A high-temperature inducible plant promoter pAcHSR and its application

Abstract

The present invention provides a high-temperature inducible plant promoter pAcHSR, the nucleotide sequence of which is shown in SEQ ID NO.1. The present invention also provides a recombinant expression vector and a recombinant bacterium containing the above promoter pAcHSR, respectively. Meanwhile, the present invention provides an application of the promoter pAcHSR in cultivating plants resistant to high-temperature stress and a method for cultivating plants resistant to high-temperature stress. The present invention screened and finally determined the high-temperature inducible plant promoter pAcHSR in the kiwifruit gene bank; the promoter pAcHSR of the present invention drives the transcription and translation of the heat shock transcription factor AcHSFA2-1 through high-temperature induction, improves the high-temperature stress resistance of plants, and has great application prospects for cultivating plants resistant to high-temperature stress and creating plant germplasm resources with high resistance to cope with global climate change.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and particularly to a heat-induced plant promoter pAcHSR and its application. Background Art

[0002] The change of the global environmental temperature has increased the frequency of extreme high-temperature events, and the resulting high-temperature stress has greatly restricted the growth of plants. In this context, breeding new plant varieties resistant to high-temperature stress is of great significance.

[0003] According to existing research reports, when encountering high-temperature stress, plants will rapidly produce various physiological changes to respond to the stress and reduce the harm of high-temperature stress to growth. Heat shock transcription factors (HSFs) are the transcription factor family most closely related to the response to high-temperature stress in plants. Therefore, based on the function of heat shock transcription factors and combined with transgenic technology to cultivate germplasm materials resistant to high-temperature stress, there is great feasibility.

[0004] However, in the operation of transgenic technology, it is necessary to select a suitable promoter to drive the expression of the target gene (heat shock transcription factor). A promoter is a DNA sequence and related regulatory elements that provide recognition and binding sites for RNA polymerase, usually located upstream of the gene and driving gene transcription.

[0005] According to the working principle of promoters, they can be generally divided into three categories: constitutive promoters, tissue-specific promoters, and inducible promoters. Among them, the target genes driven by constitutive promoters can theoretically be highly expressed in all developmental stages and all tissues and organs of plants, and are commonly used in plant gene function research; however, the continuous overexpression of target genes by constitutive promoters will, to a large extent, increase the metabolic burden of transgenic plants, hinder plant growth and reduce yield. Tissue-specific promoters and inducible promoters can drive the expression of target genes only in specific tissue sites or under the condition of the presence of inducer factors, thus avoiding the continuous overexpression of the target protein from affecting the normal growth of plants. In particular, inducible promoters are promoters formed during the evolution of plants that can respond to special biological, physical or chemical signals; in the absence of inducer factors, the target genes driven by inducible promoters do not express or have background expression; when inducer factors appear, the activity of inducible promoters is enhanced, and the expression level of the driven target genes increases significantly.

[0006] Therefore, seeking a suitable heat-induced plant promoter has become the key to cultivating germplasm materials resistant to high-temperature stress by combining transgenic technology. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a high-temperature inducible plant promoter pAcHSR and its application.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A high-temperature inducible plant promoter pAcHSR, the nucleotide sequence of the promoter pAcHSR is shown in SEQ ID NO.1.

[0010] As one of the preferred embodiments of the present invention, the promoter pAcHSR is derived from Actinidia chinensis.

[0011] As one of the preferred embodiments of the present invention, the promoter pAcHSR is used to drive the transcription and translation of the heat shock transcription factor AcHSFA2-1; and the transcriptional abundance of AcHSFA2-1 driven by the promoter pAcHSR is significantly increased after induction at 35-55 °C for 1 h, and there is no transcriptional abundance at normal temperature; meanwhile, the transcriptional activity of the promoter pAcHSR is enhanced within 30 min of induction at 35-55 °C.

[0012] A recombinant expression vector containing the above high-temperature inducible plant promoter pAcHSR.

[0013] As one of the preferred embodiments of the present invention, the recombinant expression vector is specifically pGreen II 0800-LUC-pAcHSR, and the construction method is as follows: the promoter pAcHSR sequence is carried on the pGreen II 0800-LUC vector digested with HindIII and NcoI.

[0014] A recombinant bacterium containing the above high-temperature inducible plant promoter pAcHSR.

[0015] As one of the preferred embodiments of the present invention, the construction method of the recombinant bacterium is as follows: the recombinant expression vector pGreen II 0800-LUC-pAcHSR constructed with the promoter pAcHSR is introduced into Agrobacterium tumefaciens GV3101.

[0016] An application of the above high-temperature inducible plant promoter pAcHSR in cultivating plants resistant to high-temperature stress.

[0017] A method for cultivating plants resistant to high-temperature stress, the above high-temperature inducible plant promoter pAcHSR is introduced into a target plant to obtain a transgenic plant; the transgenic plant has higher resistance to high-temperature stress than the target plant.

[0018] As one of the preferred embodiments of the present invention, the target plant is Actinidia chinensis or tobacco.

[0019] The advantages of the present invention compared with the prior art are as follows:

[0020] In the kiwifruit gene bank, the present invention screens various genes that can significantly respond to high-temperature stress treatment, and finally determines the high-temperature inducible plant promoter pAcHSR; the promoter pAcHSR of the present invention drives the transcription and translation of the heat shock transcription factor AcHSFA2-1 through high-temperature induction, improving the high-temperature stress resistance of plants; among them, the transcriptional activity of the promoter pAcHSR can be rapidly and extremely significantly induced and enhanced by high temperature; the transcriptional abundance of AcHSFA2-1 driven by the promoter pAcHSR significantly increases after 1 h of high-temperature induction and has no expression under normal temperature conditions. Therefore, the research and discovery of the high-temperature inducible plant promoter pAcHSR of the present invention have great application prospects for cultivating plants resistant to high-temperature stress and creating plant germplasm resources with high resistance to cope with global climate change. Brief Description of the Drawings

[0021] Figure 1 It is the change of transcriptional abundance of AcHSFA2-1 driven by pAcHSR during the response of kiwifruit to different temperature treatments;

[0022] Figure 2 It is a schematic diagram of the expression vector containing the pAcHSR sequence;

[0023] Figure 3 It is the result diagram of verifying the activity of the high-temperature inducible promoter pAcHSR based on dual luciferase (in the figure, "**" indicates p < 0.01, and "***" indicates p < 0.001);

[0024] Figure 4 It is the result diagram of verifying the activity of the high-temperature inducible promoter pAcHSR based on LUC luciferin signal imaging. Detailed Embodiments

[0025] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, for the reagent products and experimental methods used in the following embodiments, unless otherwise specified, they are all conventional reagents or methods in the art and will not be elaborated.

[0026] Example 1

[0027] A high-temperature inducible plant promoter pAcHSR in this example:

[0028] The nucleotide sequence of the promoter pAcHSR is shown in SEQ ID NO.1.

[0029] The promoter pAcHSR is derived from Actinidia chinensis, and the cloning steps are as follows:

[0030] (1) Extraction of Actinidia chinensis genomic DNA

[0031] Using the leaves of 'Donghong' Actinidia chinensis as the research material, genomic DNA (gDNA) was extracted by the CTAB method. Weighed 0.1 g of the sample powder ground in liquid nitrogen into a 2 mL centrifuge tube, added 1 mL of CTAB extraction solution preheated at 65 °C and 50 μL of β-mercaptoethanol, and vortexed thoroughly; incubated in a water bath at 65 °C for 10 min, centrifuged at 10000 rpm for 10 min; aspirated the supernatant, added 1 mL of 24:1 (chloroform:isoamyl alcohol), vortexed thoroughly and then centrifuged at 4 °C and 10000 rpm for 10 min; repeated once; aspirated 600 μL of the supernatant into a 1.5 mL centrifuge tube, added 600 μL of isopropanol, placed at -20 °C for 1 h and then centrifuged at 4 °C and 10000 rpm for 10 min; added 500 μL of 75% ethanol to suspend the precipitate, then centrifuged at 10000 rpm for 5 min, poured off and aspirated the remaining liquid at the bottom of the tube; after standing for 10 min, added 100 μL of DEPC water to dilute and mix evenly, and stored at -20 °C.

[0032] (2) Cloning of the pAcHSR sequence

[0033] The template used for cloning the promoter pAcHSR sequence was the gDNA of the 'Donghong' Actinidia chinensis plant, and the primers used were primer 1 (SEQ ID NO.2) and primer 2 (SEQ ID NO.3). The sequence cloning was completed with reference to the instructions of Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China). The target fragment was purified using 1.2% agarose gel electrophoresis in combination with a gel DNA recovery kit (SIMGEN, China).

[0034] After the purified target fragment was ligated to the pClone007 vector with reference to the instructions of the pClone007 Versatile Simple Vector Kit, it was transformed into DH5α Escherichia coli and spread on an LB solid medium containing ampicillin (150 mg / L) and cultured overnight at 37 °C.

[0035] Single colonies were picked for positive identification, and the positive single colonies were sent to Shangya Biotechnology (Hangzhou) for sequencing. Then, the obtained sequence was compared with the downloaded reference sequence through the software DNAMAN to confirm the correctness of the cloned sequence.

[0036] Example 2

[0037] Construction of a recombinant expression vector pGreen II 0800-LUC-pAcHSR in this example:

[0038] Using the pClone007 plasmid containing the correct pAcHSR promoter sequence as a template, the pAcHSR sequence was subcloned with reference to the instructions of Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China). The primers used were primer 3 (SEQ ID NO.4) and primer 4 (SEQ ID NO.5). The purified pAcHSR sequence was ligated into the pGreen II 0800-LUC (LUC) vector digested with HindIII and NcoI. Monoclonal colonies were picked for positive identification, and the positive monoclonal colonies were sent to Shangya Biology (Hangzhou) for sequencing verification.

[0039] Example 3

[0040] Construction of a recombinant bacterium in this example:

[0041] The recombinant expression vector pGreen II 0800-LUC-pAcHSR plasmid was introduced into Agrobacterium tumefaciens GV3101 by the freeze-thaw method.

[0042] Example 4

[0043] Obtaining of a pAcHSR-transformed plant in this example:

[0044] The recombinant Agrobacterium tumefaciens of Example 3 was spread on an LB solid medium containing kanamycin (50 mg / L) and gentamicin (25 mg / L) and cultured at 28 °C for 2 d. A small amount of bacteria was scraped and re-spread on a new LB solid medium containing the same antibiotics and cultured at 28 °C for 12 h. The bacteria activated through two rounds were suspended with an infiltration solution (containing 10 mM MES, pH 5.6; 10 mM MgCl2; 150 μM AS) and then injected into Nicotiana benthamiana.

[0045] Experimental Example 1

[0046] This experimental example was used to analyze the expression pattern of AcHSFA2-1 driven by pAcHSR in kiwifruit:

[0047] I. Experimental method:

[0048] (1) Treatment of kiwifruit plants

[0049] Select tissue culture seedlings of 'Donghong' (A. chinensis cv. Donghong) with consistent growth as the research materials. After the plants grew to 5 leaves under the conditions of 25°C and a light:dark cycle of (16 h:8 h), they were respectively treated at 35°C, 45°C, and 55°C for 1 h and 2 h. Tissue culture seedlings that had been growing at 25°C were used as the control. Each treatment included 3 biological replicates, and each biological replicate included at least 5 tissue culture seedlings.

[0050] After the plants were treated at different temperatures, leaves were immediately collected into 5 mL cryotubes, frozen with liquid nitrogen, and then stored in an ultra-low temperature freezer at -80°C.

[0051] (2) Construction of transcriptome library

[0052] Collect the treated kiwifruit leaves, grind them into powder, weigh 0.1 g, add 4 mL of CTAB extraction buffer (containing 5% β-mercaptoethanol) preheated at 65°C, vortex evenly, and heat in a 65°C water bath for 10 min. After centrifuging at 10000 rpm at room temperature for 10 min, transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (V:V = 24:1), vortex for 1 min, and then centrifuge at 15°C and 10000 rpm for 10 min. Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (V:V = 24:1), vortex evenly, and repeat centrifugation. After transferring the supernatant to a new centrifuge tube again, add 1 / 5 volume of 12 M lithium chloride, invert and mix evenly, and then place it in a 4°C refrigerator overnight, for 12 - 16 h. Centrifuge the mixture at 4°C and 10000 rpm for 30 min. Wash the precipitate twice with 500 μL of 75% ethanol (diluted with DEPC water), dry for 10 min, and dissolve and mix evenly with 30 μL of DEPC water. Use a NanoDrop One micro ultraviolet-visible spectrophotometer (Thermo Scientific, USA) to detect the concentration and purity of RNA, use 1.2% agarose gel electrophoresis to detect the quality of RNA, and store it in an ultra-low temperature freezer at -80°C.

[0053] For kiwifruit samples with four temperatures, two treatment durations, and three biological replicates, a total of 24 libraries were subjected to transcriptome sequencing. After accurately detecting that the RNA integrity was qualified by an Agilent 2100 bioanalyzer, use the kit Ultra TM RNA Library Prep Kit for (NEB, USA) to construct the library. Sequencing and bioinformatics analysis were completed by Beijing Novogene Bioinformatics Technology Co., Ltd., and the sequencing platform was illumina NovaSeq 6000 (illumina, USA).

[0054] To ensure the quality and reliability of data analysis, the raw data was filtered, and the filtering content is as follows: reads with adapters were removed; reads containing N (where N represents undetermined base information) were removed; low-quality reads were removed. After quality control, qualified sequences (clean reads) were obtained, and the qualified sequences were aligned with the Red5 (A. chinensis, Red5) genome using the Hisat2 software. The gene transcript abundance was estimated by fragments per kilobase of exon per million mapped reads (FPKM). Genes with a fold change (FC) greater than or equal to 2 and a false discovery rate (FDR) less than 0.01 were screened as differentially expressed genes (DEGs).

[0055] II. Experimental results:

[0056] The nucleotide sequence of the heat shock transcription factor AcHSFA2-1 is shown in SEQ ID NO.6, and the encoded amino acid sequence is shown in SEQ ID NO.7.

[0057] Based on the transcriptome sequencing results, it was found that the transcriptional abundance of the heat shock transcription factor AcHSFA2-1 driven by pAcHSR in kiwifruit leaves was significantly affected by temperature changes, as Figure 1 shown: Under the growth condition of 25 °C, AcHSFA2-1 was hardly expressed, and its transcriptional abundance value could not be detected; after treatment at 35 °C for 1 h, the expression level of AcHSFA2-1 increased significantly; while after treatment at 45 °C and 55 °C for 1 h, the expression level of AcHSFA2-1 increased extremely significantly, and the transcriptional abundance value at 45 °C for 1 h reached 13,000 times that at 25 °C for 1 h.

[0058] This indicates that the activity of the promoter pAcHSR can respond rapidly and strongly to the induction of high temperature.

[0059] Experimental Example 2

[0060] This experimental example was used to analyze the cis-acting elements in the pAcHSR sequence:

[0061] I. Experimental method:

[0062] The pAcHSR sequence cloned in Example 1 was used to predict the cis-acting elements contained therein and their functions using the online software PlantCARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).

[0063] II. Experimental results:

[0064] The analysis results of representative cis - acting elements in the pAcHSR sequence are shown in Table 1.

[0065] Table 1 Representative cis - acting elements in the pAcHSR sequence

[0066]

[0067]

[0068] Based on the analysis results of cis - acting elements, it was found that: the pAcHSR sequence contains various cis - acting elements that respond to various stress signals, such as temperature and light, as well as cis - acting elements that respond to various phytohormone signals, such as abscisic acid, methyl jasmonate, gibberellin, etc.

[0069] Experimental Example 3

[0070] This experimental example is used to verify the activity of the high - temperature - induced promoter pAcHSR of the present invention:

[0071] I. Experimental method:

[0072] (1) Dual - luciferase assay

[0073] The tobacco plants injected with GV3101 Agrobacterium containing pGreen II 0800 - LUC - pAcHSR (obtained in Example 4) were placed in an artificial climate chamber with constant temperature and humidity (light intensity was 300 μmol m -2 s -1 , temperature was 25 °C, humidity was 80%, and light - dark cycle was 16h:8h) and cultivated for 72 h. Then, high - temperature conditions of 45 °C were set to treat the tobacco plants for 30 min, 60 min, 90 min, and 120 min respectively. Referring to the Reporter Assay System (Promega, USA) manual, the activities of firefly luciferase (LUC) and renilla luciferase (REN) in the leaves were detected using GloMax96 (Promega, USA), and the ratio was calculated. Each result included three biological replicates.

[0074] (2) LUC luciferin signal imaging analysis

[0075] The tobacco plants injected with GV3101 Agrobacterium containing pGreen II 0800 - LUC - pAcHSR (obtained in Example 4) were placed in an artificial climate chamber with constant temperature and humidity (light intensity was 300 μmol m -2 s -1, cultivate for 72 h at a temperature of 25 °C, a humidity of 80%, and a light-dark cycle of 16 h:8 h. Then, set a high temperature condition of 45 °C to treat the tobacco plants for 90 min, and then inject 0.2 mM luciferin at the position where the Agrobacterium is injected. After placing it in the dark for half an hour, use the NightSHADE LB 985 instrument to detect the LUC fluorescence signal.

[0076] II. Experimental results:

[0077] (1) The present invention successfully constructed an expression vector "pGreen II 0800-LUC-pAcHSR" containing the pAcHSR sequence ( Figure 2 ): The vector is based on the pGreen II 0800-LUC vector and contains the CaMV 35S::REN expression cassette and the pAcHSR::LUC expression cassette. Among them, REN driven by the promoter CaMV 35S has a stable expression level and can be used as an internal reference; while the change in the expression level of LUC indicates the change in the activity of the promoter pAcHSR.

[0078] (2) The results of verifying the activity of the high-temperature-induced promoter pAcHSR based on dual luciferase are as Figure 3 shown. From the ratio of firefly luciferase (LUC) to renilla luciferase (REN) Figure 3 : After treatment at 45 °C for 30 min, the activity of pAcHSR can be significantly enhanced by 5 times; after treatment for 90 min, the activity of pAcHSR can be enhanced by 40 times.

[0079] (3) The imaging results of the LUC luciferin signal are as Figure 4 shown. From the visualization results of the luciferin signal Figure 4 : Compared with the tobacco grown under the condition of 25 °C, the LUC luciferin signal in the tobacco leaves treated at 45 °C for 90 min is extremely significantly enhanced.

[0080] This confirms that high temperature can rapidly and strongly induce the activity of the promoter pAcHSR.

[0081] In summary, the present invention provides a high-temperature inducible promoter pAcHSR derived from kiwifruit. The transcriptional abundance of the heat shock transcription factor AcHSFA2-1 driven by pAcHSR is hardly detectable in kiwifruit leaves under normal growth conditions, while its transcriptional abundance is extremely significantly enhanced by nearly 13,000 times under the condition of 45 °C high temperature. The pAcHSR sequence contains various cis-acting elements that respond to signals such as temperature, light, and hormones. The transcriptional abundance and protein expression level of the firefly luciferase-encoding gene LUC driven by pAcHSR are extremely low in tobacco leaves under normal growth conditions, while after treatment at 45 °C for 30 min, the transcriptional abundance of LUC is significantly increased by nearly 5 times; after treatment for 90 min, the transcriptional abundance of LUC is significantly increased by nearly 40 times, and the signal intensity of luciferin is extremely significantly enhanced. The experimental results confirm that the transcriptional activity of pAcHSR can be rapidly and extremely significantly induced and enhanced by high temperature, and it can be used for plant-derived inducible expression vectors, which has great application prospects for creating high-resistant plant germplasm resources to cope with global climate change.

[0082] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A high temperature inducible plant promoter pAcHSR , characterized in that, The promoter pAcHSR The nucleotide sequence is shown in SEQ ID NO.

1.

2. The high temperature inducible plant promoter according to claim 1 pAcHSR , characterized in that, The promoter pAcHSR Derived from kiwi fruit.

3. The high temperature inducible plant promoter according to claim 1 pAcHSR , characterized in that, The promoter pAcHSR Used to drive heat shock transcription factors AcHSFA2-1 Transcription and translation of pAcHSR Driven AcHSFA2-1 The transcription abundance of the promoter increased significantly after induction at 35-55℃ for 1h, but not at room temperature. pAcHSR The transcriptional activity was enhanced within 30 minutes of induction at 35~55℃.

4. A recombinant expression vector, characterized in that: Containing the high temperature inducible plant promoter according to any one of claims 1 to 3 pAcHSR .

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector is specifically pGreen II 0800-LUC- pAcHSR , the construction method is as follows: the promoter pAcHSR The sequence was loaded into the pGreen II 0800-LUC vector which was double-digested with HindIII and NcoI.

6. A recombinant bacterium, characterized in that: Containing the high temperature inducible plant promoter according to any one of claims 1 to 3 pAcHSR .

7. The recombinant bacterium according to claim 6, characterized in that The construction method of the recombinant bacteria is as follows: pAcHSR The constructed recombinant expression vector pGreen II 0800-LUC- pAcHSR Introduce Agrobacterium GV3101.

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