High-temperature inducible plant promoter pAcHSR and application thereof

Abstract

The invention provides a high-temperature inducible plant promoter pAcHSR. The nucleotide sequence of the high-temperature inducible plant promoter pAcHSR is shown as SEQ ID NO. 1. The invention further provides a recombinant expression vector containing the promoter pAcHSR and a recombinant bacterium containing the promoter pAcHSR. Meanwhile, the invention provides application of the promoter pAcHSR in the aspect of cultivating the high-temperature-stress-resistant plant and a method for cultivating the high-temperature-stress-resistant plant. According to the invention, the high-temperature inducible plant promoter pAcHSR is screened and finally determined in a kiwi fruit gene bank; the promoter pAcHSR drives transcription and translation of a heat shock transcription factor AcHSFA2-1 through high-temperature induction, the high-temperature stress resistance of plants is improved, and the promoter pAcHSR has great application prospects in cultivation of high-temperature stress resistant plants and creation of high-resistance plant germplasm resources so as to cope with global climate changes.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to a high temperature inducible plant promoter pAcHSR and an application thereof. Background Art

[0002] Changes in global environmental temperature have increased the frequency of extreme high temperature events, and the resulting high temperature stress has greatly restricted plant growth. In this context, it is of great significance to cultivate new plant varieties that are resistant to high temperature stress.

[0003] According to existing research reports, when encountering high temperature stress, plants will quickly produce a variety of physiological changes to respond to the stress and reduce the harm of high temperature stress to growth. Heat shock transcription factors (HSF) are the transcription factor family that is most closely related to the response to high temperature stress in plants. Therefore, it is feasible to cultivate germplasm materials resistant to high temperature stress based on the functions of heat shock transcription factors and combined with transgenic technology.

[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). The promoter is a DNA sequence and related regulatory elements that provide RNA polymerase recognition and binding, usually located upstream of the gene, to drive gene transcription.

[0005] According to the working principle of the promoter, it can be roughly divided into three categories: constitutive promoter, tissue-specific promoter, and inducible promoter. Among them, the target gene driven by the constitutive promoter can theoretically be highly expressed in all developmental stages and all tissues and organs of the plant, and is often used in plant gene function research; but the continuous overexpression of the target gene by the constitutive promoter will greatly increase the metabolic burden of the transgenic plant, hinder plant growth and reduce yield. Tissue-specific promoters and inducible promoters can drive the target gene to be expressed only in specific tissue parts or under the condition of the presence of inducing factors, thereby avoiding the continuous overexpression of the target protein affecting the normal growth of the plant. In particular, the inducible promoter is a promoter that responds to special biological, physical or chemical signals formed during the evolution of plants; in the absence of inducing factors, the target gene driven by the inducible promoter is not expressed or expressed at the background; when the inducing factor appears, the activity of the inducible promoter is enhanced, and the expression of the driven target gene is significantly increased.

[0006] Therefore, seeking a suitable high temperature-inducible 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 application thereof.

[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 as SEQ ID NO.1.

[0010] As one of the preferred embodiments of the present invention, the promoter pAcHSR is derived from kiwi fruit (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 transcription abundance of AcHSFA2-1 driven by the promoter pAcHSR significantly increases after induction at 35-55°C for 1 hour, but not at room temperature; at the same time, the transcription activity of the promoter pAcHSR is enhanced within 30 minutes of induction at 35-55°C.

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

[0013] As one of the preferred embodiments of the present invention, the recombinant expression vector is specifically pGreen II0800-LUC-pAcHSR, and the construction method is as follows: the promoter pAcHSR sequence is loaded into the pGreen II0800-LUC vector double-digested with HindIII and NcoI.

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

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

[0016] An application of the 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, comprising introducing the high temperature inducible plant promoter pAcHSR into a target plant to obtain a transgenic plant; the transgenic plant has a 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 kiwi fruit or tobacco.

[0019] The advantages of the present invention compared to the prior art are:

[0020] The present invention screens various genes that can significantly respond to high temperature stress treatment in the kiwifruit gene library, 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, thereby improving the plant's resistance to high temperature stress; wherein, the transcription activity of the promoter pAcHSR can be rapidly and significantly induced and enhanced by high temperature; the transcription abundance of AcHSFA2-1 driven by the promoter pAcHSR significantly increases after 1 hour of high temperature induction, and there is no expression under normal temperature conditions. Therefore, the research and discovery of the high temperature inducible plant promoter pAcHSR of the present invention has great application prospects for cultivating plants resistant to high temperature stress, creating highly resistant plant germplasm resources, and coping with global climate change. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The transcriptional abundance changes of AcHSFA2-1 driven by pAcHSR in kiwifruit in response to different temperature treatments;

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

[0023] Figure 3 This is a graph showing the activity of the high temperature-induced promoter pAcHSR based on dual luciferase (in the graph, "**" indicates p<0.01, and "***" indicates p<0.001;)

[0024] Figure 4 This is a graph showing the activity of the high temperature-induced promoter pAcHSR verified based on LUC fluorescein signal imaging. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the following embodiments, unless otherwise specified, are conventional reagents or methods in the art and will not be repeated.

[0026] Example 1

[0027] A high temperature inducible plant promoter pAcHSR of this embodiment:

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

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

[0030] (1) Extraction of kiwifruit genomic DNA

[0031] The leaves of 'Donghong' kiwifruit were used as research materials, and genomic DNA (gDNA) was extracted by CTAB method. Weigh 0.1g of the sample powder ground with liquid nitrogen into a 2mL centrifuge tube, add 1mL of 65℃ preheated CTAB extract and 50μL of β-mercaptoethanol, and vortex thoroughly; heat in a 65℃ water bath for 10min, and centrifuge at 10000rpm for 10min; aspirate the supernatant, add 1mL of 24:1 (chloroform:isoamyl alcohol), vortex thoroughly, and centrifuge at 4℃, 10000rpm for 10min; repeat once; aspirate 600μL of supernatant into a 1.5mL centrifuge tube, add 600μL of isopropanol, place at -20℃ for 1h, and centrifuge at 4℃, 10000rpm for 10min; add 500μL of 75% ethanol to suspend the precipitate, then centrifuge at 10000rpm for 5min, pour out and clean the remaining liquid at the bottom of the tube; after standing for 10min, add 100μL of DEPC water to dilute and mix, and store at -20℃.

[0032] (2) pAcHSR sequence cloning

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

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

[0035] Single clones were selected for positive identification and sent to Shangya Biotechnology (Hangzhou) for sequencing. The cloned sequence was then compared with the downloaded reference sequence using the software DNAMAN to confirm the correctness of the sequence.

[0036] Example 2

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

[0038] The pClone007 plasmid containing the correct pAcHSR promoter sequence was used as a template, and the pAcHSR sequence was subcloned according 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 loaded into the pGreen II 0800-LUC (LUC) vector double-digested with HindIII and NcoI. Single clones were picked for positive identification, and the positive single clones were sent to Shangya Biotechnology (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 GV3101 by freeze-thaw method.

[0042] Example 4

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

[0044] The recombinant Agrobacterium of Example 3 was spread on LB solid medium containing kanamycin (50 mg / L) and gentamicin (25 mg / L) and cultured at 28°C for 2 days, and 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 hours. The bacteria after two rounds of activation were suspended in an infiltration solution (containing 10 mM MES, pH 5.6; 10 mM MgCl2; 150 μM AS) and injected into Nicotiana benthamiana.

[0045] Experimental Example 1

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

[0047] 1. Experimental methods:

[0048] (1) Kiwifruit plant treatment

[0049] The tissue culture seedlings of 'Donghong' (A.chinensis cv.Donghong) with consistent growth were selected as research materials. After the plants grew to 5 leaves under 25℃ and light:dark cycle (16h:8h), they were placed at 35℃, 45℃, and 55℃ for 1h and 2h respectively. The tissue culture seedlings that were always grown at 25℃ were used as controls. Each treatment contained 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 cryovials, frozen with liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator.

[0051] (2) Transcriptome library construction

[0052] Collect the treated kiwifruit leaves, grind them into powder, weigh 0.1g, add 4mL 65℃ preheated CTAB extract (containing 5% β-mercaptoethanol), vortex and heat in a 65℃ water bath for 10min. Centrifuge at room temperature at 10000rpm for 10min, aspirate the supernatant to a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (V:V=24:1), vortex for 1min, and centrifuge at 15℃ and 10000rpm for 10min. Aspirate the supernatant to a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (V:V=24:1), vortex and repeat centrifugation. After aspirating the supernatant to a new centrifuge tube again, add 1 / 5 volume of 12M lithium chloride, invert and mix, and place in a 4℃ refrigerator overnight, 12 to 16h. The mixture was centrifuged at 4°C, 10000 rpm for 30 min, the precipitate was washed twice with 500 μL 75% ethanol (diluted with DEPC water), dried for 10 min, and dissolved and mixed with 30 μL DEPC water. The concentration and purity of RNA were detected by NanoDrop One micro-volume UV-visible spectrophotometer (Thermo Scientific, USA), the quality of RNA was detected by 1.2% agarose gel electrophoresis, and the RNA was stored in an ultra-low temperature refrigerator at -80°C.

[0053] A total of 24 libraries were sequenced for kiwifruit samples at four temperatures, two treatment durations, and three biological replicates. After the RNA integrity was accurately tested by Agilent 2100bioanalyzer, the kit was used to Ultra TM RNA Library Prep Kit for The library was constructed by Beijing Novogene Technology Co., Ltd. (NEB, USA). Sequencing and bioinformatics analysis were completed by Beijing Novogene Technology Co., Ltd., and the sequencing platform was Illumina NovaSeq 6000 (Illumina, USA).

[0054] In order to ensure the quality and reliability of data analysis, the raw data were filtered as follows: remove reads with adapters; remove reads containing N (N means that the base information cannot be determined); remove low-quality reads. After quality control, qualified sequences (clean reads) were obtained, and the qualified sequences were aligned with the Red5 (A. chinensis, Red5) genome using Hisat2 software. Gene transcript abundance was estimated as exon fragments per kilobase per million chromatographic fragments (FPKM). Genes with a fold change (FC) greater than or equal to 2 in gene expression and a false discovery rate (FDR) less than 0.01 were screened as differentially expressed genes (DEGs).

[0055] 2. 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, such as Figure 1 As shown: Under the growth condition of 25℃, AcHSFA2-1 is almost not expressed and its transcription abundance value cannot be detected; after being treated at 35℃ high temperature conditions for 1h, the expression level of AcHSFA2-1 is significantly increased; and after being treated at 45℃ and 55℃ strong high temperature conditions for 1h, the expression level of AcHSFA2-1 is extremely significantly enhanced, among which the transcription abundance value at 45℃ and 1h is 13,000 times that at 25℃ and 1h.

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

[0059] Experimental Example 2

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

[0061] 1. Experimental methods:

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

[0063] 2. 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 pAcHSR sequence

[0066]

[0067]

[0068] Based on the results of cis-acting element analysis, it was found that the pAcHSR sequence contains a variety of cis-acting elements that respond to various stress signals, such as temperature and light, as well as cis-acting elements that respond to various plant hormone 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] 1. Experimental methods:

[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 of 300 μmol m -2 s -1 , temperature 25°C, humidity 80%, light-dark cycle 16h:8h) for 72h. Then, the tobacco plants were treated at 45°C for 30min, 60min, 90min and 120min respectively. The activities of firefly luciferase (LUC) and renilla luciferase (REN) in leaves were detected using GloMax96 (Promega, USA) according to the instructions of Reporter Assay System (Promega, USA), and the ratio was calculated. Each result included three biological replicates.

[0074] (2) LUC fluorescence 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 of 300 μmol m -2 s -1, temperature 25°C, humidity 80%, light-dark cycle 16h:8h) for 72h. Then, after the tobacco plants were treated with 45°C high temperature conditions for 90min, 0.2mM luciferin was injected at the location where the Agrobacterium was injected, and after being placed away from light for half an hour, the LUC fluorescence signal was detected using the NightSHADE LB 985 instrument.

[0076] 2. 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 uses pGreen II 0800-LUC as the backbone, and contains the CaMV 35S::REN expression cassette and the pAcHSR::LUC expression cassette. Among them, the expression level of REN driven by the promoter CaMV 35S is stable 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 dual luciferase-based verification of the activity of the high temperature-induced promoter pAcHSR are as follows Figure 3 As shown. Figure 3 The ratio of firefly luciferase (LUC) to renilla luciferase (REN) showed that after high temperature treatment at 45°C for 30 minutes, the activity of pAcHSR could be significantly enhanced to 5 times; and after treatment for 90 minutes, the activity of pAcHSR could be enhanced to 40 times.

[0079] (3) LUC fluorescence signal imaging results are as follows Figure 4 As shown. Figure 4 The visualization results of the fluorescence signal show that compared with tobacco grown at 25°C, the LUC fluorescence signal in tobacco leaves treated at 45°C for 90 minutes was significantly enhanced.

[0080] This confirmed 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 almost undetectable in kiwifruit leaves under normal temperature growth conditions, while its transcriptional abundance is extremely significantly enhanced by nearly 13,000 times under 45°C high temperature conditions. The pAcHSR sequence contains a variety of cis-acting elements that respond to signals such as temperature, light, and hormones. The firefly luciferase encoding gene LUC driven by pAcHSR has extremely low transcriptional abundance and protein expression in tobacco leaves under normal temperature growth conditions, but after being treated at 45°C for 30 minutes, the transcriptional abundance of LUC increased significantly by nearly 5 times; after being treated for 90 minutes, the transcriptional abundance of LUC increased significantly by nearly 40 times, and the signal intensity of luciferin was extremely significantly enhanced. The experimental results confirmed that the transcriptional activity of pAcHSR can be rapidly and extremely significantly induced and enhanced by high temperature, and can be used for plant-derived inducible expression vectors, which has great application prospects for creating highly resistant plant germplasm resources to cope with global climate change.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

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

1.

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

3. The high temperature inducible plant promoter pAcHSR according to claim 1, characterized in that The promoter pAcHSR is used to drive the transcription and translation of the heat shock transcription factor AcHSFA2-1; and the transcription abundance of AcHSFA2-1 driven by the promoter pAcHSR significantly increases after induction at 35-55°C for 1 hour, but not under normal temperature conditions; at the same time, the transcription activity of the promoter pAcHSR is enhanced within 30 minutes of induction at 35-55°C.

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

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

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

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

8. Use of the high temperature inducible plant promoter pAcHSR as claimed in any one of claims 1 to 3 in cultivating plants resistant to high temperature stress.

9. A method for cultivating plants resistant to high temperature stress, characterized in that: The high temperature inducible plant promoter pAcHSR according to any one of claims 1 to 3 is introduced into a target plant to obtain a transgenic plant; the transgenic plant has a higher resistance to high temperature stress than the target plant.

10. The method for cultivating plants resistant to high temperature stress according to claim 9, characterized in that: The target plant is kiwi fruit or tobacco.

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