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Promoter with properties of inducing and organizing specific expression

A technology of inducible expression and sequence listing, applied in the field of promoters

Inactive Publication Date: 2010-09-29
WUXI RES INST OF APPLIED TECH TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about how these animal MRE-like elements play their regulatory roles in plants, and what are the corresponding transcription factors, and further research and analysis are needed

Method used

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  • Promoter with properties of inducing and organizing specific expression
  • Promoter with properties of inducing and organizing specific expression
  • Promoter with properties of inducing and organizing specific expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the discovery of OsMT-I-4b gene promoter

[0040] According to the full-length cDNA of the rice type I MT gene OsMT-I-4b (NCBI sequence number: NM_001073598), using the PCR method, using the rice Zhonghua No. 10 (Oryza sativa L.cv.Zhonghua 10) genomic DNA as a template, the Amplified and cloned to obtain a DNA fragment of 1730 bp upstream of the OsMT-I-4b gene, and named POsMT-I-4b (OsMT-I-4b gene promoter). Analysis of cis-acting elements in POsMT-I-4b see figure 1 , the A base in the translation initiation codon ATG is defined as +1, and there are two basic promoter elements in this sequence, TATA box and CAAT box, which are located in the -109 / -103 and -214 / -211 regions respectively , these elements function as essential elements in eukaryotic gene transcription.

[0041] Using PLACE (http: / / www.dna.affrc.go.jp / htdocs / PLACE / ) software to search and predict the cis-acting elements in the OsMT-I-4b promoter, it was found that the two basic promoter elem...

Embodiment 2

[0043] Embodiment 2, the acquisition of transgenic plants

[0044]1. Construction of recombinant expression vector A

[0045] 1. Using the genomic DNA of rice Zhonghua 10 as a template, use the specific primer pair R1 (R1-F: 5'-CA GGATCC CCCCTCAAAAACTG-3' / R1-R:5'-GC ACTAGT CTTGATCTTCTGGGTC-3', the underlined sequences represent the restriction sites of BamH I and Spe I respectively) for PCR amplification. After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis, and about 1.7kb of DNA was recovered and purified. (promoter; -1730 / -1; denoted as R1; DNA fragment shown in sequence 1 of the sequence listing).

[0046] 2. Digest the PCR product recovered in step 1 with restriction endonucleases BamH I and Spe I.

[0047] 3. Digest the plant expression vector pCAMBIA-1381 with restriction endonucleases BamH I and Spe I, and recover the vector skeleton.

[0048] 4. Ligate the digested product of step 2 with the vector backbone of step 3...

Embodiment 3

[0055] Embodiment 3, the tissue specificity of promoter

[0056] The T2 generation of the transgenic plants obtained in Example 2 and the T2 generation of the empty vector control were subjected to GUS histochemical staining to determine their tissue specificity. Arabidopsis seeds were surface sterilized, spread on 1 / 2MS (pH 5.8, 1.5% sucrose, 0.75% agar) plates, after vernalization at 4°C for 2 days, placed in a 22°C incubator, light for 16h, dark for 8h nourish. After two weeks of growth on medium, Arabidopsis seedlings were transplanted into nutrient soil to continue growing.

[0057] Histochemical detection of GUS in transgenic Arabidopsis according to Jefferson et al. .) method. Concrete steps: get seedling or each organ of its development process, fix 30min in 0.1M sodium phosphate buffer solution (pH 7.0) containing 0.5% paraformaldehyde; Then place in GUS reaction solution (containing 0.1M sodium phosphate buffer solution, pH 7.0 10mM EDTA, 5mM potassium ferricyani...

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Abstract

The invention discloses a promoter with the properties of inducing and organizing the specific expression. The promoter provided by the invention comprises DNA molecules or the partial fragment thereof shown as the sequence 1 in a sequence table. The promoter provided by the invention can be used as the promoter for metal response, applied in the transgenic plant engineering, and used for obtaining new species of crops with high tolerance to heavy metals, or be used for cultivating plants which can lead the heavy metals to be greatly accumulated. The promoter provided by the invention can be used as the promoter for stress response, applied in the transgenic plant engineering and used for obtaining the new species of the crops with the stress tolerance. The promoter provided by the invention can be used as the promoter for leading target proteins to organize the specific expression, applied in the transgenic plant engineering and used for obtaining the new species of the crops with the specific promotion of the target gene expression in roots.

Description

technical field [0001] The present invention relates to promoters having inducible and tissue-specific expression properties. Background technique [0002] Heavy metals, such as copper and zinc, can be used as cofactors or structural components of enzymes or other proteins, and play a vital role in the normal growth and development of plants. However, when the concentration of these heavy metal ions in the cells increases, it will have a toxic effect on the cells and lead to the inhibition of plant growth. During the long-term evolution of plants, a series of effective molecular mechanisms have been developed for detoxification of heavy metals, thereby improving the tolerance of plants to heavy metals, such as the use of some biomolecules for highly specific chelation of metal ions is the most important one of the mechanisms. These specific molecules include amino acids, organic acids, and two types of cysteine-rich polypeptides, phytochelatin (PC) and metallothionein (met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 刘进元董春娟王云余世实
Owner WUXI RES INST OF APPLIED TECH TSINGHUA UNIV
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