Promoter responding to abiotic stress in corn and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a promoter responding to abiotic stress in corn and application of the promoter. The invention relates to a corn ZmSKIP gene promoter responding to abiotic stress. The base sequence of the corn ZmSKIP gene promoter is as shown in SEQ ID NO. 6. The corn ZmSKIP gene upstream sequence is truncated and constructed to a reporter gene GUS gene, and the GUS gene expression is driven to research the response condition of the promoter to abiotic stress. The promoter of the 744bp fragment has the advantages that the promoter of the 744bp fragment has higher GUS gene starting capacity than other fragments, is equivalent to an existing induced promoter rd 29A for starting GUS gene expression under drought stress, and can be used as a drought-induced promoter in plant genetic engineering, so that an important theoretical basis is provided for improving the abiotic stress capacity of corn by utilizing the genetic engineering, and the application prospect is broad. Meanwhile, an important tool is provided for regulating the expression of plant genes by utilizing the promoter.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter in corn that responds to abiotic stress and an application thereof. Background Art

[0002] Improving the ability of plants to resist abiotic stress has always been a hot topic in the field of botany. Existing technologies have reported that the SKIP (Ski-interacting protein) gene in plants can significantly improve the abiotic stress resistance of plants. In rice, SKIP homologous genes can positively regulate cell stability and resistance; in Arabidopsis, the putative transcription factor in the ABA signaling pathway of the SKIP gene crop can improve the ability to resist abiotic stress.

[0003] In the study "Cloning of the Maize SKIP Homologous Gene ZmSKIP and Functional Analysis in Abiotic Stress" (Wang Xiaomin, 2012, PhD thesis of Chongqing University), specific primers were designed based on the ZmSKIP sequence in GenBank, total RNA of maize was extracted, the ZmSKIP sequence was isolated and cloned using TR-PCR technology, and the ZmSKIP sequence was analyzed using bioinformatics methods to obtain the location and evolutionary relationship of the ZmSKIP gene. The expression pattern of the ZmSKIP gene under stress conditions was analyzed using fluorescent quantitative PCR to determine the induction of the gene under stress conditions and the response between the gene and the promoter under stress conditions. It was finally found that overexpression of ZmSKIP in tobacco can improve plant resistance to abiotic stress, and the ZmSKIP gene can be used as a good candidate gene to improve the resistance of transgenic plants, providing an important reference for genetic engineering breeding of maize stress resistance.

[0004] The promoter is the most important cis-acting element for gene expression regulation. It interacts with trans-acting factors such as RNA polymerase and other protein auxiliary factors to regulate the expression of genes at the transcriptional level. It can be said that without the promoter gene, gene transcription cannot be completed. According to the mode of action of the promoter, it can be divided into constitutive promoters and specific promoters. Among them, the constitutive promoter can constantly regulate the expression of structural genes at a certain level, such as the cauliflower mosaic virus CaMV 35S promoter. However, the use of constitutive promoters has certain limitations and may cause some side effects. In addition, researchers have pointed out that the repeated use of a constitutive promoter to drive the expression of two or more exogenous genes may cause rearrangement and instability of gene expression.

[0005] Specific promoters can be divided into tissue-specific expression promoters and inducible expression promoters. Tissue-specific expression promoters express genes in specific tissues, which is mainly manifested in the obvious tissue and organ specificity of the expression of its regulatory genes, which is regulated by the development of plant organs. Under normal circumstances, inducible expression promoters basically do not initiate gene transcription or have low transcription activity. When plants are stimulated by external stimuli, they initiate the massive expression of response genes in plants, which ultimately leads to responses at the physiological and biochemical levels. This type of promoter includes light-induced promoters, ethanol-induced promoters, stress-induced promoters (such as rd 29A promoters) and pathogen-induced promoters (such as PR promoters).

[0006] Promoters are key elements that determine the frequency and efficiency of exogenous gene transcription. It is crucial to select appropriate promoters to regulate the expression of target genes at the required time and space according to the research objectives. Specific promoters can quickly induce gene expression in specific plant tissues and organs or at specific developmental stages as needed, quickly regulate the "on" and "off" of gene transcription, and overcome some shortcomings of constitutive promoters. Therefore, studying the specific promoter of the maize ZmSKIP gene is conducive to understanding the role of the maize ZmSKIP gene in maize stress resistance, and studying maize genetic engineering has very important practical application significance.

[0007] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] The purpose of the present invention is to provide a promoter responsive to abiotic stress in corn and its application, which is helpful to understand the role of corn ZmSKIP gene in corn stress resistance and has very important practical application significance for studying corn genetic engineering and cultivating plants resistant to abiotic stress.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The first object of the present invention is to provide a maize ZmSKIP gene promoter that responds to abiotic stress. The maize ZmSKIP gene promoter is obtained by using maize DNA as a template, the base sequence shown in SEQ ID NO.2 as a forward primer, the base sequence shown in SEQ ID NO.4 as a reverse primer, using a high-fidelity DNA polymerase for PCR reaction, and performing gel electrophoresis and gel excision to recover the PCR product. The base sequence is shown in SEQ ID NO.6.

[0011] The second object of the present invention is to provide the use of the corn ZmSKIP gene promoter in improving the ability of plants to resist abiotic stress.

[0012] The third object of the present invention is to provide a recombinant vector, which comprises the maize ZmSKIP gene promoter.

[0013] The fourth object of the present invention is to provide the use of the recombinant vector in improving the ability of plants to resist abiotic stress.

[0014] The fifth object of the present invention is to provide a transgenic plant cell line or a recombinant bacterium, wherein the transgenic plant cell line or the recombinant bacterium comprises the maize ZmSKIP gene promoter.

[0015] The sixth object of the present invention is to provide the use of the transgenic plant cell line or recombinant bacteria in improving the ability of plants to resist abiotic stress.

[0016] A seventh object of the present invention is to provide a method for improving the abiotic stress resistance of plants or a method for cultivating abiotic stress-resistant plants, comprising the following steps:

[0017] S1. Constructing a recombinant vector containing a maize ZmSKIP gene promoter, wherein the base sequence of the maize ZmSKIP gene promoter is shown in SEQ ID NO.6;

[0018] S2. The recombinant vector obtained in S1 was transferred into competent Agrobacterium to obtain Agrobacterium carrying the maize ZmSKIP gene promoter;

[0019] S3. Infect plants with the Agrobacterium obtained in S2 to obtain plants carrying the maize ZmSKIP gene promoter.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention truncates the upstream sequence of the maize ZmSKIP gene and constructs it into the reporter gene GUS gene to drive the expression of the GUS gene to study the response of the promoter to abiotic stress. It is found that the 744bp fragment promoter has a stronger ability to start the GUS gene than other fragments, and is comparable to the existing inducible promoter rd29A in starting the GUS gene expression under drought stress, and can be used as a drought-induced promoter in plant genetic engineering, which is conducive to using genetic engineering to improve the abiotic stress ability of maize and provide an important theoretical basis, and also provides an important tool for using promoters to regulate the expression of plant genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of GUS staining identification of different tissues of transgenic plants;

[0023] Figure 2 The results of GUS activity determination of the three promoters of the present invention under different stress modes;

[0024] Figure 3 The results are the GUS activity determination results of the 744 bp fragment promoter of the present invention and the existing promoter under different stress modes. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solution of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0026] 1. Construction of pLP100-pZmSKIP:GUS

[0027] 1.1 Maize DNA extraction

[0028] TaKaRa MiniBEST Plant Genomic DNA Extraction Kit was used for extraction. 2-5 g of corn seedlings or leaves were chopped and ground into powder in a mortar with liquid nitrogen, and then the operation was performed according to the instructions of the kit.

[0029] 1.2 PCR amplification of promoter fragment

[0030] Using corn DNA as a template, primers were designed, and PrimeSTARTM HS DNA Polymerase high-fidelity DNA polymerase was used for PCR reaction. In this example, three sets of primers were designed, and the downstream primers of the three sets of primers were the same, as shown in Tables 1-3 below.

[0031] Table 1 Primer 1

[0032] Primer name Base sequence Sequence Listing Number Remark 1F <![CDATA[5'-CCC AAGCTT AATCATAGGTGCTCCACATCAT-3']]> SEQ ID No.1 Hind III sites in italics R <![CDATA[5'-CGC GGATCC TACCTAGGGTTCCGGCGAGTCG-3]]> SEQ ID No.4 Italicized BamHI sites

[0033] Table 2 Primer 2

[0034] Primer name Base sequence Sequence Listing Number Remark 2F <![CDATA[5'-CCC AAGCTT AGAACGATTGAGTGGTTTTGCC-3']]> SEQ ID No.2 Hind III sites in italics R <![CDATA[5'-CGC GGATCC TACCTAGGGTTCCGGCGAGTCG-3]]> SEQ ID No.4 Italicized BamHI sites

[0035] Table 3 Primer 3

[0036] Primer name Base sequence Sequence Listing Number Remark 3F <![CDATA[5'-CCC AAGCTT ATCCATTGGCGGACAGCAG-3']]> SEQ ID No.3 Hind III sites in italics R <![CDATA[5'-CGC GGATCC TACCTAGGGTTCCGGCGAGTCG-3]]> SEQ ID No.4 Italicized BamHI sites

[0037] The PCR program was: 98°C, 2 min; 98°C 10 s, 55°C 15 s, 72°C 2.5 min, 32 cycles; 72°C, 10 min.

[0038] 1.3 Gel electrophoresis to recover promoter fragments

[0039] After gel electrophoresis of the PCR products, the target bands with the expected sizes were cut and recovered. The base sequences of the recovered promoter fragments are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively, and their lengths are 1967 bp, 744 bp and 351 bp, respectively.

[0040] 1.4 Construction of the intermediate vector pEASY-pZmSKIP

[0041] According to the instructions of the pEASY-Blunt Cloning Kit, the three recovered promoter fragments were connected to the pEASY-Blunt fragments, transformed into Trans1-T1 competent cells, coated with plates containing kanamycin, and cultured in a 37°C incubator overnight. After colony PCR, restriction digestion, and sequencing verification, they were named pEASY-pZmSKIP-1F vector, pEASY-pZmSKIP-2F vector, and pEASY-pZmSKIP-3F vector, respectively.

[0042] 1.5 Construction of plant expression vector pLP100-pZmSKIP:GUS

[0043] The pLP100-pZmSKIP series vectors and pLP100 plasmid were extracted using TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0; the pLP100 plasmid and pEASY-pZmSKIP series intermediate vectors were digested with Hind Ⅲ and BamH Ⅰ restriction endonucleases, respectively. The digestion system is shown in Table 4.

[0044] Table 4 Enzyme digestion system

[0045] Reagents Addition amount 10×FistDigest Green Buffer 5.0μL Hind III restriction endonuclease 2.5μL BamH Ⅰ restriction endonuclease 2.5μL Plasmid (pLP100 or pEASY-pZmSKIP) 15.0μL <![CDATA[ddH2O]]> 25.0μL

[0046] After enzyme digestion and gel electrophoresis, pLP100 digestion fragments and pEASY-pZmSKIP digestion fragments were recovered and then connected with T4 DNA Ligase. The connection system is shown in Table 5. The molar ratio of the connected fragments in Table 5 is between 1:2-6 (vector: fragment; that is, the fragment should be more) and the connection is carried out at 4°C overnight.

[0047] Table 5 Connection system

[0048] Reagents Addition amount 10×T4 DNA ligase Buffer 2.0μL <![CDATA[T4 DNA ligase]]> 1.0μL pLP100 fragment 5.0μL pEASY-pZmSKIP fragment 10.0μL <![CDATA[ddH2O]]> 2.0μL

[0049] The ligated vector was transformed into competent E. coli JM109 cells. After plating and screening, it was verified by PCR. A single colony was inoculated in LB medium for shaking culture and plasmid was extracted. After enzyme digestion verification, it was named pLP100-pZmSKIP: GUS plasmid. The pLP100-pZmSKIP: GUS plasmid was transformed into competent Agrobacterium LBA4404 cells by freeze-thaw method. For details, please refer to "Cloning of Maize SKIP Homologous Gene ZmSKIP and Functional Analysis in Abiotic Stress" (Wang Xiaomin, 2012, PhD thesis of Chongqing University), which will not be repeated here.

[0050] A single colony of Agrobacterium tumefaciens from a positive transformant was inoculated into 20 mL of YEB liquid medium supplemented with 50 mg / L kanamycin and 50 mg / L rifampicin, and cultured in the dark at 28°C, 250 rpm for 48 h for activation. 1 mL of the activated Agrobacterium was taken and cultured until OD 600 =0.5. Centrifuge at 4°C, 1000 rpm for 15 min, remove the supernatant, add 20 mL of MS liquid culture medium to resuspend, and use for infection.

[0051] 2. Tobacco genetic transformation

[0052] 2.1 Infection

[0053] Take the sterile tobacco seedlings and cut the leaves (remove the main veins and edges) into 4cm 2 The explants were placed in the Agrobacterium infection solution containing pLP100-pZmSKIP:GUS plasmid and infected at 80 rpm for 10-15 min. The explants were removed, the excess bacterial solution was washed off with filter paper, and the explants were placed in MS solid medium again and cultured at 25°C in the dark for 2 days.

[0054] 2.2 Screening of resistant seedlings

[0055] The cultured explants were transferred to the selective differentiation medium and cultured under 18h light / 6h dark, 25°C, and 60% relative humidity until differentiation and budding. During this process, the culture rack was replaced every 2 weeks. The selective differentiation medium was MS basic medium + 3mg / L 6-BA + 0.05mg / L IAA + 50mg / L Kanamycin + 400mg / L Carbenicillin. MS basic culture was MS with 3% sucrose, 0.8% sucrose, pH 5.8.

[0056] 2.3 Rooting culture

[0057] When the buds grow to about 3-5cm, they are cut off and transferred to the selective rooting medium for cultivation. After rooting, they are transplanted to the greenhouse. The selective rooting medium is MS basic medium + 0.5mg / L IAA + 100mg / L Kanamycin + 400mg / L Carbenicillin. MS basic medium is MS, plus 3% sucrose, 0.8% sucrose, pH 5.8.

[0058] 3. GUS staining of different tissues of transgenic plants

[0059] Different tissues of the resistance-screened transgenic plants were taken as materials, stained with the GUS staining kit (SL7160) from Beijing Coolbo Technology Co., Ltd. and photographed under a microscope. The results are shown in Figure 1 .

[0060] Depend on Figure 1 It can be seen that through tissue staining, the promoters of the three fragments can drive the expression of the GUS reporter gene and can be expressed in various tissues of the plant, but there are certain differences in expression. Among them, the expression of the GUS reporter gene driven by the promoter of the 351bp fragment is very weak in the leaves.

[0061] 4. GUS activity determination of promoter under different stress modes

[0062] 4.1 Treatment of transgenic tobacco plants

[0063] The T1 transgenic tobacco seedlings grown for 4 weeks were transferred to MS medium, which was supplemented with 200mM NaCl, 300mM mannitol, and 15% PEG-6000. At the same time, the treatment of spraying 100mM ABA was set as the positive control, and the treatment of spraying the transgenic tobacco seedlings with water was set as the blank control. After 24 hours of treatment, the third complete leaf of each treatment was taken for the determination of GUS enzyme activity.

[0064] 4.2 GUS enzyme activity assay

[0065] GUS enzyme is a hydrolase that can hydrolyze derivatives of β-glucuronide with optical activity genes. It uses 4-methylumbelliferyl-D-glucuronide (4-MUG) as a substrate, and the resulting 4-methylumbelliferyl (4-MUB) is fluorescent. Based on this principle, the fluorescence assay method was used to quantitatively detect GUS activity after leaves were treated with each treatment method.

[0066] 4.2.1. Extraction of GUS enzyme solution

[0067] Use wet paper towels to wipe off the dust and residual liquid on the surface of tobacco seedling leaves, and take samples by punching. Quickly place the material in liquid nitrogen and grind it into powder, add 3 times the volume of GUS extraction buffer (GUS extraction buffer: measure 0.1M sodium phosphate buffer (pH7.0) 100mL, 0.1M EDTA 20mL, 1% sarcosyl 20mL, TritonX-100200μL, β-mercaptoethanol 140μL, mix and add distilled water to 200mL), grind for another 2min, add to a new 1.5mL EP tube, shake and mix; centrifuge at 4℃, 12000rpm for 10min, and take the supernatant and save it for later use.

[0068] 4.2.2 Determination of protein content in GUS extract

[0069] S1. Draw the standard curve

[0070] 250μg / mL BSA standard solution (Shanghai Biotechnology, NO. C500642) was diluted to 25μg / mL, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, and 250μg / mL in sequence. 10μL of the dilution was added to the sample wells of the ELISA plate, and then 190μL of Coomassie Brilliant Blue solution was added to mix thoroughly. The mixture was placed at room temperature for 2 minutes. For the blank control, water was used instead of the BSA dilution solution. The light absorbance at 595nm was measured and a standard curve was drawn. Each concentration was repeated 3 times.

[0071] S2. Determination of sample protein content

[0072] Dilute the GUS extract (i.e. the extracted GUS enzyme solution) 10 times and 100 times with GUS extraction buffer, respectively, pipette 10 μL of the dilution into the sample well of the ELISA plate, then add 190 μL of Coomassie Brilliant Blue solution and mix thoroughly, place at room temperature for 2 minutes, and replace the GUS extract with GUS extraction buffer for the blank control, and measure the light absorption value at 595 nm. Calculate the protein content of the sample according to the protein standard curve.

[0073] 4.2.3 GUS fluorescence assay

[0074] S1. Preparation of standard curve

[0075] Prepare 4-MU gradient concentration solution (1mM 4-MU diluted with 0.2M Na2CO3 reaction stop solution): 100μM, 10μM, 1μM, 100nM. Measure the fluorescence of each sample under the conditions of excitation light 365nm, emission light 455nm, and slit 3nm. For the blank control, replace 4-MU with the reaction stop solution and draw a standard curve.

[0076] S2. Enzyme reaction

[0077] a. Take 7 centrifuge tubes and add 900 μL of reaction stop solution to each tube. Number them 1-7.

[0078] b. Add 50-100 μL (adjusted according to the protein content) of GUS protein extract to the reaction buffer (add 1 mM MUG to the above GUS extraction buffer) preheated to 37°C and mix thoroughly;

[0079] c. Immediately take out 100 μL and add it to tube 1 containing 900 μL of reaction termination solution and mix well (blank control at time 0);

[0080] d. Incubate at 37°C and strictly time the reaction. Take out 100 μL of the reaction mixture at 5, 10, 20, 30, 45, and 60 minutes, add it to tube 2, mix well, and place at room temperature.

[0081] e. Take tube 1 (sample at time 0) as blank, measure the fluorescence intensity at different time points under the conditions of excitation light 365nm, emission light 455nm, and slit 3nm, calculate the 4-MU content of each tube according to the standard curve, and then calculate the enzyme activity in units of 4-MU pmol•min -1 •μg -1 The results are shown in Figure 2 .

[0082] Depend on Figure 2 It can be seen that through the GUS protein fluorescence measurement of transgenic tobacco seedlings, it was found that the 1967bp fragment promoter and the 744bp fragment promoter have response functions to drought (mannitol, PEG-6000), high salt (NaCl) and ABA, while the 351bp fragment promoter has a significantly lower response to drought, high salt and ABA than other fragment promoters, or even no response.

[0083] Comparison of the 1967 bp fragment promoter and the 744 bp fragment promoter revealed that the 744 bp fragment promoter had a better response than the 1967 bp fragment promoter, indicating that the 1967 bp fragment promoter was redundant. In summary, the 744 bp fragment promoter was selected as the complete promoter of the maize ZmSKIP gene.

[0084] 5. Comparison between the 744 bp fragment promoter of the present invention and existing promoters

[0085] The GUS activity of the 744 bp fragment promoter, the inducible promoter rd 29A, and the constitutive promoter CaMV 35S under abiotic stress (mannitol, high salt NaCl) and ABA was determined. The specific method is shown in "4. Determination of GUS activity of promoters under different stress modes". The results are shown in Figure 3 .

[0086] Depend on Figure 3 It can be seen that under ABA treatment, the 744 bp fragment promoter of the present invention and the inducible promoter rd 29A promote the expression of GUS gene at a similar level and lower than the constitutive promoter CaMV 35S;

[0087] Under high salt stress, the inducible promoter rd 29A promoted GUS gene expression higher than the constitutive promoter CaMV35S and the 744 bp fragment promoter of the present invention, and the constitutive promoter CaMV 35S was higher than the 744 bp fragment promoter of the present invention;

[0088] Under drought stress, the 744 bp fragment promoter of the present invention and the inducible promoter rd 29A are comparable in promoting the expression of the GUS gene, and are higher than the constitutive promoter CaMV 35S.

[0089] In summary, the present invention found that the 744bp fragment promoter has a stronger ability to promote the GUS gene than other fragments, and is comparable to the existing inducible promoter rd 29A in promoting the expression of the GUS gene under drought stress, and can be used as a drought-induced promoter in plant genetic engineering, which is conducive to using genetic engineering to improve the abiotic stress resistance of corn and provides an important theoretical basis, and also provides an important tool for regulating the expression of plant genes using promoters.

[0090] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A maize ZmSKIP gene promoter responsive to abiotic stress, characterized in that: The base sequence of the maize ZmSKIP gene promoter is shown in SEQ ID NO.

6.

2. Use of the maize ZmSKIP gene promoter according to claim 1 in improving the ability of plants to resist abiotic stress.

3. A recombinant vector, characterized in that: The recombinant vector comprises the maize ZmSKIP gene promoter according to claim 1.

4. Use of the recombinant vector according to claim 3 in improving the ability of plants to resist abiotic stress.

5. A transgenic plant cell line or recombinant bacterium, characterized in that: The transgenic plant cell line or recombinant bacteria comprises the maize ZmSKIP gene promoter according to claim 1.

6. Use of the transgenic plant cell line or recombinant bacteria according to claim 5 in improving the ability of plants to resist abiotic stress.

7. A method for improving the resistance of plants to abiotic stress or a method for cultivating plants resistant to abiotic stress, characterized in that: The following steps are involved: S1. Constructing a recombinant vector containing a maize ZmSKIP gene promoter, wherein the base sequence of the maize ZmSKIP gene promoter is shown in SEQ ID NO.6; S2. The recombinant vector obtained in S1 was transferred into competent Agrobacterium to obtain Agrobacterium carrying the maize ZmSKIP gene promoter; S3. Infect plants with the Agrobacterium obtained in S2 to obtain plants carrying the maize ZmSKIP gene promoter.

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