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Plant osmotic stress inducible promoter and application thereof

A promoter and sequence technology, applied in the field of molecular biology, can solve problems such as hindering the normal growth and development of plants, destroying the metabolic balance of plants, etc., to achieve the effect of improving gene expression and enhancing ability

Inactive Publication Date: 2015-03-04
SHIJIAZHUANG ACADEMY OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a plant osmotic stress-inducible promoter, which solves the problem that in the current stress-resistant molecular breeding process, the use of constitutive promoters makes the continuous expression of exogenous genes in large quantities, thereby destroying the metabolic balance of plants and hindering Problems with normal plant growth and development

Method used

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  • Plant osmotic stress inducible promoter and application thereof
  • Plant osmotic stress inducible promoter and application thereof
  • Plant osmotic stress inducible promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Expression analysis of osmotic stress-induced expression gene TaDeh1 in Shimai 15

[0023] 1.1 Material Handling

[0024] Selected Shimai 15 seeds were cultured in a light incubator with tap water until the stage of two leaves and one heart, the culture temperature was 22±1°C, and the light was 14 hours a day. Select 20-25 seedlings with consistent growth and development, put them in petri dishes, and treat them with 20% polyethylene glycol (PEG8000). Leaves and roots were stored in a -70°C refrigerator after quick freezing in liquid nitrogen; the experiment was set up with 3 replicates.

[0025] 1.2 Wheat total RNA extraction

[0026] RNAiso Plus (TaKaRa) was used to extract total RNA from 15 roots and leaves of shimai, and RNase-free DNase I (TaKaRa) was used to remove residual DNA in the total RNA.

[0027] 1.3 Synthesis of the first strand of cDNA

[0028] Using PrimeScript TM 1st Strand cDNA Synthesis Kit (TaKaRa) was used for first-strand cDNA synt...

Embodiment 2

[0037] Example 2 Cloning of osmotic stress-induced expression gene TaDeh1 promoter of Shimai 15

[0038] 2.1 Plasmid extraction from Shimai 15BAC mixed pool

[0039] Prepare the BAC mixed pool plasmid extraction reagent according to the molecular cloning guide, and carry out the plasmid extraction according to the following steps.

[0040] 1) Collect the bacterial solution twice in a 50ml centrifuge tube, centrifuge at 10,000rpm at 4°C for 5min, and remove the supernatant;

[0041]2) Add 6ml of pre-cooled solution I and shake to mix;

[0042] 3) Add 6ml of solution II, mix upside down, and place at room temperature for 5 minutes;

[0043] 4) Add 6ml of pre-cooling solution III, mix well by inverting up and down, and place on ice for 10 minutes;

[0044] 5) Centrifuge at 12,000rpm at 4°C for 10min;

[0045] 6) Take the supernatant, add 0.8 times the volume of isopropanol, and place it at room temperature for 20 minutes;

[0046] 7) Centrifuge at 12,000 rpm at 4°C for 10 mi...

Embodiment 3

[0070] Example 3 Construction of osmotic stress-induced expression gene TaDeh1 promoter expression vector (pTaDeh1pro: GUS)

[0071] The pCAMBIA1831Z vector and the pMD18-T simple vector containing the TaDeh1 promoter were digested with BamHI and HidIII respectively, and the large fragment of the pCAMBIA1831Z vector and the fragment of TaDeh1pro were recovered respectively. 4 After ligation with DNA ligase (NEB), DH5α competent cells were transformed, and the constructed promoter expression vector was verified by digestion with BamHI and HidIII and sequencing; see pDeh1pro: GUS expression vector map figure 2 .

[0072] 3.1 BamHI and HidIII digestion of pCAMBIA1831Z vector and pMD18-T simple vector containing TaDeh1pro

[0073] Plasmids were extracted using the GenStar plasmid mini-extraction kit. The plasmid was digested and digested in a water bath at 37°C for 3 hours, and the digested product containing TaDeh1pro and the large fragment of the pCAMBIA1391Z vector were reco...

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Abstract

The invention discloses a promoter of which the sequence is as shown in SEQ ID NO:1 in a sequence table. The promoter can be used for culturing transgenic plants, i.e., the transgenic plants containing the promoter and target genes are obtained by firstly, establishing a recombinant expression carrier by using the promoter and the target genes, subsequently infecting a transformant by using the recombinant expression carrier, further transforming target plants by using the transformant, and detecting. Due to the osmotic stress inducible characteristic, the promoter disclosed by the invention can be used for connecting relevant anti-retroviral target genes, used for promoting the expression of the relevant anti-retroviral target genes when plants are subjected to osmotic stress, and used for improving the capability of the plants to adversity stress, and a powerful tool is provided for the plant stress resistance gene engineering.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an osmotic stress-inducible promoter derived from wheat and its application. Background technique [0002] Gene transcription is the first highly selective link in the process of genetic information transmission, and the regulation of gene transcription is the essence and basis of studying this high selectivity. The regulatory elements of gene transcription mainly include cis-acting elements and trans-acting factors. The cis-acting elements of eukaryotic genes can be divided into promoters, enhancers and negative regulatory elements silencers according to their functions. The promoter responds to the induction of the external environment, stimuli, etc., participates in tissue-specific selection, controls the temporal and spatial expression of genes, maintains the basic level of expression of genes, etc., and maintains the growth, differentiation, development, movement, metabolis...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/82A01H5/00
Inventor 李孟军郭进考李亚青史占良高欣娜张楠
Owner SHIJIAZHUANG ACADEMY OF AGRI & FORESTRY SCI
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