Intelligent orthogonal regulation and control system for low-temperature response in plant body and application of intelligent orthogonal regulation and control system

Abstract

The invention discloses a plant in-vivo low-temperature response intelligent orthogonal regulation and control system and application thereof, and belongs to the technical field of plant genetic engineering. According to the invention, the thought of synthetic biology is utilized, and a transcription factor CRF-GAL4-VP16, which can respond to low temperature, nucleoplasm shuttle and orthogonal regulation gene expression in a plant body, is reconstructed. The effect that the target gene starts expression only in a low-temperature environment and stops expression after the temperature is recovered is achieved, and material energy consumption of chassis plants is reduced. A new thought is provided for future engineering application of efficient artificial intelligence crops; the innovative development of a synthetic biological technology in the technical fields of construction of novel efficient artificial plants (crops) and related production is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to an intelligent orthogonal control system for low temperature response in plants and applications thereof. Background Art

[0002] According to the degree of low temperature, low temperature stress can be divided into chilling injury (0-20℃) and freezing injury (<0℃). Some common vegetable crops, such as tomatoes, cucumbers, peppers, etc., are very susceptible to low temperature stress, which causes damage to the chloroplast photosystem, reduced activity of carbon assimilation-related enzymes, and reduced photosynthetic capacity, which in turn leads to poor vegetative and reproductive growth, reduced yield, premature aging, and an increase in deformed fruits, which seriously reduces the planting benefits.

[0003] Exogenous expression of low-temperature resistance genes in plants is an effective strategy to improve the low-temperature resistance of plants. However, the expression of low-temperature resistance genes is currently mostly controlled by strong promoters such as CaMV 35S and UBQ10 or endogenous low-temperature inducible promoters in plants. Strong promoters lack specificity in initiating gene expression, and long-term continuous expression consumes a lot of energy and material basis; endogenous low-temperature inducible promoters in plants often have other environmental factor response elements at the same time, and the initiation of gene expression lacks orthogonality. In the future, we need smart varieties that, when they are in a certain stress environment, the corresponding series of resistance genes are orthogonally expressed to systematically enhance the resistance of the plant; and when the stress disappears, the gene expression is turned off to save materials and energy.

[0004] The GAL4-VP16 transcription module, which is composed of the yeast GAL4 binding domain and the VP16 activation domain, has been proven to be able to successfully orthogonally regulate downstream gene expression in plants. This function depends on the GAL4 binding element (CCE) in the promoter region of the downstream gene. However, the continuous orthogonal activation of downstream gene expression by the GAL4-VP16 transcription module in the cell nucleus cannot achieve the effect of "intelligent regulation". Therefore, in response to low temperature stress, it is necessary to develop an intelligent low-temperature orthogonal regulatory module that can transfer from the cytoplasm to the cell nucleus in a low temperature environment to complete the regulation of downstream target genes, and then transfer from the cell nucleus back to the cytoplasm to shut down the regulation of downstream genes after the temperature rises, so as to achieve the effect of intelligent orthogonal regulation under low temperature stress. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a plant body low temperature response intelligent orthogonal regulation system and its application. The plant body low temperature response intelligent orthogonal regulation module of the present invention can intelligently select the required functional genes for expression according to the changes in the low temperature environment, laying a foundation for the cultivation of intelligent plant varieties that can resist low temperature stress.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a CRF(KR)-GAL4-VP16 regulatory module is provided, wherein the CRF(KR)-GAL4-VP16 regulatory module is a protein as shown in (A1) or (A2):

[0008] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing;

[0009] (A2) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0010] The protein described in (A1) can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0011] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. In order to facilitate the purification of the protein in (A1), a tag can be connected to the amino terminus or carboxyl terminus of the protein in (A1). The tag can be Poly-Arg (usually 6 RRRRR), Poly-His (usually 6 HHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).

[0012] The CRF (KR) -GAL4-VP16 regulatory module of the present invention is formed by connecting GAL4-VP16 and the C-terminus of the CRF (KR) protein through 2XLinker (GGGGSGGGGS), and has the nuclear-cytoplasmic shuttling property and transcription activation ability in response to low temperature.

[0013] The second aspect of the present invention provides a gene encoding the above-mentioned CRF(KR)-GAL4-VP16 regulatory module.

[0014] Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0015] The third aspect of the present invention provides a recombinant expression vector or a genetically engineered bacterium containing the above encoding gene.

[0016] The fourth aspect of the present invention provides the use of the above-mentioned CRF (KR) -GAL4-VP16 regulatory module, encoding gene, recombinant expression vector or genetically engineered bacteria in the following (1) or (2):

[0017] (1) Construct an intelligent orthogonal control system for plant response to low temperature;

[0018] (2) Cultivate smart plant varieties that can resist low temperature stress.

[0019] The fifth aspect of the present invention provides an intelligent orthogonal regulatory system for low temperature response in plants, comprising: a CRF(KR)-GAL4-VP16 regulatory module and a promoter that specifically recognizes and upregulates expression of GAL4-VP16.

[0020] Preferably, the nucleotide sequence of the promoter specifically recognized and up-regulated by GAL4-VP16 is as shown in SEQ ID NO.3.

[0021] The sixth aspect of the present invention provides the application of the above-mentioned intelligent orthogonal control system for low temperature response in plants in any one of the following (1)-(3):

[0022] (1) Regulate the expression of exogenous genes in plants to intelligently respond to low temperature stress;

[0023] (2) Regulate plant resistance to low temperature stress;

[0024] (3) Cultivate smart plant varieties that can resist low temperature stress.

[0025] In the above application, the intelligent response to low temperature stress refers to specifically starting the intelligent low temperature orthogonal regulatory module under low temperature stress to regulate the expression of downstream target genes; and after the temperature returns to normal, shutting down the regulation of downstream target genes.

[0026] In the above application, the plant smart variety can enter the cell nucleus from the cytoplasm under low temperature environment to identify and bind to the promoter of the target gene to activate its expression, and return to the cytoplasm from the cell nucleus after the temperature is restored, thereby shutting down the expression of the target gene.

[0027] A seventh aspect of the present invention provides a method for improving plant resistance to low temperature stress, comprising the following steps:

[0028] The CRF(KR)-GAL4-VP16 regulatory module, the promoter specifically recognized and upregulated by GAL4-VP16, and the exogenous low-temperature resistance gene are transferred into chassis plants through genetic transformation.

[0029] Preferably, the promoter is connected to the 5' end of the exogenous low temperature resistance gene so that it can be specifically recognized by CRF-GAL4-VP16 and activated for expression.

[0030] In the above method, the exogenous low-temperature resistance gene can be a low-temperature resistance gene reported in the prior art, for example: the gene WRKY51 (SlWRKY51 regulates proline content to enhance chilling tolerance in tomato) that regulates low-temperature tolerance in tomato, the low-temperature resistance genes TaSnRK1α and TaPAP6L (ATaSnRK1αmodulatesTaPAP6L-mediated wheat cold tolerance through regulatingendogenous jasmonic acid) in wheat, etc.; it can also be a newly discovered low-temperature resistance gene.

[0031] Beneficial effects of the present invention:

[0032] The present invention uses the idea of ​​synthetic biology to reconstruct CRF-GAL4-VP16, a transcription factor that can respond to low temperature, shuttle between nucleus and cytoplasm, and orthogonally regulate gene expression in plants. The effect of starting the expression of the target gene only in a low temperature environment and shutting down the expression after the temperature recovers is achieved, saving the material energy consumption of the chassis plant. This provides new ideas for the future engineering application of efficient artificial intelligence crops; and promotes the innovative development of synthetic biology technology in the construction of new and efficient artificial plants (crops) and related production technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :(A) Subcellular localization analysis of GAL4-VP16 protein with nuclear localization peptide (NSL) inserted. GFP: green fluorescent protein; Chl.: chlorophyll autofluorescence; DAPI: nuclear dye; Bright field: white field; Merged: fused image. (B) Assembly construction of GAL4-NSL-VP16 adaptive promoter; gray squares are elements specifically recognized and bound by GAL4; green and blue squares are the basic promoters of Arabidopsis LHCB2.4 and tomato RBCS1 genes, respectively; orange squares are reporter genes encoding luciferase (LUC). (C) Fluorescence imaging analysis and quantitative analysis of fluorescence signals; left: Fluorescence imaging analysis of tobacco leaf epidermal cells after empty GFP and GAL4-NSL-VP16-GFP were co-transformed with two promoters. Right: Quantitative analysis of fluorescence signals after empty GFP and GAL4-NSL-VP16-GFP were co-transformed with two promoters into tobacco leaf epidermal cells.

[0034] Figure 2:(A) Subcellular localization analysis of CRF(KR)-GAL4-VP16-GFP before and after low temperature treatment. GFP: green fluorescent protein; DAPI: nuclear dye; Bright field: white field; Merged: fused image. (B) Western bolt analysis of the changes in the content of CRF(KR)-GAL4-VP16 protein in the nucleus and cytoplasm before and after low temperature treatment. ACTIN is an internal reference control. (C) LUC reporter system analysis of CRF(KR) transcriptional regulatory function.

[0035] Figure 3 :(A) Imaging analysis of CRF(KR)-GAL4-VP16-GFP regulating the expression of proSlRBCS1:LUC before and after low temperature treatment; (B) Quantitative analysis of CRF(KR)-GAL4-VP16-GFP regulating the expression of proSlRBCS1:LUC before and after low temperature treatment. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0037] Terminology Note:

[0038] Orthogonality: refers to the ability of an element to function independently of chassis cells and other genetic elements. Many elements have inconsistent functional performance in different plant chassis cells and lack sufficient orthogonality. Element reconstruction can be achieved by simplifying internal components, removing sequences that cause inefficiency and sequence parts that are regulated by endogenous sources, while still retaining the biological function of the element.

[0039] As mentioned earlier, global climate change has made the problem of low temperature damage very prominent, and low temperature stress severely limits the yield and quality of cold-sensitive crops. However, at present, the improvement of crop cold resistance from the genetic level is mostly through the continuous expression of resistance genes through strong promoters such as CaMV 35S and UBQ10, or through the induction of resistance gene expression through endogenous low temperature inducible promoters in plants. Strong promoters lack specificity in initiating gene expression, and long-term continuous expression consumes a lot of energy and material basis; endogenous low temperature inducible promoters in plants often have other environmental factor response elements at the same time, and the initiation of gene expression lacks orthogonality. Therefore, we need smart crops in the future. When they are in a certain stress environment, the corresponding series of resistance genes are orthogonally expressed to systematically improve the resistance of the plant; when the stress disappears, the gene expression is turned off to save materials and energy.

[0040] Based on this, the present invention designs and develops a low temperature responsive intelligent orthogonal regulatory module CRF(KR)-GAL4-VP16, whose amino acid sequence is shown in SEQ ID NO.1, and is as follows:

[0041] MDAKFAGKPIQAQFYSGQTNLEQMQDTPTRGARHRRAQSETFFRFPSFDDDMLLDDVVSDFSLDVQAPTLMQPANSPDSSSTGPALSAGPSDYPKPLAHYRSLSVDADFFDGLDFGGVPTEKKLMGSEPR HRHSNSMDGSFDTSFESESISVKKAMAPDRLAELSLIDPKRAKRILANRQSAARSRERKTRYTSELERKVQTLQTEATTLSAQITVLQRDTSGLNAENKELKFRLQALEQQAHLRDALNDTLREELQRL KIEAGQLPSANGNRGMHGGGGSGGGGSMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVN KDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSMGGGGGSAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG.

[0042] The module is composed of CRF (KR) and GAL4-VP16 connected by 2XLinker (GGGGSGGGGS). The present invention commissioned BGI to synthesize the optimized CRF (KR)-GAL4-VP16 sequence, the nucleotide sequence of which is shown in SEQ ID NO.2, as follows:

[0043] ATGGACGCTAAGTTCGCCGGAAAGCCCATTCAGGCCCAATTTTATTCCGGTCAAACT

[0044] AACCTCGAACAGATGCAGGATACTCCAACCCGCGGGGCACGCCACCGCCGTGCACA

[0045] ATCCGAAACCTTTTTCCGTTTTCCCAGTTTTGATGATGATATGCTTTTAGACGATGTC

[0046] GTTTCTGACTTCAGTCTCGATGTTCAGGCTCCGACCCTCATGCAGCCGGCTAATTCG

[0047] CCTGATTCTTCGTCTACTGGACCAGCGTTGTCGGCTGGACCTTCCGATTACCCTAAG

[0048] CCGCTGGCTCACTACCGTAGTCTATCTGTTGATGCTGATTTTTTCGATGGACTGGATT

[0049] TTGGTGGTGTGCCGACGGAGAAGAAACTGATGGGTTCTGAGCCACGTCATAGGCAT

[0050] AGCAATTCTATGGATGGGTCTTTTGATACGTCGTCGTTTGAGTCTGAATCGATTTCT

[0051] GTTAAGAAGGCTATGGCTCCTGATAGGCTTGCTGAGCTGTCCTTGATTGATCCCAAA

[0052] AGAGCTAAAAGGATCCTAGCAAACAGACAATCTGCTGCACGTTCCAGAGAGCGAA

[0053] AAACTCGTTACACTAGTGAGCTAGAGAGGAAAGTGCAGACTCTGCAGACTGAAGCC

[0054] ACCACTCTATCCGCTCAGATCACAGTTCTACAGAGAGACACTTCTGGACTAAATGCT

[0055] GAAAACAAAGAACTGAAATTTCGGTTGCAAGCTTTGGAACAACAAGCACACCTTAG

[0056] AGATGCTCTAAATGATACGTTGAGGGAAGAACTGCAGCGGCTTAAGATTGAAGCAG

[0057] GCCAACTTCCATCTGCAAATGGAAACAGAGGAATGCATGGGGGAGGAGGATCAGG

[0058] TGGTGGTGGATCAATGAAATTGCTTTCTTCAATAGAGCAAGCTTGCGATATTTGTAG

[0059] ACTTAAGAAGTTGAAGTGTTCTAAGGAGAAGCCTAAGTGTGCTAAGTGTCTTAAAA

[0060] ATAATTGGGAATGTAGGTATAGCCCTAAAACTAAAAGATCACCACTTACTAGAGCT

[0061] CACTTGACTGAAGTTGAATCTAGGCTTGAAAGGCTTGAACAACTTTTCCTTTTGATT

[0062] TTTCCAAGAGAGGACCTTGATATGATTTTGAAAATGGATTCTCTTCAGGATATCAAG

[0063] GCATTGCTTACAGGCTTGTTTGTGCAGGATAATGTTAATAAGGATGCTGTGACTGAT

[0064] AGATTGGCTTCTGTTGAAACTGATATGCCACTTACTCTTAGGCAGCATAGAATTTCA

[0065] GCAACTAGCTCTTCTGAAGAATCTTCTAATAAAGGTCAGAGACAACTTACTGTTTCT

[0066] ATGGGAGGAGGAGGAGGATCTGCTCCACCTACAGATGTTAGTCTTGGAGATGAACT

[0067] TCATTTGGATGGTGAAGATGTTGCAATGGCTCATGCAGATGCTCTTGATGATTTTGA

[0068] TTTGGATATGCTTGGTGACGGAGATTCTCCTGGTCCTGGTTTTACACCACATGATTC

[0069] TGCTCCATATGGTGCTCTTGATATGGCTGATTTTGAGTTTGAGCAAATGTTTACAGATGCTTTGGGTATTGATGAATATGGTGGATGA (SEQ ID NO. 2).

[0070] The low temperature response intelligent orthogonal regulatory module CRF(KR)-GAL4-VP16 of the present invention is composed of CRF(KR) with nucleocytoplasmic shuttling properties and GAL4-VP16 with transcriptional activation properties. Among them:

[0071] CRF comes from tomatoes and can respond to low temperature treatment and enter the cell nucleus to function in a low temperature environment; and return to the cytoplasm after the temperature rises. The present invention transforms CRF and replaces the key amino acid K at position 187 that performs transcriptional regulation function with R through overlap technology or artificial synthesis to obtain CRF (KR), which only has the function of nuclear-cytoplasmic shuttling, but not the transcriptional regulation function in the cell nucleus.

[0072] GAL4-VP16 is a transcriptional regulatory factor in the yeast system. In order to enable it to play an orthogonal regulatory function in plants, the present invention optimizes it according to codon preference. Then, CRF (KR) and GAL4-VP16 are connected by 2XLinker (GGGGSGGGGS) to construct a low temperature responsive intelligent orthogonal regulatory module CRF (KR) -GAL4-VP16.

[0073] Furthermore, in order to realize the expression of exogenous low-temperature resistance genes in plants in an intelligent response to low-temperature stress, based on the CRF (KR) -GAL4-VP16 regulatory module, the present invention further constructs an intelligent orthogonal regulatory system for low-temperature response in plants, including: a CRF (KR) -GAL4-VP16 regulatory module and a promoter (CRE) that specifically recognizes and upregulates expression of GAL4-VP16.

[0074] By screening the promoters, it was found that the proSlRBCS1 promoter (SEQ ID NO. 3) can be specifically recognized and activated by GAL4-VP16. The promoter was connected to the 5' end of the exogenous low temperature resistance gene so that it could be specifically recognized and activated by CRF-GAL4-VP16.

[0075] In summary, CRF(KR) can successfully carry GAL4-VP16 into the cell nucleus under low temperature conditions and activate the expression of exogenous low temperature resistance genes through the CRE promoter; under normal temperature conditions, CRF(KR)-GAL4-VP16 returns to the cytoplasm, shutting down the regulatory effect.

[0076] The CRF(KR)-GAL4-VP16 module of the present invention can be used in the field of plant genetic engineering. By adding CRE to the 5' end of the target gene and transferring it together with CRF(KR)-GAL4-VP16 into the plant, the intelligent orthogonal regulation expression of the gene in a low temperature environment can be achieved. This is very important for optimizing the cold tolerance of plants and cultivating intelligent cold-tolerant plants.

[0077] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0078] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. If the specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are usually followed, such as J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd Edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Guide, Science Press, 2001; or the conditions recommended by the manufacturer.

[0079] Example 1: Screening of CREs compatible with GAL4-VP16

[0080] 1. Test method:

[0081] The GAL4-VP16 from the yeast system was codon optimized so that it could play an orthogonal regulatory function in plants. The coding sequence of the codon-optimized GAL4-VP16 is shown in SEQ ID NO.4, which is as follows:

[0082] .

[0083] The overlap technique was used to insert a nuclear localization peptide sequence (CCTAAAAAGAAAAGGAAGGTT) between GAL4 and VP16, so that the encoded protein (GAL4-NSL-VP16) could enter the nucleus smoothly. Then the coding sequence of GAL4-NSL-VP16 was synthesized by BGI, and connected to the pSuper1300-GFP vector through SalI and KpnI digestion to construct the plant expression vector GAL4-NSL-VP16-GFP for subcellular localization analysis.

[0084] Based on the degeneracy of GAL4 recognition elements and the basic promoters of plant endogenous genes (Arabidopsis LHCB2.4 and tomato RBCS1), proSIRBCS1:LUC and proAtLHCB2.4:LUC were synthesized by BGI and ligated to the pGreen0800 vector through KpnI and SalI digestion to construct plant expression vectors (proSIRBCS1:LUC and proAtLHCB2.4:LUC) carrying LUC reporter genes. They were co-transfected into tobacco leaf epidermal cells with GAL4-NSL-VP16-GFP, and fluorescence imaging analysis was performed to detect the LUC signal intensity.

[0085] The empty GFP group was used as a negative control.

[0086] 2. Test results:

[0087] Subcellular localization analysis showed that the optimized GAL4-NSL-VP16-GFP was successfully expressed and localized in the cell nucleus ( Figure 1 A).

[0088] The promoter assembly was constructed as Figure 1 B. The fluorescence imaging and LUC signal intensity detection results after co-transfection into tobacco leaf epidermal cells are shown in Figure 1 As shown in Figure C, the results show that GAL4-NSL-VP16-GFP can successfully activate the expression of LUC through the recombinant promoter; the signal difference of proSlRBCS1 promoter (SEQ ID NO.3) is greater. Therefore, proSlRBCS1 promoter was selected for subsequent experiments.

[0089] Example 2: Construction and functional investigation of CRF(KR)-GAL4-VP16

[0090] 1. Experimental methods:

[0091] 1.1. Construction of vector

[0092] Artificially synthesized CRF(KR)-GAL4-VP16 (synthesized by BGI) was constructed into pSuper1300-GFP and pZP211-FLAG plant expression vectors by restriction digestion and ligation. CRF-GAL4-VP16 (synthesized by BGI) was used as a control and was also ligated into pSuper1300-GFP vector by restriction digestion. At the same time, the proAtMYB44 promoter was amplified from the Arabidopsis genome and constructed into the binary LUC reporter system, pGreen0800 vector by restriction digestion and ligation: the above vectors were transformed into Agrobacterium strain GV3101 by freeze-thaw method, and then co-injected into tobacco leaf epidermal cells by Agrobacterium-mediated method.

[0093] 1.2. Analysis of low temperature-induced CRF(KR)-GAL4-VP16 nuclear-cytoplasmic shuttling.

[0094] After the above tobacco was cultured under normal growth conditions for 2-3 days, the leaves of tobacco injected with CRF(KR)-GAL4-VP16-GFP were taken before low temperature treatment, 30 minutes after low temperature treatment, and 30 minutes after low temperature treatment for fluorescence observation by two-photon confocal microscope (Zeiss, Germany). The leaves of tobacco injected with CRF(KR)-GAL4-VP16-FLAG were taken before low temperature treatment, 30 minutes after low temperature treatment, and 30 minutes after low temperature treatment for extraction of nuclear protein and cytoplasmic protein, and Western blot experiments were performed using FLAG antibody to detect the changes in the content of CRF(KR)-GAL4-VP16-FLAG in the nucleus and cytoplasm.

[0095] 1.3 Analysis of transcriptional activity of CRF(KR)

[0096] Agrobacterium containing proAtMYB44:LUC and CRF(KR)-GAL4-VP16-GFP and CRF-GAL4-VP16-GFP were co-injected into tobacco epidermal cells. After the tobacco was cultured under normal growth conditions for 2-3 days, the leaves were imaged using an in vivo imaging system (Xenogene, USA) and detected using a dual-luciferase reporter gene detection kit (Full Gold, FR201-01-V2).

[0097] 2. Planting method: Tobacco (Nicotiana benthamiana) was planted in a greenhouse with 16 hours of sunshine and 8 hours of dark cultivation. The cultivation temperature was 25°C and the air humidity was 60-70%. Two E-41L2 Percival incubators were used for low temperature treatment and normal temperature recovery treatment of tobacco.

[0098] The greenhouse is located in the State Key Laboratory of Crop Biology, Shandong Agricultural University.

[0099] 3. Test results:

[0100] Fluorescence observation analysis and Western blot experiments showed that CRF(KR)-GAL4-VP16-GFP still has the characteristics of being enriched in the nucleus at low temperature (4°C) and in the cytoplasm at normal temperature (25°C). Figure 2A, B). The proAtMYB44 promoter has been shown to be specifically recognized and activated by the CRF Arabidopsis homologous gene. We used the LUC reporter system to test whether CRF(KR) had lost its own transcriptional activation function. Compared with CRF, CRF(KR) could no longer activate the expression of LUC through the proAtMYB44 promoter ( Figure 2 C).

[0101] Example 3: Analysis of the regulation of LUC reporter gene by CRF(KR)-GAL4-VP16 module in tobacco epidermal cells

[0102] 1. Experimental methods:

[0103] 1.1. Construction of CRF(KR)-GAL4-VP16 module vector

[0104] Artificially synthesized CRF(KR)-GAL4-VP16 (synthesized by BGI) was constructed into the pSuper1300-GFP plant expression vector by restriction digestion and ligation. At the same time, CRE was constructed into the binary LUC reporter system, pGreen0800 vector by restriction digestion and ligation: the above vectors were transformed into Agrobacterium strain GV3101 by freeze-thaw method, and then co-injected into tobacco leaf epidermal cells by Agrobacterium-mediated method. GFP+CRE-LUC was used as negative control.

[0105] 1.2. Expression analysis of LUC reporter gene.

[0106] After the above tobacco was cultured under normal growth conditions for 2-3 days, a portion of the leaves were imaged using an in vivo imaging system (In vivo imaging system, Xenogene, USA) and detected using a dual luciferase reporter gene detection kit (Full Gold, FR201-01-V2). Another portion of the tobacco was placed in a low temperature (4°C) light incubator for 30 minutes and then a portion was used for detection, and the other portion was transferred to a normal temperature (25°C) light incubator for recovery for 30 minutes before detection.

[0107] 2. Planting method: Tobacco (Nicotiana benthamiana) was planted in a greenhouse with 16 hours of sunshine and 8 hours of dark cultivation. The cultivation temperature was 25°C and the air humidity was 60-70%. Two E-41L2 Percival incubators were used for low temperature treatment and normal temperature recovery treatment of tobacco.

[0108] The greenhouse is located in the State Key Laboratory of Crop Biology, Shandong Agricultural University.

[0109] 3. Experimental results

[0110] Results Figure 3Imaging (A) and quantitative analysis (B) of LUC expression showed that CRF(KR)-GAL4-VP16 could induce LUC expression in large quantities in a low temperature environment, but when the temperature returned to normal, the activation effect was significantly weakened or even disappeared.

[0111] The above experimental results confirm that the CRF(KR)-GAL4-VP16 module can intelligently and orthogonally regulate the expression of target genes with CRE promoter under low temperature stress.

[0112] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A CRF(KR)-GAL4-VP16 regulatory module, characterized in that: The CRF(KR)-GAL4-VP16 regulatory module is a protein as shown below (A1) or (A2): (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; (A2) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. The coding gene of the CRF(KR)-GAL4-VP16 regulatory module according to claim 1.

3. The coding gene according to claim 1, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

4. A recombinant expression vector or genetically engineered bacteria containing the coding gene according to claim 2 or 3.

5. Use of the CRF(KR)-GAL4-VP16 regulatory module of claim 1, the coding gene of claim 2 or 3, or the recombinant expression vector or genetically engineered bacteria of claim 4 in the following (1) or (2): (1) Construct an intelligent orthogonal control system for plant response to low temperature; (2) Cultivate smart plant varieties that can resist low temperature stress.

6. An intelligent orthogonal control system for low temperature response in plants, characterized in that: include: CRF(KR)-GAL4-VP16 regulatory module and the promoter that GAL4-VP16 specifically recognizes and upregulates expression.

7. The intelligent orthogonal control system for low temperature response in plants according to claim 6, characterized in that: The nucleotide sequence of the promoter specifically recognized and up-regulated by GAL4-VP16 is shown in SEQ ID NO.

3.

8. Application of the intelligent orthogonal control system for low temperature response in plants according to claim 6 or 7 in any one of the following (1)-(3): (1) Regulate the expression of exogenous genes in plants to intelligently respond to low temperature stress; (2) Regulate plant resistance to low temperature stress; (3) Cultivate smart plant varieties that can resist low temperature stress.

9. A method for improving plant resistance to low temperature stress, characterized in that: The following steps are involved: The CRF(KR)-GAL4-VP16 regulatory module, the promoter specifically recognized and upregulated by GAL4-VP16, and the exogenous low-temperature resistance gene are transferred into chassis plants through genetic transformation.

10. The method according to claim 9, characterized in that The promoter was connected to the 5' end of the exogenous cold resistance gene.

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