of CrBZR1 are as follows:

[0081] In this example, the transcriptional repression activity of CrBZR1 was studied by transiently expressing it in Nicotiana benthamiana using a dual-luciferase assay system and GUS staining.

[0082] The dual luciferase detection system uses the pBD vector to recombinantly construct the GAL4-CrBZR1 fusion protein as the Effector of the system. After sequencing confirmation, GAL4-CrBZR1 (CrBZR1 gene is inserted between Age I and Stu I), GAL4-VP16 (positive control, the vector construction method is the same as the construction method of the GAL4-CrBZR1 recombinant vector, the VP16 gene is inserted between Age I and Stu I), and the empty pBD vector are transformed into Agrobacterium competent cells GV3101 (competent cells GV3101 containing pSoup+P19). The Effector:Reporter (the bacterial solution has been saved in the laboratory) is mixed with the two bacterial solutions at a ratio of 0.3:0.7 and transiently expressed in Nicotiana benthamiana. The determination of firefly luciferase (LUC) and Renilla luciferase (REN) is carried out using ReporterAssay System (Promega, USA) kit.

[0083] Vector construction method for GUS staining experiment: refer to "WangY, Wang L, ZouY, et al. SoybeanmiR172cTargets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40Expression and Regulate Nodule Initiation[J]. Plant Cell, 2014, 26(12)." and "Wu H, Fu B, Sun P, et al. A NAC Transcription Factor Represses Putrescine BiosynthesisandAffects Drought Tolerance[J]. Plant Physiology, 2016: 1532-1547." for construction: Chongyi wild orange cDNA was used as a template to amplify the full-length CrBZR1 gene, and the full-length CrBZR1 gene was inserted between the EcoRI and Sal I restriction sites of the pYF503 vector to obtain the recombinant GDBD-CrBZR1 plasmid as an effector. The GDBD-CrBZR1 plasmid, the empty pYF503 vector (positive control, containing GDBD), and the 35S-USA-GUS plasmid (reporter, previously published) were transformed into Agrobacterium competent GV3101 cells (competent GV3101 containing pSoup+P19). The effector:reporter mixture was mixed at a ratio of 0.3:0.7 and transiently expressed in Nicotiana benthamiana.

[0084] The results of the dual luciferase assay system are as follows Figure 4 As shown in Figure A, the LUC / REN ratio of pBD-VP16 is 2.67 times that of pBD, which is significantly higher than the pBD empty control, indicating that pBD-VP16 has transcriptional activation activity. The transcriptional activation of the positive control shows that the experimental system is reliable; the LUC / REN ratio of pBD-CrBZR1 is 0.64 times that of pBD, which is significantly lower than the pBD empty control, indicating that the CrBZR1 gene has transcriptional inhibition activity.

[0085] GUS staining was performed using a GUS staining kit (Coolaber, Beijing) according to the instructions, with the following modifications: the prepared leaves were immersed in GUS staining solution, vacuumed at 37°C for 1 h, and then placed in the dark at room temperature for several hours; finally, the leaves were transferred to a 75% by volume ethanol solution for decolorization until the negative control appeared white.

[0086] GUS staining results Figure 4As shown in B, GDBD and Reporter bacteria were co-injected and transiently expressed in tobacco leaves, and the entire leaves turned blue; while Effector and Reporter bacteria were co-injected and transiently expressed in tobacco leaves, and only part of the leaves turned blue, indicating that the CrBZR1 transcription factor has an inhibitory effect.

[0087] Example 5

[0088] The CrBZR1 gene overexpression and virus-induced gene silencing (VIGS) vectors were constructed as follows:

[0089] 1. Overexpression vector construction

[0090] According to the analysis of the multiple cloning site of 1300-EGFP vector and the restriction enzyme site of CrBZR1 gene ORF, Xba I and Sma I were selected as the restriction enzymes ( Figure 5 Middle A).

[0091] Primers SEQ ID NO. 5: 5'-TCTAGAATGACAGCGGGAGGCTCATC-3' and SEQ ID NO. 6: 5'-CCCGGGTCCACGTGCCTTGCCACTTCCAAGT-3' were designed to construct the overexpression vector.

[0092] TransStart FastPfu DNA Polymerase (Full Gold) was used to amplify the target fragment. The PCR system was as follows: 10.0 μL of 5× TransStart FastPfu Buffer, 5.0 μL of dNTPMix (2.5 mM each), 1.0 μL of the cDNA template from Example 1, 2.0 μL of the forward primer (10 μM), 2.0 μL of the reverse primer (10 μM), 1.0 μL of TransStart FastPfu DNA Polymerase, and 9.0 μL of ddH2O2.

[0093] The PCR program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for 35 cycles; and extension at 72°C for 5 min.

[0094] The above PCR products were recovered by gel excision and then subjected to double enzyme digestion. The system was 100 μL: 10×H buffer, 10 μL; Xba I and Sma I (TaKaRa), 5 μL each; recovered product, 25 μL; ddH2O, 55 μL; and recovered after enzyme digestion at 37°C overnight.

[0095] The 1300-EGFP vector plasmid was also digested and recovered using the same enzyme system.

[0096] The recovered target fragment and the vector were ligated. The total ligation reaction volume was 10 μL: 10×T4 ligation buffer, 1 μL; T4 ligase, 1 μL; 1300-EGFP vector, 0.5 μL; target fragment, 4.5 μL; ligation was carried out at 16°C for 16 h.

[0097] The ligation product was then transformed into Escherichia coli DH5α and screened on LB solid plates containing 50 mg / L kanamycin. Single colonies that tested positive for PCR were sent for sequencing at Wuhan Qingke Xinye Biotechnology Co., Ltd. Sequencing confirmed that the reading frame was correct, indicating that the 1300-EGFP-CrBZR1 recombinant vector was successfully constructed. The recombinant vector was introduced into Agrobacterium tumefaciens GV3103 using the freeze-thaw method (referring to the Molecular Cloning Laboratory Manual, 4th edition, Science Press, 2017), and the culture was stored at -80°C (containing 25% by volume glycerol).

[0098] 2. VIGS vector construction

[0099] In order to obtain CrBZR1 gene silenced transgenic plants, pTRV2 vector was used for recombinant construction. First, primers were designed to amplify the 399 bp fragment at the 5' end of the CrBZR1 gene and inserted it between the BamH I and Sma I sites on the pTRV2 vector ( Figure 5 (B) The vector primer sequences are as follows:

[0100] Forward primer (SEQ ID NO. 9): 5′-GGATCCATGACAGCGGGAGGCTCATCA-3′; Reverse primer (SEQ ID NO. 10): 5′-CCCGGGATTTCCCTCGAAACGAGTGGGACT-3′;

[0101] PCR amplification system: 25 μL of 1-5TM2× High-Fidelity Master Mix (Qingke Xinye Biotechnology Co., Ltd.), 2 μL of forward primer and reverse primer, 2 μL of Chongyi wild orange cDNA in Example 1, and 19 μL of ddH2O.

[0102] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 5 min after the cycle was completed.

[0103] The above PCR products were recovered by gel excision and then subjected to double enzyme digestion. The system was 100 μL: 10×T buffer, 10 μL; 0.1% BSA, 10 μL; BamH I and Sma I (TaKaRa), 5 μL each; recovered product, 25 μL; ddH2O, 45 μL; and recovered after enzyme digestion at 37°C overnight.

[0104] The pTRV2 vector plasmid was also digested with the same enzyme system and recovered.

[0105] The recovered target fragment and the vector were ligated. The total ligation reaction volume was 10 μL: 10×T4 ligation buffer, 1 μL; T4 ligase, 1 μL; pTRV2 vector, 0.5 μL; target fragment, 4.5 μL; ligation was carried out at 16°C for 16 h.

[0106] The ligation product was then transformed into Escherichia coli DH5α and screened on LB solid plates containing 50 mg / L kanamycin. Single colonies that tested positive for PCR were sent for sequencing at Wuhan Qingke Xinye Biotechnology Co., Ltd. Sequencing confirmed that the reading frame was correct, indicating that the pTRV2-CrBZR1 recombinant vector was successfully constructed. The recombinant vector and the helper plasmid pTRV1 were separately introduced into Agrobacterium tumefaciens GV3103 using the freeze-thaw method, and the bacterial suspension was stored at -80°C (containing 25% glycerol).

[0107] Example 6

[0108] Application of CrBZR1 gene in improving cold resistance of tobacco

[0109] 1. The steps for Agrobacterium tumefaciens-mediated tobacco genetic transformation are as follows:

[0110] (1) Cultivation of Agrobacterium tumefaciens: Take fresh Agrobacterium tumefaciens culture, streak on LB solid plate (containing 50 mg / L kanamycin), scrape the streaked plaque, add it to liquid MS minimal medium, and culture at 28°C and 200 rpm. 600 =0.4~0.6 when impregnation is carried out;

[0111] (2) Infection: Take healthy wild-type detached tobacco leaves, remove the main veins, cut into 0.5 cm × 0.5 cm squares, and immerse in the prepared Agrobacterium tumefaciens solution for 9-12 minutes with intermittent shaking;

[0112] (3) Co-cultivation: Take the infected tobacco leaves, dry them on sterile filter paper, and evenly arrange them on the co-cultivation medium with the back of the leaves facing downwards. Figure 6 Middle A, a), cultured at 25°C for 3 days;

[0113] (4) Screening culture: After co-cultivation, wash once with cephalosporin solution (500 mg / L), then wash 3 to 5 times with sterile water, and then transfer to screening culture medium ( Figure 6 (a, b).

[0114] (5) Rooting: When the adventitious buds on the screening medium grow to about 1 cm, they are cut and transferred to the rooting medium ( Figure 6 In Ac).

[0115] Table 1 Tobacco transformation medium formula

[0116] name Components and content MS minimal medium 30g / L sucrose; 7.5g / L agar; pH 5.8 Co-culture medium MS basic medium + 2.0 mg / L 6-BA + 0.3 mg / L NAA Screening medium Co-culture medium + 50 mg / L hygromycin + 500 mg / L cephalosporin rooting medium MS basic medium + 0.3 mg / L NAA + 50 mg / L hygromycin + 500 mg / L cephalosporin

[0117] 2. Identification of positive transgenic tobacco

[0118] When the rooted seedlings had 2 to 3 leaves, DNA was extracted using the CTAB method.

[0119] Using the obtained DNA as a template, two pairs of primers, kanamycin gene primers (forward primer SEQ ID NO.11: 5'-CGCAGAAGGCAATGTCATACCA-3'; reverse primer SEQ ID NO.12: 5'-CCTTTGCTCGGAAGAGTATGAA-3') and 35S promoter sequence forward primer (SEQ ID NO.13: 5'-CAAAGCAAGTGGATTGATGTGAT-3') and reverse primer (SEQ ID NO.6), were used to identify plants overexpressing the CrBZR1 gene and empty-loaded positive plants.

[0120] The target fragment was amplified using Fermentas Taq enzyme. The PCR system consisted of 2.0 μL of 10× PCR buffer, 0.4 μL of 10 mM dNTP mix, 1.0 μL of tobacco DNA, 0.3 μL each of the forward and reverse primers (10 μM), 0.2 μL of polymerase, and 15.8 μL of ddH₂O. The PCR program was as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 2 min; and finally, extension at 72°C for 5 min.

[0121] PCR verification electrophoresis results Figure 6 As shown in B. Two overexpression lines OE2 and OE14 and empty vector EV were obtained. B2, B14, and EV were used as separate transgenic lines and then used as maternal plants for seed collection.

[0122] 3. Identification of Cold Resistance of Overexpressed Tobacco

[0123] Tobacco seeds from two overexpression lines B2 and B14, empty vector (EV), and wild-type (WT) from the same batch were collected and sown aseptically: the seeds were first soaked in 70% alcohol for 1 min, then washed three times with sterile double-distilled water, and then disinfected with 1 mL of 2.5% NaClO for 8 min, shaken several times in between, and then washed three times with sterile ddH2O. Finally, the seeds were spread flat on MS minimal medium containing 50 mg / L kanamycin using a sterile inoculation needle. After germination and growth into seedlings, they were used for subsequent cold resistance phenotype and related index determination.

[0124] (1) Phenotype: Overexpressing CrBZR1 tobacco B2, B14 and their wild-type control WT and empty control EV were treated at 4°C for 2 h and then recovered at room temperature for 2 h. The phenotypic data after no treatment, treatment at 4°C and recovery at room temperature for 2 h are shown in the following table. Figure 7 As shown in A.

[0125] Depend on Figure 7 Figure A shows that, without low-temperature treatment, the growth conditions of the overexpressing CrBZR1 gene and the control plants were similar. After low-temperature treatment, the WT and EV plants wilted severely, with most leaves losing water and drying up, while the overexpressing plants OE2 and OE14 wilted relatively mildly. After returning to room temperature, the growth conditions of the control plants partially improved, while the leaves of the overexpressing plants generally improved. These results indicate that tobacco plants overexpressing CrBZR1 have stronger cold tolerance and recover faster after low-temperature treatment.

[0126] (2) Conductivity: Take the leaves of transgenic and control plants treated at 4°C for 0 h and 2 h, remove the midrib and leaf edges, cut into small pieces of about 0.5 cm × 0.5 cm with scissors, weigh 0.25 g, put them into a glass test tube with 15 mL of deionized water, shake at 120 r / min, and shake at 25°C for 2 h. Then use a conductivity meter (DDS-307) to measure the conductivity and record it as C1. Then put the test tube into boiling water and boil it for 1 min. After cooling to room temperature, measure the conductivity and record it as C2. Use blank ultrapure water as a control for the two measurements and record them as CK1 and CK2. The calculation formula is:

[0127] Relative conductivity (%) = (C1-CK1) / (C2-CK2)×100%.

[0128] like Figure 7 As shown in Figure B, before low temperature stress treatment, there was no significant difference in the relative conductivity of WT, EV, B2 and B14. However, after low temperature treatment, WT and EV were significantly higher than the two transgenic lines, indicating that under low temperature stress, WT and EV had more electrolyte extravasation and their cell membranes were more damaged.

[0129] (3) Malondialdehyde content: Malondialdehyde content was determined using a malondialdehyde assay kit (Nanjing Jiancheng Bioengineering Institute). Overexpressing CrBZR1 tobacco OE2, OE14, their wild-type control WT, and empty control EV were treated at 4°C for 2 h. 0.2 g of plant samples before and after low-temperature treatment were weighed and added to 4 mL of 0.1 mol / L PBS (phosphate buffer, pH = 7.4) for grinding to prepare plant tissue homogenate. The procedure was then performed according to the kit instructions. Finally, the absorbance of the supernatant at 532 nm was measured using a spectrophotometer, and the MDA content was calculated according to the formula provided in the instructions.

[0130] The results are as follows Figure 7 As shown in Figure C, before low temperature stress, there was no significant difference in the MDA content of WT, EV, B2, and B14. However, after low temperature stress, the MDA content of WT, EV, B2, and B14 increased, but the increase in WT and EV was significantly higher than that in B2 and B14, indicating that the cell membranes of WT and EV were more severely damaged under low temperature stress, thereby proving the drought tolerance of the CrBZR1 gene in the present invention.

[0131] Example 7

[0132] Application of CrBZR1 gene in improving cold resistance of lemon

[0133] In order to provide more abundant and powerful experimental evidence to illustrate the role of the CrBZR1 gene in cold resistance, the overexpression vector in Example 5 was transformed into lemon by Agrobacterium-mediated citrus epicotyl transformation to verify whether its overexpression would enhance the cold resistance of lemon.

[0134] 1. The steps for Agrobacterium tumefaciens-mediated transformation of citrus epicotyls and identification of positive transgenic plants are as follows:

[0135] (1) Sowing: Take lemon seeds, soak them in 1M NaOH for 20 min, wash them with clean water, soak them in 2% NaClO solution on a clean bench for 15-20 min, wash them three times with sterile water, peel off the seed coat under sterile conditions, sow them on MT solid medium, culture them in the dark for 3-4 weeks, and then culture them in the light for 1 week before use for transformation.

[0136] (2) Preparation of bacterial suspension: Streak the Agrobacterium tumefaciens culture medium containing the recombinant vector overexpressed in Example 5 onto solid LB medium containing 100 mg / L kanamycin and culture in the dark at 28°C for 2 days; pick a single colony and streak it onto a new plate again and culture in the dark at 28°C for 2-3 days; scrape the grown bacteria with a scalpel and inoculate them into liquid MT medium without antibiotics, and culture at 28°C with shaking at 200 rpm until the OD 600 =0.6-0.8, add acetosyringone to a final concentration of 100 μM and set aside.

[0137] (3) Co-cultivation: Take the epicotyls of sterile lemon seedlings, cut them into 1-1.5 cm long stem segments on a clean bench, soak them in the prepared overexpression vector Agrobacterium solution and infect them for 20 minutes, shaking them several times during the process. After infection, use sterile filter paper to absorb the excess solution, spread the explants on the co-culture medium, and incubate them in the dark at 25°C for 3 days ( Figure 8 In A, a).

[0138] (4) Screening and rooting: After 3 days of co-cultivation, wash the explants 3 to 5 times with sterile water, then dry the surface water with sterile absorbent paper and transfer them to the screening medium ( Figure 8 A, b). After culturing in the dark at 25°C for 4 weeks, transfer to light conditions. When the resistant buds are >0.5 cm, cut them off and transfer them to proliferation medium to promote their differentiation ( Figure 8 When the resistant buds are >1.5 cm long, the lemon resistant buds are grafted onto the rootstock lemon ( Figure 8 A, d), then soil culture ( Figure 8 In A, e).

[0139] Commonly used culture medium formula:

[0140] LB solid medium: peptone 10 g / L + yeast extract 5 g / L + NaCl 10 g / L + agar 15 g / L;

[0141] Lemon co-culture medium: MT medium + 1 mg / L BA + 20 mg / L ASA;

[0142] Lemon screening medium: MT medium + 1 mg / L BA + 400 mg / L Cef + 50 mg / L Km;

[0143] Lemon bud elongation and proliferation medium: MT medium + 0.5 mg / L BA + 0.5 mg / L GA3 + 0.5 mg / L IAA + 400 mg / L Cef;

[0144] Lemon grafting medium: 1 / 2MT medium + 0.5mg / L NAA + 0.1mg / L IBA + 0.5g / L activated carbon

[0145] During the transformation process, each culture medium was supplemented with 7.5 g / L agar and 35 g / L sucrose, and the pH was adjusted to 5.8.

[0146] (5) Positive plant identification:

[0147] DNA extraction was the same as that of tobacco.

[0148] PCR detection: The PCR reaction system and procedure for lemon are the same as those for tobacco.

[0149] Real-time fluorescence quantitative PCR and semi-quantitative analysis were used to analyze the expression of the CrBZR1 gene in overexpressed lemons and controls. Primer3Plus was used to design real-time quantitative primers for the CrBZR1 gene, and citrus actin was used as the internal reference gene:

[0150] Forward primer (SEQ ID NO. 14): 5'-CATCCCTCAGCACCTTCC-3'; Reverse primer (SEQ ID NO. 15): 5'-CCAACCTTAGCACTTCTCC-3';

[0151] Internal reference gene:

[0152] Forward primer (SEQ ID NO. 16): 5'-ACCTTGAGCTCATTTCGCCA-3'; reverse primer (SEQ ID NO. 17): 5'-CCCAACGACCAGAATCCACA-3'.

[0153] The quantitative PCR system is 10 μL, including iTaq TM Universal 5.0 μL of Green Supermix (Roche), 0.2 μL each of real-time quantitative forward and reverse primers (10 mol / L), 0.5 μL of the cDNA template in Example 1, and 4.1 μL of ddH 2 O.

[0154] The semi-quantitative system and PCR program were the same as those for tobacco, except that the number of cycles in the PCR program was changed to 30 cycles.

[0155] Instrument use 480II (Roche). The reaction program was set as 95°C for 30 s of initial denaturation; 95°C for 10 s of denaturation; 60°C for 10 s of annealing; and 72°C for 10 s of extension for 40 cycles. Quantitative PCR data were analyzed using the instrument's built-in software.

[0156] The test results of lemon are as follows Figure 8 B. a in B is the PCR identification result; b and c in B are the expression analysis of CrBZR1 gene in overexpressed lemon and control, respectively. M is a 2000bp marker, CK L is the lemon control, OE30 and OE42 are two lemon overexpression lines, Figure 8 It can be seen that the expression level of CrBZR1 gene is significantly upregulated in OE30 and OE42, which can be used for subsequent resistance identification.

[0157] 2. Identification of cold resistance of transgenic lemons

[0158] Lemon CK L, OE30, and OE42 were treated at -4°C for 8 hours. Figure 9 As shown, without low temperature treatment, OE30, OE42 and CK L There was no significant difference; after low temperature treatment, CK L The whole plant wilted, while OE30 and OE42 wilted to a lesser extent. After returning to room temperature, CK L Some leaves still did not recover, while OE30 and OE42 had basically recovered. This result shows that lemons overexpressing CrBZR1 have stronger cold resistance and suffer less damage under low temperature stress.

[0159] Example 8

[0160] Application of transcription factor CrBZR1 in improving cold resistance of Chongyi wild orange

[0161] The VIGS vector in Example 5 was introduced into Chongyi wild orange through Agrobacterium-mediated transfection to verify whether silencing the CrBZR1 gene would reduce the cold resistance of Chongyi wild orange.

[0162] 1. The steps for Agrobacterium-mediated VIGS infection and positive plant identification are as follows:

[0163] (1) Sowing

[0164] Chongyi wild orange seeds were soaked in 1M NaOH solution for 15 min to remove pectin, sterilized with 2.5% NaClO solution for 15 min, washed three to four times with sterile water, spread on gauze, and placed in a 28°C incubator for germination. Maintain a moist environment and use the seeds for VIGS silencing experiments when they germinate to 2-3 cm.

[0165] (2) Preparation of Agrobacterium infection solution

[0166] The activation, expansion culture, collection and cleaning of pTRV1, pTRV2 and pTRV2-CrBZR1 Agrobacterium culture were the same as those in Example 5. An appropriate amount of cleaning solution was added to resuspend the culture solution and the OD value of the culture solution was adjusted to 0. 600 The expression of pTRV1 in the presence of pTRV2 is approximately 1.0. Then, mix pTRV1 and pTRV2 in a 1:1 ratio, and pTRV1 and pTRV2-CrBZR1 in the same ratio. The pTRV1 solution assists the expression of the pTRV2 solution. Let it stand in the dark at room temperature for 2-3 hours before use for infection.

[0167] (3) Agrobacterium infection of Chongyi wild orange

[0168] Use a needle to poke some small holes in the germinated Chongyi wild orange seeds to facilitate Agrobacterium infection, then soak them in the prepared bacterial solution; vacuum evacuate for 1 minute, quickly deflate, place them on a shaker at 80 rpm for 10 minutes, and repeat this process two to three times; remove the seeds, dry the bacterial solution on the surface with filter paper, spread them flat on moist filter paper, and place them in a 25°C incubator for dark culture (see Figure 10 A, a), planted in the soil three days later. Positive identification can be carried out after one month (see Figure 10 (a, b).

[0169] (4) Identification of positive plants

[0170] DNA extraction was the same as that of tobacco.

[0171] PCR detection: Two pairs of primers were used to identify positive plants. The primers for identifying the auxiliary plasmid pTRV1 are as follows:

[0172] Forward primer: (SEQ ID NO. 18): 5'-ATTGAGGCGAAGTACGATGG-3', reverse primer: (SEQ ID NO. 19): 5'-CCATCCACAATTATTTTCCGC-3';

[0173] The primers for identifying the pTRV2-CrBZR1 recombinant plasmid are as follows:

[0174] The forward primer is (SEQ ID NO. 20): 5'-ATTCACTGGGAGATGATACGCT-3', and the reverse primer is (SEQ ID NO. 10). The PCR reaction system and procedure for Chongyi wild orange are the same as those for tobacco.

[0175] The test results of Chongyi wild orange are as follows Figure 10 Middle B. M: Marker, +: positive plasmid, -: wild-type Chongyi wild orange, 1-10: ten positive Chongyi wild oranges, and subsequent resistance identification was performed.

[0176] 2. VIGS Cold Resistance Evaluation of Chongyi Wild Orange

[0177] In order to explore the effect of silencing the CrBZR1 gene on the resistance of Chongyi wild orange to low temperature stress, the present invention subjected two-month-old pTRV-CrBZR1-silenced Chongyi wild orange and pTRV control to low temperature stress.

[0178] The silenced plants and controls were placed at -5°C for 12 hours and their expression changes were observed. Figure 11As shown in Figure A, before low temperature treatment, the growth status of pTRV2-CrBZR1 plants and pTRV controls were relatively consistent. After 12 hours of treatment in a -5°C low-temperature incubator, it was found that pTRV-CrBZR1 plants showed more severe growth inhibition and leaf wilting, while the leaves of the pTRV control showed a slight waterlogged appearance. After two days of recovery to room temperature, the pTRV2-CrBZR1 plants had died of dehydration, while the pTRV control plants only had some yellowing leaves and were not dead.

[0179] The results showed that silencing the CrBZR1 gene reduced the cold tolerance of Chongyi wild orange. Subsequently, the relative conductivity and malondialdehyde (MDA) of the silenced plants and the control were measured under -2℃ for 6h low temperature stress. Figure 11 BC), there was no significant difference in the relative conductivity and malondialdehyde content of the silenced plants compared with the control at -2℃0h, but after -2℃6h, the relative conductivity and malondialdehyde content of the silenced plants were significantly higher than those of the control, indicating that the cell membrane system of the silenced plants was more seriously damaged under low temperature stress.

[0180] From the above results, it can be concluded that the CrBZR1 gene described in the present invention can improve the cold resistance of plants and can be used to cultivate cold-resistant plants.

[0181] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of the CrBZR1 gene in improving plant cold resistance and / or breeding cold-resistant plants, wherein the nucleotide sequence of the CrBZR1 gene is shown in SEQ ID NO.1; the plant is at least one of tobacco, citrus and lemon.

2. A method for cultivating cold-resistant plants, characterized in that: include: Promoting the expression of the CrBZR1 gene in target plants; the nucleotide sequence of the CrBZR1 gene is shown in SEQ ID NO.1; The plant is at least one of tobacco, citrus and lemon.

3. The method according to claim 2, characterized in that The method of promoting the expression of the CrBZR1 gene in the target plant is to introduce the CrBZR1 gene or a recombinant expression vector containing the CrBZR1 gene into the target plant.

4. The method according to claim 3, characterized in that The initial vector of the recombinant expression vector includes a plasmid vector.

5. The method according to claim 4, characterized in that The CrBZR1 gene was inserted between the XbaI and SmaI sites of the initial vector.

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