CN114854746A - Adversity stress inducible promoter TIP1-6-pro and expression vector construction method and application thereof - Google Patents
Adversity stress inducible promoter TIP1-6-pro and expression vector construction method and application thereof Download PDFInfo
- Publication number
- CN114854746A CN114854746A CN202210418024.3A CN202210418024A CN114854746A CN 114854746 A CN114854746 A CN 114854746A CN 202210418024 A CN202210418024 A CN 202210418024A CN 114854746 A CN114854746 A CN 114854746A
- Authority
- CN
- China
- Prior art keywords
- stress
- tip1
- adversity
- pro
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
- C12N15/8227—Root-specific
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8291—Hormone-influenced development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8291—Hormone-influenced development
- C12N15/8293—Abscisic acid [ABA]
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of agricultural biology, and provides an adversity stress inducible promoter TIP1-6-pro and a construction method and application of an expression vector thereof, aiming at solving the problems that a promoter and a functional gene which can be used for plant stress-resistant molecular design breeding are few, wherein the nucleotide sequence of the TIP1-6-pro is shown as SEQID NO: 1, a DNA fragment of a vegetable soybean promoter TIP1-6-pro is obtained by cloning, and the function of the DNA fragment is verified, so that the promoter drives the expression of a target gene in a whole seedling plant under the conditions of drought, high salt, ABA and MeJA, and can be applied to plant stress resistance genetic engineering.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to an adversity stress inducible promoter TIP1-6-pro and an expression vector construction method and application thereof.
Background
TIPs (protop intracytoplasmic proteins) are aquaporins positioned on vacuolar membranes or vacuolar formers, are connected by 5 hydrophilic rings to form 6 transmembrane helices, mediate transmembrane transport of substances such as water and the like, and play an active role in plant response to adversity stress.
Vegetable soybeans refer to special soybean varieties harvested for fresh pod eating and serve as an important bean vegetable. The nutritional food is rich in protein, carbohydrate, vitamins, isoflavone, saponin, phospholipid and the like, has extremely high nutritional value, rich active ingredients and wide market prospect. The growth, yield and quality of vegetable soybeans are seriously influenced by the frequent abnormal drought climate and salinization of land in recent years. At the beginning of the domestic research on adverse stress such as vegetable soybean drought, a few promoters and functional genes can be used for vegetable soybean stress-resistant molecule design breeding.
The promoter is an important component of a functional gene, controls the initiation time, space and strength of gene transcription, and is regulated and controlled by the growth and development stage of plants, internal and external environmental stimulation and hormone level. Constitutive, inducible and tissue-specific promoters are classified according to their transcription patterns. The constitutive promoter is not limited by time and space, can efficiently promote the continuous, stable and high expression of exogenous genes in all tissues of plants, and can cause resource waste. Inducible and tissue-specific promoters are used for driving target genes to be efficiently expressed in specific tissues under specific conditions, can effectively solve the problems of constitutive promoters, and are developed and utilized as hot spots of genetic engineering research.
The adversity stress inducible promoter can receive an inducing signal under an adversity condition, so that target gene expression is specifically started, and external environment stress is resisted. The hormone plays an important role in the signal transmission process of plant adversity stress, and regulates and controls the processes of plant growth, stomata opening and closing, photosynthesis, permeation regulation substance, antioxidant enzyme accumulation and the like. The vegetable soybean adversity stress inducible promoter is excavated, and a plant expression vector is constructed for driving the target gene to be efficiently expressed under the adversity condition, so that the vegetable soybean adversity stress inducible promoter has important significance for researching the stress resistance regulation and control capability of vegetable soybeans.
Disclosure of Invention
In order to solve the problem that a promoter and functional genes which can be used for plant stress-resistant molecular design breeding are few, the invention provides an adversity stress inducible promoter TIP1-6-pro and a construction method and application of an expression vector thereof, a DNA fragment of a vegetable soybean promoter TIP1-6-pro is obtained by cloning, and the function of the DNA fragment is verified, so that the promoter can drive target gene expression in whole seedling plants under the conditions of drought, high salt, ABA and MeJA, and can be applied to plant stress-resistant genetic engineering.
The invention is realized by the following technical scheme: an adversity stress inducible promoter TIP1-6-pro nucleotide sequence is shown as SEQ ID NO: 1, nucleotide sequence 1-1483.
A construction method of a transgenic plant with adversity stress induced expression transgene comprises the following steps:
(1) constructing a plant expression vector of the adversity stress inducible promoter;
preferably, the vector backbone is pCAMBIA1391z plasmid.
(2) Inserting a gene to be expressed by the stress induction into a plant expression vector in the step (1), and starting expression by the stress-inducible promoter TIP 1-6-pro;
the gene of the adversity stress induced expression is GUS, and the gene product is GUS protein (beta-glucuronidase); GUS protein is stable and not easy to degrade, the detected material is soaked in a buffer solution containing a substrate X-Gluc, and if GUS gene transformation occurs in tissue cells and the GUS protein is expressed, the tissue or the cell with GUS activity can be blue, and can be observed by naked eyes or a microscope, and the degree of the GUS activity can be reflected by the depth of staining to a certain degree. Therefore, GUS can be used for observing the expression condition of the exogenous gene in specific organs, tissues and even single cells, and the method is very visual and convenient;
(3) transforming agrobacterium with the plant expression vector constructed in the step (2);
(4) infecting the plant with the agrobacterium transformed in the step (3) to obtain transgenic plant with adversity stress induced expression transferred gene.
The stress includes abiotic stress and hormone influence, wherein the abiotic stress includes drought and high salinity, and the hormone influence includes ABA (abscisic acid) and MeJA (methyl jasmonate). ABA and MeJA are both called stress hormones or stress hormones (stress hormons) and can increase drought resistance and salt resistance of plants.
The promoter TIP1-6-pro can start the target gene to be obviously expressed in the whole seedling plant, particularly in vascular tissues, root hairs and lateral roots, can obviously improve the expression level of the target gene under abiotic stress such as drought and high salt and hormone conditions such as ABA, MeJA and the like, and has good application value in plant stress resistance genetic engineering breeding.
An application of the method for constructing transgenic plant with adversity stress induced expression transferred gene in drought resistance and salt resistance of plant. Namely, the seedling plant is planted in the environment of adversity stress, and the promoter TIP1-6-pro can ensure that the introduced target gene can be stably expressed in the whole seedling plant under the environment of adversity stress.
The plant comprises leguminous plants and arabidopsis thaliana; the leguminous plant is preferably vegetable soybean.
Compared with the prior art, the invention has the beneficial effects that: the promoter TIP1-6-pro can start the target gene to be obviously expressed in the whole seedling plant, particularly in vascular tissues, root hairs and lateral roots, can obviously improve the expression level of the target gene under the conditions of drought, high salt content, ABA and MeJA, and has good application value in plant stress-resistant genetic engineering breeding.
Drawings
FIG. 1 is a schematic diagram showing the expression pattern of TIP1-6 gene of the present invention under various treatment conditions;
wherein: a and B are respectively PEG and NaCl abiotic stress treatment, C and D are respectively ABA and MeJA hormone treatment;
FIG. 2 is a schematic diagram showing the distribution of abiotic stress and hormone responsive elements in the TIP1-6-pro promoter of the present invention;
FIG. 3 is a schematic diagram of construction of GUS fusion expression vector of TIP1-6-pro promoter of the present invention;
FIG. 4 is a schematic representation of GUS staining of the TIP1-6-pro promoter transgenic Arabidopsis in different tissues of the present invention;
wherein: a is the result of dyeing the overground part of the seedling, and B is the result of dyeing the underground part (main root and lateral root) of the seedling;
FIG. 5 is a schematic diagram of GUS activity detection of a TIP1-6-pro promoter transgenic Arabidopsis thaliana under different concentrations of PEG and NaCl treatment conditions according to the present invention;
wherein: a and C are GUS staining results, B and D are GUS activity assay results, asterisks indicate significant differences in treated versus control plants (. about.p < 0.01);
FIG. 6 is a schematic diagram of GUS activity detection of TIP1-6-pro promoter transgenic Arabidopsis under different concentrations of ABA and MeJA treatment conditions according to the present invention;
where a and C are GUS staining results, B and D are GUS activity assays, and asterisks indicate significant differences between treated and control plants (. about.p < 0.01).
Detailed Description
The present invention will be described in further detail with reference to the following examples and accompanying drawings, in which the materials used in the examples are commercially available or may be processed by conventional methods.
The primer list is shown in table 1:
table 1:
note: restriction enzyme recognition sites are underlined.
Example 1
(1) Material treatment: seeds of vegetable soybeans (seed quality number: GMLN 012012012017) are sown in a flowerpot (nutrient soil: vermiculite: 3:1), and the flowerpot is placed in a light incubator, the temperature is set to be 22 ℃ in the daytime/20 ℃ in the nighttime, the light cycle is set to be 16h of light/8 h of dark, and the relative humidity is set to be 60%. Culturing for 3 weeks, taking out from the flowerpot, washing off soil, respectively performing drought (20 wt% PEG6000), high salt (250mM NaCl), abscisic acid (100 muM ABA) and methyl jasmonate (100 muM MeJA) solution root soaking treatment, respectively treating for 0h, 0.5h, 1.5h, 6h, 12h and 24h, setting the same time period for clean water root soaking treatment as test control, finally respectively taking the whole seedling and wrapping the seedling with tinfoil paper, placing the seedling in liquid nitrogen for quick freezing, and storing the seedling in an ultra-low temperature refrigerator at-80 ℃.
(2) Analysis of gene expression pattern: RNA was extracted and cDNA was synthesized by reverse transcription according to the instructions of RNAprep Pure Plant Kit and FastQuant RT Kit (Tiangen Biochemical technology Co., Ltd.). The cDNA of vegetable soybean is taken as a template, TIP1-6F and TIP1-6R are taken as primer combinations, real-time fluorescence quantitative PCR amplification is carried out on TIP-6, an Actin gene is set as an internal reference, and the primer sequences are shown in Table 1.
Fluorescent quantitative PCR (qRT-PCR) PCR amplification was performed using an Applied Biosystems StepOnePlusTMreal-Time System. The PCR reaction conditions were set as follows: 15min at 95 ℃; 95 ℃ for 10s, 52 ℃ for 20s, 72 ℃ for 30s, 40 cycles. The PCR reaction system was 20. mu.l, according to the instructions of SuperReal Premix Plus (SYBR Green) (Tiangen Biochemical technology Co., Ltd.). After the reaction, the change in the primer melting curve and fluorescence value was analyzed by 2 –ΔΔCt The method analyzes the mRNA expression amount.
The results show that: the TIP1-6 gene responds to drought, high salt, ABA and MeJA, the expression level is highest after PEG and MeJA treatment for 1.5h, and the expression level is highest after NaCl and ABA treatment for 6.0h, as shown in figure 1.
(3) Promoter cloning and analysis of the constituent elements: DNA was extracted according to the instructions of the Rapid plant genomic DNA extraction System (Tiangen Biochemical technology Co., Ltd.). Using vegetable soybean DNA as a template, high fidelity enzyme PrimeSTAR Max (TaKaRa) and TIP1-6-proF and TIP1-6-proR as primer combinations, the primer sequences were PCR amplified as shown in Table 1. The PCR reaction conditions were set as follows: 3min at 95 ℃; at 95 ℃ for 30s, at 52 ℃ for 30s, at 72 ℃ for 1min, for 35 cycles; 5min at 72 ℃. The PCR reaction system was 20. mu.l, according to the instructions for the high fidelity enzyme PrimeSTAR Max (TaKaRa). The PCR amplified fragment was subjected to fragment recovery using a DNA gel recovery kit (TaKaRa), and ligated into a cloning vector pEASY-Blunt (all-grass Biotech Co., Ltd.) to transform E.coli competent cell DH 5. alpha. After screening and sequencing comparison, the correct bacterial liquid is extracted with plasmid DNA extraction kit and stored at-80 deg.c for further use. The abiotic stress and cis-acting elements of the hormone signal contained in the promoter sequence were analyzed using the online software plantacare (http:// bioinformatics. psb. content. be/western tools/plantacare/html /).
The results show that: the DNA molecule from-1483 to-1 is a nucleotide sequence of promoter TIP1-6-pro shown in sequence 1, and comprises drought and multiple hormone response elements, specifically, 1 MBS (drought), 5 ABRE (abscisic acid), 2 CGTCA-motif (jasmonic acid), 2 TGACG-motif (jasmonic acid), 1 TCA-element (salicylic acid), 1 TGA-element (auxin), 1P box (gibberellin), and 1 TATC box element, as shown in FIG. 2.
Example 2
(1) Constructing a promoter GUS gene fusion expression vector: PCR amplification was performed as shown in Table 1 using the high fidelity enzyme PrimeSTAR Max with TIP1-6-proGUSF and TIP1-6-proGUSR as primer combinations based on the restriction sites (Pst I and Sma I) of the expression vector pCAMBIA1391 z. And carrying out double enzyme digestion (TaKaRa) on the PCR amplification fragment and the pCAMBIA1391z vector at the same time, recovering and connecting the amplified fragment subjected to enzyme digestion and the vector (TaKaRa), and sequencing the constructed recombinant vector pCAMBIA1391z-TIP 1-6-pro. After sequencing comparison, the correct bacterial liquid is extracted with plasmid DNA extraction kit and stored at-80 deg.c for further use.
The results show that: the sequence between restriction sites Pst I and Sma I of the pCAMBIA1391z vector was replaced with a DNA molecule shown in FIG. 2 from position-1483 to position-1, and GUS of the recombinant vector was expressed by TIP1-6-pro as shown in FIG. 3.
(2) Obtaining an arabidopsis genetic transformation plant: transforming a recombinant vector pCAMBIA1391z-TIP1-6-pro into arabidopsis thaliana according to an agrobacterium-mediated inflorescence dip-dyeing method, disinfecting the harvested T1 generation transgenic arabidopsis thaliana seeds, sowing the seeds into a 40mg/ml HYG (hygromycin) MS culture medium, screening resistant seedlings, and transplanting the seedlings into soil. Extracting DNA of T1 generation resistant vaccine, and PCR amplifying by using TIP1-6-proF and TIP1-6-proR as primer combination. By the same method, T3 generation transgenic arabidopsis homozygous lines are obtained by screening.
Test example 1
Transgenic Arabidopsis plants were immersed in GUS staining solution (0.1M phosphate buffer, 0.5mM potassium ferrocyanide, 0.5mM potassium ferricyanide, X-Gluc 0.5mg/ml) and treated overnight at a constant temperature of 37 ℃ for 12 hours, then decolorized with 70% by volume ethanol, and the distribution of GUS expression was observed by a stereomicroscope and photographed as shown in FIG. 4.
The results show that: TIP1-6-pro has promoter activity, blue color is detected in root, stem and leaf tissues of transgenic Arabidopsis seedlings, and the blue color is particularly obviously expressed in vascular tissues, root hairs and lateral roots.
Test example 2
Abiotic stress and hormone treatment of transgenic arabidopsis plants: the obtained T3 generation transgenic Arabidopsis seeds are sown in MS culture medium, after the seedlings normally grow for 5 days, the seedlings are respectively transferred to MS culture medium with different concentrations (PEG, NaCl, ABA, MeJA) for treatment and culture for 5 days, GUS staining is photographed, and GUS activity is measured. The GUS enzyme activity determination method mainly refers to a Jefferson method, 4-methylumbelliferyl-beta-D-glucuronide ester (4-MUG) is used as a substrate, and the GUS enzyme catalyzes the hydrolysis of the 4-methylumbelliferyl-beta-D-glucuronide ester into 4-methylumbelliferone (4-MU) and beta-D-glucuronic acid. The hydroxyl in the 4-MU molecule is dissociated and then excited by 365nm light to generate 455nm fluorescence, which can be quantified by a fluorescence spectrophotometer.
PEG simulates drought stress; NaCl simulates salt stress; the ABA can adjust the stomatal aperture of the plant, and the content of the ABA in the plant leaves is increased under the condition of water shortage, so that stomatal closure is caused, because the ABA promotes outflow of potassium ions, chloride ions, malic acid ions and the like, and stomatal closure is promoted; MeJA can regulate and control stomata opening and closing and photosynthesis intensity of plants, promote the content of permeation regulating substances in the plants to be increased, regulate an antioxidant enzyme system of the plants, and relieve the damage of drought and salt stress to the plants.
The results show that: as shown in FIGS. 5 and 6, TIP1-6-pro is a drought, high-salt, ABA, MeJA inducible promoter, transgenic Arabidopsis seedlings are all darkened in blue after treatment with different concentrations of PEG, NaCl, ABA, and MeJA, and GUS activity is strongest after treatment with 2 wt% PEG, 50mM NaCl, 25. mu.M ABA, and 50. mu.M MeJA.
According to the test examples, the application of the method for constructing the transgenic plant for inducing expression of the transgene by the adversity stress in drought resistance and salt resistance of the plant is realized, namely that the adversity stress inducible promoter TIP1-6-pro can start the efficient expression of the target gene under the conditions of drought, high salt, ABA and MeJA.
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> stress-inducible promoter TIP1-6-pro and expression vector construction method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1483
<212> DNA
<213> stress-inducible promoter TIP1-6-pro
<400> 1
AGCCGTCCAT AATAAACAAA GAACGAAACA GATAAGCTTA AAAATTATGA TAATTTAAAT 60
ACTGTTGGAG GTGGCAATGG GATTTGAAAA TACTTGTGGC TCCTGATGCA TCATACTGAA 120
AGACAAATAC AGACGAGTAG CAATTTGTAG ATTATATTCG AGGCCCAGAT TGAATTGATA 180
GGCCTTGGCA CTTTTATTAT TATTTTAAGC AAATCACTTG AGCAGTTTGA TACATGATTA 240
GTTGATTTAA GAATTAAAAT CGAATCAACT GAAACCCTCA TTTTCACAAC ACTACCTGCT 300
GAATTTTCAA TTAGCTCTAA CAAATCATTG AACATTGTCA TAAAAAAAAA AAAAAAAAAA 360
ACTTAGGCTG TGTTTGTCTG ATCTGTAGTC GGATCCATGA AGAAAACTAG TGAAACTTTT 420
AGTAACAAAT TTATTATCTT AATTGACGTC AAGTGGCCAC TATCTATGGA CACAAAGTGA 480
TTAATTTATC CATTCGTAAC AACTAACAAA ATAAACATGA CAATGTTATA GCGCAGGGAC 540
TCTTTGAATA AAGCATTAAC CTTCGACCAT CAAATGATCG TATTCACGTT GACACCACTC 600
CAAACAACAT TGTTATCCCA CAGCTAGGAA TGAAGCTGGC ACACCTCCAA TATCACAACC 660
AATAAGAGCC AATAATTTTG TGGGCCATCC TCACCTTTCA AATTCTGGAG CTGACTTGAA 720
AAAAAAATAT CATTATTCAT TAGACCTTTG CTCTTATCAT CTCCACGAAA CACATGGTAT 780
TCTTTACGAT TGATTTTTGG TAACAAAAGT CGGAAAAGAT AAATTAATTA GTCTTTGAAT 840
AATTTGGTAT TCTCTGAAGA GGAGTGCTAT CAGCATTGAT TGAAGGATCA ATGCACCTAA 900
ATAAATTTAT TAACTACATT TGCAACACTA GTAAAAGACT GTAAAACCAT ATTTGATCTT 960
ATGAAAAGTA AAAGAATAAA CTAATCGTTA CAGGAATTAA TAATGAAACT TTGTTTGCAA 1020
GACAAATTAG TTGACTAGCT CCCTAGGGGG TGTGGTAATA ACTAATAATA ACTATGAATA 1080
ATAATGAATC CAACATTATT GGGCCGGTCC ACATCACCGC CCAGTAAAAG AATTCAAAGG 1140
GTAGGTTTGG TCCAAAAGGT ACACATTGAT TGAAGGACCC ACCAGCCCAC AGGCAATTGG 1200
TCGGTGACCA GTGATTAGTC CACATCATGT TGTACACGTG GCATCACAAG AAGGACCGGA 1260
AGGCCCGCCC CTCCGCCACC CTAAGCAATA GACAGGTGGC AAAGAGTTTG AATTTTCTTC 1320
CTTATCTTTG TCCCTCGTGT CTTAAACTCC GCTAGCTATA GTGTTGTGTA ATACTATATA 1380
ACACCCGTAA CAATTGCACA AAAGTTCCTA ACAACGACTT AAGGCATTCT CTCTTCTATT 1440
CTATTCTAAA CTCGAAACAA TCTTAGAGAA AGAAGCAGAA GAA 1483
Claims (9)
1. An adversity stress inducible promoter TIP1-6-pro, wherein the nucleotide sequence of the adversity stress inducible promoter TIP1-6-pro is shown as SEQ ID NO: 1 is shown.
2. The adversity stress inducible promoter TIP1-6-pro according to claim 1, wherein said adversity stress comprises abiotic stress, hormonal effect.
3. The adversity stress inducible promoter TIP1-6-pro according to claim 2, wherein said abiotic stress comprises drought, salt stress and the hormonal effect comprises ABA, MeJA.
4. A construction method of a transgenic plant with adversity stress induced expression transgene is characterized by comprising the following steps:
(1) constructing a plant expression vector of the adversity stress inducible promoter;
(2) inserting a gene to be expressed by the stress induction into a plant expression vector in the step (1), and starting expression by the stress-inducible promoter TIP 1-6-pro;
(3) transforming agrobacterium with the plant expression vector constructed in the step (2);
(4) infecting the plant with the agrobacterium transformed in the step (3) to obtain a transgenic plant with adversity stress induced expression transgene.
5. The method for constructing a transgenic plant with an adversity stress induction expression transgene according to claim 4, wherein the vector skeleton in step (1) is pCAMBIA1391z plasmid.
6. The method for constructing transgenic plant with transgene expression induced by adversity stress as claimed in claim 4, wherein said adversity stress includes abiotic stress and hormonal influence.
7. The method for constructing transgenic plant with transgene induced by adversity stress as claimed in claim 6, wherein said abiotic stress includes drought and salt stress, and the hormonal influence includes ABA and MeJA.
8. An application of the method for constructing transgenic plant with adversity stress induced expression transferred gene in drought resistance and salt resistance of plant.
9. The application of the method for constructing transgenic plants with transgene expressed by inducing stress to be transferred to plants in drought resistance and salt resistance of plants according to claim 6 is characterized in that the plants comprise vegetable soybeans and arabidopsis thaliana.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210418024.3A CN114854746B (en) | 2022-04-20 | 2022-04-20 | Adversity stress inducible promoter TIP1-6-pro and construction method and application of expression vector thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210418024.3A CN114854746B (en) | 2022-04-20 | 2022-04-20 | Adversity stress inducible promoter TIP1-6-pro and construction method and application of expression vector thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114854746A true CN114854746A (en) | 2022-08-05 |
| CN114854746B CN114854746B (en) | 2023-07-11 |
Family
ID=82631855
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210418024.3A Active CN114854746B (en) | 2022-04-20 | 2022-04-20 | Adversity stress inducible promoter TIP1-6-pro and construction method and application of expression vector thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114854746B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090282582A1 (en) * | 2006-10-20 | 2009-11-12 | Riken | Environmental stress responsive promoter and method of tissue-specific gene expression using the same |
| US20120023614A1 (en) * | 2010-04-01 | 2012-01-26 | Gendocs, Inc. | Environmental stress-inducible 557 promoter isolated from rice and uses thereof |
| CN105524925A (en) * | 2016-01-11 | 2016-04-27 | 北京市农林科学院 | Plant induction type promoter and application of plant induction type promoter |
| CN105985956A (en) * | 2015-02-04 | 2016-10-05 | 中国科学院遗传与发育生物学研究所 | Promoter for inducible expression of GhiWRKY40 under stresses and application of promoter |
| CN107881172A (en) * | 2016-09-30 | 2018-04-06 | 江汉大学 | A kind of adverse circumstance inducible promoter, adverse circumstance inducible promoter plant expression vector and induction target gene expression |
| US20180312856A1 (en) * | 2015-10-23 | 2018-11-01 | Universidad De Talca | Synthetic promotor induced by abiotic stress, genetic construct containing same and plant cells transformed therewith |
| CN114214358A (en) * | 2021-11-17 | 2022-03-22 | 中国农业科学院生物技术研究所 | Inducible expression vector and application thereof in cultivation of sentinel crops |
-
2022
- 2022-04-20 CN CN202210418024.3A patent/CN114854746B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090282582A1 (en) * | 2006-10-20 | 2009-11-12 | Riken | Environmental stress responsive promoter and method of tissue-specific gene expression using the same |
| US20120023614A1 (en) * | 2010-04-01 | 2012-01-26 | Gendocs, Inc. | Environmental stress-inducible 557 promoter isolated from rice and uses thereof |
| CN105985956A (en) * | 2015-02-04 | 2016-10-05 | 中国科学院遗传与发育生物学研究所 | Promoter for inducible expression of GhiWRKY40 under stresses and application of promoter |
| US20180312856A1 (en) * | 2015-10-23 | 2018-11-01 | Universidad De Talca | Synthetic promotor induced by abiotic stress, genetic construct containing same and plant cells transformed therewith |
| CN105524925A (en) * | 2016-01-11 | 2016-04-27 | 北京市农林科学院 | Plant induction type promoter and application of plant induction type promoter |
| CN107881172A (en) * | 2016-09-30 | 2018-04-06 | 江汉大学 | A kind of adverse circumstance inducible promoter, adverse circumstance inducible promoter plant expression vector and induction target gene expression |
| CN114214358A (en) * | 2021-11-17 | 2022-03-22 | 中国农业科学院生物技术研究所 | Inducible expression vector and application thereof in cultivation of sentinel crops |
Non-Patent Citations (4)
| Title |
|---|
| LI SONG等: "SoybeanTIPGeneFamilyAnalysisandCharacterizationofGmTIP1;5andGmTIP2;5WaterTransportActivity", FRONTIERSINPLANTSCIENCE * |
| ZHIJUAN FENG等: "Promoter of Vegetable Soybean GmTIP1;6 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis", INT. J. MOL. SCI * |
| 冯志娟等: "干旱和高盐胁迫下菜用大豆GmTIP2;6启动子的功能分析", 分子植物育种 * |
| 吕兆勇;赵春梅;薛仁镐;: "葡萄逆境胁迫诱导启动子的克隆及表达分析", 华北农学报 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114854746B (en) | 2023-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113025626B (en) | Application of tumorous stem mustard BjuEAR1 gene in regulation of plant stress resistance | |
| CN105198976B (en) | A kind of and plant adversity resistance related protein GsERF6 and its encoding gene and application | |
| CN107881172B (en) | Stress inducible promoter, stress inducible promoter plant expression vector and method for inducing expression of target gene | |
| CN105505932B (en) | A kind of plant inducible promoter and its application | |
| CN110004154B (en) | Application of tea tree CsJAZ1 gene | |
| CN112522283B (en) | Pollen development related gene and application thereof | |
| CN103243096B (en) | Plant tissue specific expression promoter and application of plant tissue specific expression promoter | |
| CN108486112B (en) | Promoter with anther tissue specificity | |
| CN111621504A (en) | Stress-resistant gene BjuIBS of tumorous stem mustard and application thereof | |
| CN114703199B (en) | Plant drought resistance related gene TaCML46 and application thereof | |
| CN113774059B (en) | Dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof | |
| CN114854746B (en) | Adversity stress inducible promoter TIP1-6-pro and construction method and application of expression vector thereof | |
| CN113061617B (en) | Chinese cabbage B-type response regulatory factor gene BrRR10 and application thereof | |
| CN102965374B (en) | Preparation method and applications of rape BnRabGDI3 promoter | |
| CN103788187B (en) | Flowering of plant associated protein GmSOC1-like and encoding gene thereof and application | |
| CN109234290B (en) | Brassica napus BnKAT2 gene and promoter and application thereof | |
| CN108795942B (en) | Rice exogenous stress induced expression promoter Psubs3 and application thereof | |
| CN105274106A (en) | Peanut AhWRI-1 promoter and preparation method and application | |
| CN112251439B (en) | Arabidopsis thaliana high-temperature induction promoter pHTG1 and recombinant vector thereof | |
| CN115927310B (en) | China rose skin thorn specific expression promoter proRcLAC15 and application thereof | |
| CN108823221A (en) | Larch in Xinanlin area LgUGPase gene promoter sequence and its application in genetic modification of plants | |
| CN111718938B (en) | High-temperature inducible promoter specifically expressed by plant green tissue and application thereof | |
| CN114921466A (en) | Inducible promoter pDI19-1 and expression vector construction method and application thereof | |
| CN116769797B (en) | Application of methyl jasmonate and PpyMYC2 gene in germination | |
| CN115927309B (en) | China rose root specific expression promoter proRcPRX10 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |