CN116376911B - Plant drought, low temperature and osmotic stress induced promoter and application thereof - Google Patents
Plant drought, low temperature and osmotic stress induced promoter and application thereof Download PDFInfo
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Abstract
The invention relates to the fields of biotechnology and plant genetic engineering, in particular to a promoter for drought, low temperature and osmotic stress induction of plants and application thereof. The nucleic acid sequence of the promoter is a DNA sequence shown as SEQ ID NO.1. The promoter can drive the high expression of an Arabidopsis thaliana C2H 2-type zinc finger transcription factor protein 5 (AtZAT 5) gene under drought and low temperature stress, can be used for improving and improving the growth characteristics and stress tolerance of plants, and has good application prospects in stress-tolerant genetic improvement of plants.
Description
Technical Field
The invention relates to the fields of biotechnology and plant genetic engineering, in particular to a promoter for drought, low temperature and osmotic stress induction of plants and application thereof.
Background
The promoter is a DNA sequence which is positioned in the upstream region of the 5' end of the gene, can bind RNA polymerase and ensure the accurate and effective initiation of gene transcription, and plays an important role in the expression regulation of the gene. The activity of a promoter determines the expression efficiency of an endogenous or exogenous gene. Three general categories can be distinguished based on the expression characteristics of the promoters: constitutive promoters, tissue-specific promoters, and inducible promoters. The constitutive promoter can effectively drive the excessive expression of exogenous genes, but can cause resource waste, even influence the growth and development of plants and normal physiological metabolism, for example, dwarf deformity of plants can be caused when the CaMV35S promoter is used for improving the stress resistance of plants. And the inducible promoters such as the adversity stress inducible promoters do not affect the growth metabolism of plants under normal conditions, and only when the plants are subjected to external stress, the over-expression of the adversity resistance genes is started, so that the plants show a certain tolerance or adversity resistance. Therefore, isolation and identification of stress-inducible promoters in plants and utilization thereof has become one of the research hotspots of plant genetic engineering in recent years.
Adverse stress such as drought, low temperature, high osmotic pressure and the like seriously affects quality and yield of crops. The transcription factors are driven by the stress-induced promoter, so that the expression of a plurality of stress-induced genes is activated, and the method is an effective means for improving the stress resistance of crops. Most of the currently reported adversity-induced promoters are induced by single adversity stress, but the researches on the promoters induced by multiple adversity stresses are relatively few. Because the plant is not only stressed by a single adversity, but also stressed by multiple adversity in different growth periods in the whole growth process of the plant, the single adversity stress inducible promoter shows a certain limitation in application, and the multiple adversity stress inducible promoters have more advantages in application of improving the comprehensive adversity resistance of the plant.
Chinese patent CN107099535a discloses a promoter induced by low temperature, high salt, drought or ABA, which can be induced and started by various adversity factors of low temperature, high salt, drought or ABA; chinese patent CN114921466A discloses an inducible promoter pDI19-1, and an expression vector construction method and application thereof, which can obviously improve the expression level of a target gene under the conditions of low temperature, drought, ABA (abscisic acid) and MeJA (methyl jasmonate). However, only these promoters can not meet the needs of research and application, and the invention aims to provide a novel plant drought, low temperature and osmotic stress induced promoter so as to enrich the variety of the promoters and meet the needs of application.
Disclosure of Invention
The invention aims to solve the technical problems that the number of the promoters obtained by current research is small, and the requirements of research and application cannot be met.
In order to solve the problems, the invention discovers a promoter induced by various stress through long-term research, in particular to a promoter ProAtZAT5 which is derived from Arabidopsis and is induced by drought, low temperature and high osmotic stress, and has good application prospect in genetic engineering improvement and genetic improvement of comprehensive stress resistance of plants.
In order to achieve the above purpose, the invention is mainly realized by the following technical means: a promoter for drought, low-temp and osmotic stress induction of plant has the nucleic acid sequence of SEQ ID NO.1.
SEQ ID NO.1：
aagtgatatgattatcatactaatatttagaagtggttaaaaataaatatattttattattattcttctttctttttcttttctttttttttgacagggatttttgttttttaaaaagagtccttgacaaggattttgtggtgtggtttagccactatgcattataccttatgcatagatatgggttcaagcctcaatctcccactgtgagactttgaaagtatgttgacaaaaaaaggtgtataatttttccagaataaattattttaatctagtttgcctagctaaattaaataattattttgaggtccctaaacttcataattttccggtctctcaatcttgacataacttattctctgtctatagtttttttttttttgaacttttctctatagattacttattctataatctataaagtattatttttggatgtaccgtgtaaactcgttttaatggttagttgtcacttgtccatacaaaaatgtaacattccaatatttgacaataaagtaatgttacataaaaaatttctataatttgagtatgttttttttttttttttgtcggcctataatttgagtatgttaattaatctcaattgtaaaaatagatctctaattatcttaaaactgtttccagtcttaagattagtagataaccaattgagcactagctttcaactctctctacaaatgttaatcaagatatgattaaaaaatgagagaaaaaaaaaagtcgtggtgttgtctcttcacattaacaaagactaagcttaatattgtttttagcctaacatgccaccgatgaatgcacctaatcctataaacaccggccactgcatgtcgcctcctaattatgaaccatgtatgtctgtgaatcattatttccctttgataatcttattagtatatttcaaaacatttaatcaaaagttttcttgtagtcgtcacgaccattaataatatgtaatatattctttttatttgctgtcgtgaacatttttatctttagttctatctatttcggttctcattgaaagcgtcgatttgatgacgtggacctaaagtgcgtgaaagtaaaacctttgctttgtccttagaactgaatgttcacactcccaaaaattaaattcgaaactaaataaataaaaacaaataacaaaaacgtgaattggtggcctaagtgccactcaacactactaatgaacctttgcctaattcagtggttaactcttcacgggtttcactttcccttttatttctcctacttttgcttgaaacaatattaatcattttcactcggaattaataacaattctctgttaattatggaatatgaatgattcaagagtttgattaatcacaccaattgatattaatatgtttgttattagaaagtatgtactagaagaagctgtctaatacaatactaataaatgaaaaaacgtcatgaagggttgacttctggtatatagatctaggtagagatcagattttgaagatatggattaaaagtagatacaacagaacagaaactagaaaagaagaaaaattatttacatgtcatatttttttcatcattgaattataaatttgttgattgattttattgttatggaatttggcagagaaaacgcagaagtgtcaggtaatgggtatagagtgaaccgtcaaaatgttattggttagctaaaaacattataataatgtaaagtggatcgatgtgtaaaatcttttgtggagtaaactaaaaagattaacaaccaccaaacttataacctctctctttttttttccgataaatatatatataagcagccccaccatcgtcttcttcactgcctctcttctctcttctcttctgtctctctcttgttagttcactccacataataaacaccaaagatttcattctcttctccataatttcgaagtttcttgaattgggtttgtttcttgatttgtttcttgaattgggttttggtcttcttttcttactatatttggat
The promoter disclosed by the invention can drive the high expression of an Arabidopsis C2H 2-type zinc finger transcription factor protein 5 (AtZAT 5) gene under drought and low-temperature stress, so that the promoter is named as ProAtZAT5. The promoter is a promoter of a section of gene obtained by cloning from an arabidopsis genome, then a recombinant vector for driving the expression of a reporter gene beta-glucosidase Gene (GUS) by the promoter is constructed, the constructed recombinant vector is transformed into arabidopsis, and a single copy inserted transgenic homozygous strain is obtained through multi-generation resistance screening; GUS histochemical staining experiments are carried out on the obtained transgenic homozygous plants, and the GUS histochemical staining experiments show that the promoter drives GUS reporter genes to be expressed at high level under drought, cold and high osmotic stress induction.
A primer pair for cloning the promoter ProAtZAT5, which comprises a first primer and a second primer, wherein the DNA sequence of the first primer is shown as SEQ ID NO. 2; the DNA sequence of the second primer is shown as SEQ ID NO. 3.
SEQ ID NO.2：AAGTGATATGATTATCATAC
SEQ ID NO.3：ATCCAAAATATAGTAAGAAAAG
A primer pair for amplifying the promoter ProAtZAT5 comprises a first primer and a second primer, both of which contain homologous arm sequences of enzyme cutting sites, and amplification products can be connected into a plant expression vector in a homologous recombination mode. The DNA sequence of the first primer is shown as SEQ ID NO. 4; the DNA sequence of the second primer is shown as SEQ ID NO. 5.
SEQ ID NO.4：caagcttggctgcaggtcgacAAGTGATATGATTATCATAC
SEQ ID NO.5：tcttagaattcccggggatccATCCAAAATATAGTAAGAAAAG
An inducible promoter eukaryotic expression vector for plant transformation, which comprises a promoter with a nucleotide sequence of SEQ ID NO.1, and further comprises pCAMBIA1391-ProAtZAT 5; the expression vector is recombinant expression vector, and can make the exogenous target gene express in high efficiency. The method is realized by the following steps: the recombinant expression vector is used for transforming plants, and the obtained transformed plants can induce the efficient expression of exogenous genes through drought and/or low-temperature treatment.
A transformant which is a plant expression vector, a host into which a promoter having a nucleotide sequence of SEQ ID NO.1 is introduced or a host into which the above recombinant eukaryotic expression vector is introduced. Further, the transformant is a cell line or a callus or a transgenic plant. The host at the time of transformation is Agrobacterium.
The promoter ProAtZAT5 is applied to plant stress resistance gene research or stress resistance genetic improvement. The specific method is to transform plants with a recombinant expression vector containing the promoter, which is used for improving tolerance or stress resistance of the plants or treat the obtained transformed plants through drought and/or low temperature and/or osmotic stress, and is used for inducing efficient expression of exogenous genes.
The invention has the beneficial effects that:
the promoter provided by the invention specifically drives exogenous genes to express in plants under drought, low temperature and osmotic stress, so that the promoter can be used for improving and improving the growth characteristics and stress tolerance of plants, and has good application prospects in stress-tolerant genetic improvement of plants.
Drawings
Fig. 1: the promoter ProAtZAT5PCR amplification electrophoretogram of the invention;
fig. 2: plasmid PCR electrophoresis diagram of the promoter ProAtZAT5 connection expression vector pCAMBIA 1391;
fig. 3: the plant expression vector pCAMBIA1391-ProAtZAT5 constructed by the invention is shown in GUS schematic diagram;
fig. 4: in the invention, the ProAtZAT5 drives GUS gene to carry out GUS histochemical staining under drought and low temperature stress conditions.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: cloning of promoters
1.1 preparation of Arabidopsis genome DNA
Taking arabidopsis planted in a greenhouse, taking tender leaves of the arabidopsis, extracting genome DNA by using a plant genome DNA extraction kit, detecting by agarose gel electrophoresis, and preserving at-20 ℃ for later use.
1.2 cloning of promoter ProAtZAT5
The arabidopsis genome DNA is taken as a template, and the upstream primer is as follows: AAGTGATATGATTATCATAC (SEQ ID NO. 2), the downstream primer is: ATCCAAAATATAGTAAGAAAAG (SEQ ID NO. 3) the ProAtZAT5 promoter candidate region was obtained by PCR amplification under the following conditions: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15min, annealing at 50℃for 15s, extension at 72℃for 2min, 30 cycles total, and storage at 4 ℃. The PCR product was electrophoretically detected (FIG. 1) and sequenced to obtain the 2014bp ProAtZAT5 promoter, which was SEQ ID NO.1.
Example 2: construction of plant expression vectors
The pCAMBIA1391 vector was digested with SalI/BamHI, and the large fragment was recovered for use. The PCR was again performed using the PCR product of example 1 as a template and caagcttggctgcaggtcgacAAGTGATATGATTATCATAC (SEQ ID NO. 4) and tcttagaattcccggggatccATCCAAAATATAGTAAGAAAAG (SEQ ID NO. 5) under the same amplification conditions as in example 1. The PCR product was then purified and recovered, and the promoter-order fragment was ligated into pCAMBIA1391 vector by means of homologous recombination, and positive recombinants were determined by PCR sequencing identification (FIG. 2). Thus, a plant recombinant expression vector pCAMBIA1391-ProAtZAT5:: GUS (FIG. 3) capable of expressing GUS gene was obtained.
Example 3: GUS Activity assay
3.1 Agrobacterium-mediated plant transformation
GUS was transformed into GV3301 in Agrobacterium by electric shock transformation.
Taking Arabidopsis seeds, sterilizing by sodium hypochlorite, sowing the Arabidopsis seeds on a seedling culture medium, and transferring the Arabidopsis seeds into soil after one week. After flowering, the inflorescence infection method is adopted, and the agrobacterium transferred into pCAMBIA1391-ProAtZAT 5:GUS is used for infection. Specifically, agrobacterium cultured at 28 ℃ is resuspended by using 5% sucrose solution after being cultured to OD600 = 0.8, and infection transformation is carried out by a flower dipping mode, and plants grow normally in a tissue incubator (24 ℃ for 16h/8h day/night cycle) after infection. After harvesting the seeds, resistance selection was performed on hygromycin medium containing 15 mg/L.
3.2 analysis of GUS expression Activity in transgenic plants
Seedlings of transgenic Arabidopsis T3 generation homozygous material were planted for GUS histochemical staining analysis, and fluorescence detection of GUS activity was performed in accordance with Jefferson method. Seedlings grown in soil for about 2 weeks are respectively subjected to stress treatment and drought treatment: placing the seedlings on a culture dish for water shortage for 8 hours, and then rehydrating overnight; high-permeability treatment of PEG 6000: the seedlings were placed in petri dishes containing 25% peg6000 in water overnight; and (3) low-temperature treatment: the seedlings were placed in an aqueous petri dish for overnight treatment at 4 ℃; control: seedlings were placed in an aqueous petri dish and cultured under normal conditions. The control and stress treated seedlings were then immersed in GUS staining solution, evacuated overnight at 37℃and decolorized with 75% ethanol, the staining observed under an inverted microscope, and photographed for recording.
The results showed that promoter proatat 5 was able to drive strong high level expression of GUS gene in transgenic plants under drought, low temperature, PEG6000 stress conditions (fig. 4).
The foregoing description of the preferred embodiments of the present invention is provided for the purpose of illustration only, and is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, alternatives, and alternatives falling within the spirit and scope of the invention.
Claims (8)
1. The application of a promoter in drought resistance, low temperature and osmotic stress resistance gene research or genetic improvement of plants is provided, and the nucleic acid sequence of the promoter is a DNA sequence shown as SEQ ID NO.1.
2. Use of a primer pair for cloning the promoter of claim 1 in drought, low temperature, osmotic stress gene research or genetic improvement of plants, characterized in that: the primer pair comprises a first primer and a second primer, and the DNA sequence of the first primer is shown as SEQ ID NO. 2; the DNA sequence of the second primer is shown as SEQ ID NO. 3.
3. Use of a primer pair for amplifying the promoter of claim 1 in drought, low temperature, osmotic stress gene research or genetic improvement of plants, characterized in that: the primer pair comprises a first primer and a second primer, and the DNA sequence of the first primer is shown as SEQ ID NO. 4; the DNA sequence of the second primer is shown as SEQ ID NO. 5.
4. The use according to claim 1, wherein: when in use, a recombinant expression vector containing a promoter with a nucleotide sequence of SEQ ID NO.1 is constructed.
5. The use according to claim 1, wherein: when in use, a transformant which is introduced with a promoter with a nucleotide sequence of SEQ ID NO.1 is constructed.
6. The transformant according to claim 5, wherein: the transformant is a cell line or a callus or a transgenic plant.
7. The transformant according to claim 5, wherein: the host at the time of transformation is Agrobacterium.
8. The use according to claim 1, wherein: transforming plants with a recombinant expression vector comprising said promoter, and using the obtained transformed plants under drought and/or low temperature and/or osmotic conditions for inducing efficient expression of exogenous genes.
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| Lin,X等.Arabidopsis thaliana C2H2-type zinc finger family protein (AT2G28200), mRNA.NCBI Genbank.2022,第1-2页. * |
| Overexpression of MdZAT5, an C2H2-Type Zinc Finger Protein, Regulates Anthocyanin Accumulation and Salt Stress Response in Apple Calli and Arabidopsis;Da-Ru Wang等;Int J Mol Sci;第23卷(第3期);第1-16页 * |
| 大豆两个C2H2型转录因子基因序列特征及表达分析;侯思宇等;植物生理学报;第665-674页 * |
| 烟草C2H2锌指蛋白转录因子家族成员的鉴定与表达分析;杨明磊;晁江涛;王大伟;胡军华;吴华;龚达平;刘贯山;;遗传(第04期);第337-349页 * |
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