CN107881172B - Stress inducible promoter, stress inducible promoter plant expression vector and method for inducing expression of target gene - Google Patents
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Abstract
The invention relates to a stress inducible promoter sequence, which comprises a 1388bp DNA nucleotide sequence of SEQ ID NO. 1 and belongs to the technical field of plant genetic engineering. The invention also provides a stress inducible promoter plant expression vector and a method for expressing a target gene by a plant under the induction of low temperature, drought and salt, and the target gene can be replacedGUSInserting the gene into a plant expression vector containing the promoter of the invention, and introducing the recombinant vector into a target plant; SEQ ID NO 2 and SEQ ID NO 3 are PCR primers, each of which comprisesEcoRI andHina diiii restriction site, said primer being suitable for amplifying a DNA sequence comprising SEQ ID No. 1; the invention can be used for starting the high-efficiency expression of exogenous genes under cold, drought and salt stress, and is suitable for the improvement research of plant genetic breeding.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a stress inducible promoter, a stress inducible promoter expression vector and an induced target gene expression method.
Background
Low-temperature cold damage, drought and soil salinization are important disasters which limit agricultural production and threaten grain safety, and particularly the large-area damage of crops can be caused by cold tides in spring and autumn and drought in key periods of crop growth, so that serious yield reduction is caused. Therefore, in addition to strengthening the research on the defense technology of low temperature, drought and salt damage, the plant genetic engineering technology means is adopted to express the stress resistance genes in the crops so as to improve the resistance of the crops to the stress and reduce the serious damage of the low temperature, the drought and the high salt to the crops, and the plant genetic engineering technology means becomes an important means for crop breeding.
Currently, regulation of gene expression in plants mainly includes DNA level regulation, transcription level regulation, post-transcription level regulation, translation level regulation, and post-translation level regulation. The promoter is a DNA sequence located in front of a transcription initiation site, can activate RNA polymerase, enables the RNA polymerase to be accurately combined with template DNA, and accordingly initiates the transcription of related genes, and is provided with a plurality of cis-acting elements, can regulate and control the expression of downstream target genes through the interaction with trans-acting factors, and is a key step in the regulation and control of transcription level.
Researches show that plant promoters mainly comprise a constitutive promoter, a tissue-specific promoter and an inducible promoter. Wherein the constitutive promoter can stably start gene expression in the whole development stage of the plant and is not induced by external environment to generate violent change; a tissue-specific promoter is capable of promoting gene expression in a specific tissue at a specific time; the inducible promoter can induce the expression of genes according to different environmental stresses or hormone stimulation and the like, so that the defense capability of the plant to adverse environmental conditions is enhanced, and the normal physiological metabolism and growth development of the plant are protected. At present, the constitutive promoter is most commonly used in plant transgenic research, and the common promoters include a cauliflower mosaic virus (CaMV) 35S promoter, a rice Actin promoter and a maize Ubiquitin promoter, wherein the CaMV 35S promoter is mainly applied to transgenic research of dicotyledonous plants, and the Actin and Ubiquitin promoters are mostly applied to monocotyledonous plants, and particularly, the Ubiquitin promoter is widely applied to driving exogenous gene expression in transgenic research of rice and maize. Although the constitutive promoter has the advantages of stability, high efficiency, broad spectrum and the like when starting the expression of an exogenous gene, the overexpression of protein is easily caused due to the high-efficiency and continuous expression of a target gene in the whole growth and development process of a plant, so that not only is the energy waste caused, but also the physiological balance of the plant is easily influenced, and further the yield is possibly influenced. Therefore, more and more researchers have focused on inducible promoters that induce expression under specific conditions and tissue-specific promoters that express at specific sites and at specific periods of time to mitigate the damage to plant growth caused by over-expression of heterologous proteins.
There are many reports on the research of plant tissue specific promoters: for example, Arabidopsis thaliana specifically expressed in leavesatslAGene and cornC4PdKA gene promoter; expressed specifically in fruitE4、E8、ACCA synthase gene promoter; phaseolus vulgaris with specific expression in vascular bundlePAL、GRP118A gene promoter; arabidopsis thaliana specifically expressed in rootsPyk10Gene promoters, and the like. The research reports on the inducible promoter mainly comprise abiotic adversity stress inducible type, biotic stress inducible type, light inducible type, hormone inducible type and the like. The inducible promoter does not express or expresses in a low amount under normal conditions, and a large amount of related protein begins to be expressed when corresponding inducing conditions appear, so that damage to plants caused by adverse environments is reduced. Research has shown that some inducible promoters can be induced by multiple factors simultaneously, and are called multiple inducible promoters, for example: arabidopsis thalianaCor15aThe gene promoter has low temperature and drought induction characteristics; arabidopsis thalianard29AThe promoter has low-temperature, drought and high-salt induction activities; rice plasma membraneCaATPaseThe promoter has drought, cold, MeJA and ABA inducing properties.
The inducible promoter is utilized to drive the high-efficiency expression of the stress-resistant gene under specific conditions, so that the resistance of the plant to adverse environmental conditions is enhanced, and the inducible promoter gradually becomes a hot point of research in recent years. However, the number of inducible promoters that can be widely used in transgenic research is still relatively small, especially the number of inducible promoters that are multi-element.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides an adversity inducible promoter sequence capable of inducing the expression of exogenous genes under low temperature, drought and salt stress.
The technical scheme provided by the invention for solving the technical problems is as follows: an stress-inducible promoter comprising a 1388bp DNA nucleotide sequence of SEQ ID NO. 1.
Further, the sequence of the stress inducible promoter is from brachypodium distachyon AP2/EREBP family geneBdDREB-47。
The technical problem (II) to be solved by the invention is as follows: provides a plant expression vector of a plant stress inducible promoter, which can be applied to genetic transformation.
The technical scheme provided by the invention for solving the technical problems is as follows: the plant expression vector comprises the stress inducible promoter sequence.
The technical problem (III) to be solved by the invention is as follows: provides a method for inducing and expressing target genes of plants under low temperature, drought and salt stress.
The technical scheme provided by the invention for solving the technical problems is as follows: target genes are inserted into a plant recombinant vector containing the promoter, the recombinant vector is introduced into target plants, and the target genes are induced to express in the plants under the conditions of low temperature, drought and salt stress.
The invention has the beneficial effects that:
the inducible promoter provided by the invention belongs to a multi-element inducible promoter, and can start the expression of an exogenous gene under the conditions of cold, drought and salt stress. According to the technical scheme provided by the invention, exogenous genes are replacedGUSThe gene is inserted into the stress inducible promoter plant expression vector, and the expression of the exogenous gene can be controlled by artificially controlling environmental conditions (temperature, humidity and salt content), so that energy waste and possible damage caused by continuous expression of a target gene are avoided. In addition, the stress-resistant gene is used as an exogenous gene, and the inducible promoter provided by the invention can efficiently start the expression of the stress-resistant gene under cold, drought and salt stress, so that the resistance of the plant is enhanced.
Drawings
FIG. 1 is a PCR amplification electrophoretogram of a promoter of the present invention, wherein M is DL2000 Marker, and the band sizes are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom; 1 is the promoter pBdDREB-47 of the invention.
FIG. 2 shows the sequence of the pCAMBIA1381-GUS vector and the promoter of the present invention linked to the T vectorEcoRI andHina dii double-enzyme digestion electrophoretogram, wherein M is a DNA Marker III, and the sizes of the bands are 4500bp, 3000bp, 2000bp, 1200bp, 800bp, 500bp and 200bp from top to bottom; 1 is pBdDREB-47-T plasmid; 2 is pCAMBIA1381-GUS plasmid.
FIG. 3 is PCR electrophoresis of bacterial liquid after promoter is connected to pCAMBIA1381-GUS plant expression vector, wherein M is DL2000 Marker; 1-10 are monoclonals.
FIG. 4 shows the promoter ligated to pCAMBIA1381-GUS plant expression vectorEcoRI andHina dii double enzyme digestion identification electrophoretogram, wherein M is a DNA Marker III; 1 is pC1381-pBdDREB-47-GUS plasmid.
FIG. 5 is an electrophoresis diagram of PCR detection of Agrobacterium tumefaciens bacteria liquid, wherein M is DL2000 Marker; 1-3 is agrobacterium monoclone; 4 is negative; 5 is pC1381-pBdDREB-47-GUS plasmid.
FIG. 6 is a partial T0 generation transgenic tobacco positive detection electrophoretogram, wherein M is DL2000 Marker; 1-7 is a transgenic tobacco T0 generation individual plant; 8 is wild tobacco; 9 is pC1381-pBdDREB-47-GUS plasmid.
FIG. 7 is a schematic diagram of the construction of the promoter plant expression vector of the present invention.
FIG. 8 is a graph of GUS staining of transgenic tobacco before and after cold, drought and salt stress treatments.
Detailed Description
Example one
This example relates to the grass of Bisui brachypodiumBdDREB-47Cloning of the gene promoter.
1.1 preparation of Bisui brachypodium genome DNA
Extracting leaves of the normally growing brachypodium distachyon by using a CTAB method, detecting the concentration and the quality of the extracted DNA by using an ultramicro nucleic acid protein determinator Q5000, and diluting the DNA concentration to 100 ng/mul for later use.
1.2 cloning of the promoter
The extracted brachypodium distachyon DNA is used as a template, and primers of SEQ ID NO. 2 and SEQ ID NO. 3 are used for carrying out PCR amplification to obtain a sequence of SEQ ID NO. 1.
The PCR reaction system is as follows:
| reagent | Volume (μ l) |
| ddH2O | 5.8 |
| 2×Prime Star |
10 |
| dNTP mix（2.5mM） | 2 |
| Upstream primer (10. mu.M) | 0.5 |
| Downstream primer (10. mu.M) | 0.5 |
| Takara Prime Star Taq enzyme | 0.2 |
| |
1 |
| General System | 20 |
PCR reaction procedure: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 30s, annealing at 55 ℃ for 20s (annealing temperature increase of 0.5 ℃ per cycle), extension at 72 ℃ for 90s, and amplification for 20 cycles; denaturation at 98 ℃ for 30s, annealing at 65 ℃ for 20s, extension at 72 ℃ for 90s, and amplification for 15 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.
The PCR product is absorbed by 10 mul and detected by 1% agarose gel electrophoresis, and the specific result is shown in figure 1, wherein Marker is DL2000, and the size of the target band is 1388 bp.
The remaining PCR product was ligated to a T-Vector using Takara Prime Star Taq high fidelity enzyme, the amplification product was Blunt ended, the T-Vector was pEASY-Blunt Cloning Vector (purchased from Beijing Quanyujin Biotechnology Co., Ltd.), according to the kit instructions, and the ligation system was 5. mu.l (4. mu.l PCR product + 1. mu. l T Vector).
The ligation product is transferred into Escherichia coli competent cells Trans-T1 (purchased from Beijing Quanji Biotech Co., Ltd.) by heat shock method, and the specific operation method is described in the kit instruction. Selecting a single clone to carry out colony PCR detection, selecting a positive clone to carry out sequencing, and displaying that the amplification sequence is completely coincided with SEQ ID NO. 1 by a sequencing result.
Cloning with correct sequencing is carried out for propagation, plasmids are extracted by a small plasmid extraction kit (purchased from Tiangen Biochemical technology Co., Ltd.), and the concentration of the extracted plasmids is detected by an ultra-quantitative nucleic acid protein determinator Q5000.
Example two
This example relates to the construction of a plant expression vector for a stress-inducible promoter (pBdDREB-47).
By usingEcoRI andHindiill (purchased from NEB company) double enzyme digestion recombinant plasmid pBdDREB-47-T and vector pCAMBIA1381-GUS, the enzyme digestion result is shown in figure 2, target fragment (1388 bp) and pCAMBIA1381-GUS vector fragment are respectively recovered and connected by T4 DNA ligase (purchased from NEB company), the target fragment and pCAMBIA are transferred into escherichia coli competent cell Trans-T1 by heat shock method, the result of positive detection of single clone is shown in figure 3, the amplification propagation extraction plasmid is obtained, the positive plasmid is further subjected to enzyme digestion identification (( EcoRI andHindiiII bisEnzyme cutting) results are shown in figure 4, and the sequencing result is correct, which indicates that the plant recombinant expression vector pC1381-pBdDREB-47-GUS is successfully constructed, pC1381-pBdDREB-47-GUS recombinant plasmid can be further transferred into agrobacterium-infected cell EHA105 (purchased from Shanghai Fenghe Biotech limited company) by a heat shock method, positive detection is carried out, the result is shown in figure 5, and the correct plasmid can be used for subsequent plant transgenosis.
A schematic diagram for constructing a plant expression vector containing a pBdDREB-47 promoter is shown in figure 7, wherein 35S Pro and pBdDREB-47 are promoters, 35S Ter and Nos Ter are terminators, a selection marker is hygromycin resistance, a GUS gene is a reporter gene, and the activity of the pBdDREB-47 promoter can be judged according to the expression condition of the GUS gene.
EXAMPLE III
This example relates to the transformation of tobacco with the pC1381-pBdDREB-47-GUS recombinant plasmid by Agrobacterium-mediated method.
3.1 preparation of transgenic recipient Material
Sowing seeds of wild tobacco in a flowerpot, placing the flowerpot in a 25-degree culture room (12 h light/12 h dark), covering a preservative film for preserving moisture, and gradually removing the preservative film after seedlings come out of the soil. Transplanting is carried out after two weeks, and the leaves can be used for transgenic experiments when the tobacco grows to 2 months.
3.2 tobacco leaf Sterilization and Pre-culture
Selecting tobacco with good growth condition, cutting off tender leaves of the tobacco with scissors, and soaking and sterilizing the tobacco leaves with 75% ethanol for 50s on an ultra-clean workbench. Soaking in 2.5% sodium hypochlorite solution for 10min for sterilization, shaking for sufficient sterilization, and washing tobacco leaf with sterile water for 5 times.
Placing the sterilized tobacco leaf on sterile filter paper, sufficiently drying surface water, and cutting the tobacco leaf into pieces of about 0.5 × 0.5 cm with sterile blade2The cut leaves were spread on a tobacco co-culture medium (MS + 6-BA (2 mg/L) + NAA (0.2 mg/L) + Agar (8 g/L) + sucrose (30 g/L) pH 5.8), and pre-cultured in the dark at 25 ℃ for 3 days.
3.3 preparation of Agrobacterium solutions
The agrobacterium liquid containing pC1381-pBdDREB-47-GUS recombinant plasmid is streaked on an LB solid plate containing 50mg/L kan, after dark culture for 1 day at 28 ℃, the agrobacterium liquid is suspended in 50ml of MS liquid culture medium, shake culture is carried out for 30min at 200r/min of a shaking table at 28 ℃, and next step of experiment is immediately carried out.
3.4 Agrobacterium infection
And (3) placing the tobacco leaves after being pre-cultured for 3 days into the fully-vibrated agrobacterium liquid, and soaking for 10 minutes while continuously shaking. After infection, the leaves are placed on sterile filter paper so as to fully absorb the residual bacterial liquid on the leaves, and then the impregnated leaves are dispersedly placed on a co-culture medium paved with the sterile filter paper and cultured in dark at 25 ℃ for 3 days.
3.5 differentiation and rooting of tobacco leaves
The tobacco leaves after 3 days of dark culture are transferred to a differentiation medium (MS + 6-BA (2 mg/L) + NAA (0.2 mg/L) + Cb (500 mg/L) + Kan (100 mg/L) + Agar (8 g/L) + sucrose (30 g/L) pH 5.8), and cultured under the conditions of 25 ℃ and 12h of light/12 h of dark until buds are differentiated.
When the buds grow to 2-3 cm, buds are cut off, inserted into a rooting medium (1/2 MS + Kan (100 mg/L) + Cef (250 mg/L) + Agar (8 g/L) + sucrose (30 g/L) pH 5.8), and cultured under the conditions of 25 ℃ and 12h of light/12 h of dark. The explants can root after two weeks generally, and when the root systems grow well, the explants are transplanted into flowerpots and continuously cultured in a greenhouse. Extracting genome DNA of transgenic T0 tobacco, performing positive detection by using GUS gene primer, wherein the detection result of partial plant is shown in figure 6, and SEQ ID NO. 4 and SEQ ID NO. 5 are GUS gene PCR detection primers.
Example four
This example relates to the identification of the Activity of stress-inducible promoters
The promoter disclosed by the invention is mainly characterized in that cold (4 ℃), drought and salt (300 mM NaCl) stress treatment is carried out on T1 generation transgenic tobacco seedlings, and the expression condition of a GUS reporter gene is detected by GUS staining, so that the promoter disclosed by the invention is identified to have cold, drought and salt induction characteristics.
4.1 preparation of the test materials
Respectively taking a proper amount of wild type and transgenic tobacco T0 generation seeds in a 2ml centrifuge tube, adding water to soak for 1-2h to ensure that the seeds are completely settled at the bottom of the tube, and washing for 2-3 times to remove impurities in the seeds. Transferring the soaked seeds to a sterile 2ml centrifuge tube on a super clean bench, adding sterile water for washing twice, then sterilizing with 70% ethanol for 50s, sterilizing with 1% sodium hypochlorite solution for 8-10min, and finally washing with sterile water for 3-5 times (the centrifuge tube is continuously shaken in the whole sterilizing and washing process). And (3) sowing the disinfected T0 generation seeds on an MS culture medium containing 50mg/L hygromycin, sowing wild tobacco seeds on the MS culture medium, growing under the conditions of 25 ℃ and 12h of light/12 h of darkness, germinating after about 5-7 days, and transplanting seedlings into a small pot for later use after two weeks.
4.2 promoter Activity identification under Cold, drought and salt stress treatment
Putting 1-month-old wild type and transgenic T1 tobacco-substituted seedlings into a 4-DEG C artificial climate box, respectively shearing leaves subjected to cold treatment for 0h, 12h and 24h into a10 ml centrifuge tube, adding a proper amount of non-diffusion GUS dye solution (purchased from Beijing Oobolae technology, Ltd.), fully immersing the leaves in the GUS dye solution, vacuumizing for 10min to enable the GUS dye solution to fully penetrate the leaves, dyeing in a dark place at 37 ℃ for 12h, and decoloring with absolute ethyl alcohol. The drought stress and salt stress treatment comprises the steps of carrying out water control drought on 1-month-old wild type and transgenic T1 generation tobacco seedlings and pouring 300mM NaCl solution once a day, and sampling at 0d and 6d of the treatment to carry out GUS staining. The black part is the expression part of GUS gene, and the deeper the black part is, the higher the expression level is. As shown in FIG. 8, the expression level of the GUS gene in the transgenic tobacco leaves before cold treatment (0 h) is very low (the black color is very weak), and the expression level of the GUS gene is obviously increased after cold treatment for 12h and 24h (the black color of the leaves is deepened), so that the promoter provided by the invention can be used for sufficiently explaining that the promoter does not basically start the expression of the GUS gene or the expression level is very low at normal temperature, and can quickly start the expression of a target gene once low-temperature stress is met. Meanwhile, the GUS expression quantity is very low before the treatment of drought stress and salt stress, and the GUS expression quantity is obviously increased after the treatment for 6 days. Therefore, the promoter provided by the invention has high-efficiency cold, drought and salt induction activities, and can be used for the research of plant genetic breeding improvement.
The present invention is not limited to the above embodiments, and the technical solutions of the above embodiments of the present invention may be combined with each other in a crossing manner to form a new technical solution, and all technical solutions formed by using equivalent substitutions fall within the scope of the present invention.
Sequence listing
<110> university of Jianghan
<120> stress inducible promoter, stress inducible promoter plant expression vector and method for inducing expression of target gene
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tatgcattta ttcaaggagc tggcttcttg tgttgaagtg agttgccatg aaatgcgcgg 180
tcattagtta tttgcatgct tgcactaaga gacaaaatga ttaggttgtt gatatgcttt 240
gtaaataccc tttgtaaaac agtagtaatt tcaaaatgtt ttaaatctca aaccacgtgc 300
ctttaattac ctccggattg attcgacaag atcttcaaag ctaatgggta tgtttcgatt 360
ctttcttgcc ttaaaagttt cttcatcttt ctagatatac aaggctaaat tggagggaaa 420
tagggcccaa tgatcttcat atttcgttga tactgtatat tattggggtg agaaatgaga 480
attgctgacc ggatccgtct cgtcccttgg cttgcaagga cgagctcatg gccgcttcca 540
accacgcacg agctccttcg aggttggaag gatgcaaacg agctcctcgc aagcctggat 600
ctcacttggc ggcagcggag ggcacggagc cacgaaggag aaggacatgg ctgcggtgcg 660
cgccaccgcc gagcccgaag aaacggcgat gatatttcac cggcgagctt gccggcaacg 720
tgatgaccca cgtggacaga tacggtaaca gaaatagaaa cgtgagtgca atacatagag 780
tacgatttta cttgaacagg tggcagcagc tggttgggag atggggctcg gcaatacata 840
gagtacgatt ttactagagc cggcgatcgt tgccggtata caaaccattg aagcaaacgc 900
ggcaaaaaaa tatctaatcg ttgggcctcg tgggctagta gcagctagct agtggagccc 960
aatcccctct cttgggcaga ggaagctgcc cattcgttgg gcctccatga gctagcagct 1020
agctagtgga gcccaatccc ccccccgctc ttcggcagag gaagctgcct tttttttttc 1080
tttccttaac gctgcaacct cacgccgtca cgcgtcgttc actcgtttgc cagttgccac 1140
cagcttatgc tggccaacgg gtacctggcg ttccggcagc gcgccacgtg tcaactcttc 1200
agcgcgtcac cgaaaactat aaaaagagct gccgcaaacc agactgactt actgaaccct 1260
ctccacaagc ccaatcctct catctcacac ttccgacaca ccagactgag ctactaatct 1320
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Claims (3)
1. A stress-inducible promoter, characterized in that: the stress inducible promoter is a 1388bp DNA nucleotide sequence of SEQ ID NO. 1;
the sequence of the stress inducible promoter is from brachypodium distachyon AP2/EREBP family gene BdDREB-47.
2. A stress-inducible promoter plant expression vector characterized by: the plant expression vector comprising the stress-inducible promoter sequence of claim 1.
3. A method of inducing expression of a target gene, comprising: inserting a target gene into a plant recombinant vector containing the promoter of claim 1, introducing the recombinant vector into a target plant, and inducing the target gene to express in the target plant under the conditions of low temperature, drought or salt stress, wherein the target plant is tobacco.
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| CN113151273B (en) * | 2021-04-14 | 2022-09-06 | 新疆农业大学 | Abiotic stress inducible promoter and application thereof |
| CN114854746B (en) * | 2022-04-20 | 2023-07-11 | 浙江省农业科学院 | Adversity stress inducible promoter TIP1-6-pro and construction method and application of expression vector thereof |
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Non-Patent Citations (4)
| Title |
|---|
| Expansion and stress responses of AP2/EREBP superfamily in Brachypodium Distachyon;Lihong Chen et al;《SCIENTIFIC REPORTS》;20160212;第6卷;摘要，第6-8页，图5A,图6A，Supplementary Table1/6/9 * |
| NC_016134.2;Genbank;《Genbank》;20151208;全文 * |
| 二穗短柄草MAPK和MAPKK家族的鉴定、相互作用及其表达谱分析;陈利红;《中国博士学位论文全文数据库（电子期刊）基础科学辑》;20141215;全文 * |
| 二穗短柄草逆境诱导型启动子pBdDREB-47的克隆及功能验证;李丽丽;《分子植物育种》;20161101;第15卷(第1期);全文 * |
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