Inducing reinforced composing type promoter and uses thereof

A technology of constitutive promoters and promoters, applied in the field of genetic engineering, to achieve the effect of improving the response effect

Inactive Publication Date: 2008-05-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, whether the PR promoter can still drive the expression of the reporter gene in response to the induction of SA and TMV after inserting the enhancer element or how many enhancer elements are inserted into the PR promoter has not been reported and patented.

Method used

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  • Inducing reinforced composing type promoter and uses thereof
  • Inducing reinforced composing type promoter and uses thereof
  • Inducing reinforced composing type promoter and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The percentages in the following examples are all mass percentages unless otherwise specified. Example 1 Construction of Induced Enhanced Constitutive Promoters Enh6 and Enh8

[0048] 1. Extraction of total DNA from tobacco (Nicotiana tabacum)

[0049] Take 1-2 g of young tobacco leaves in a pot in the greenhouse, and use the CTAB method ("Molecular Cloning" third edition) to extract the total DNA. Take 1-5 μl DNA sample, measure its concentration and purity with a UV spectrophotometer, and use 0.8% agarose gel electrophoresis to detect the DNA purity and integrity. Store the extracted DNA at -20°C.

[0050] 2. PCR amplification and recovery of amplified fragments

[0051] According to the sequence (GenBank accession number: AB086949), the following two primers (primer 1 and primer 2; sequence 2 and sequence 3 in the sequence listing) were designed and synthesized to amplify the sequence of the promoter PR-N, in order to facilitate future cloning and construction, R...

Embodiment 2

[0069]Example 2 Construction of Induced Enhanced Constitutive Promoter-Driven Reporter Gene GUS (Enh6::GUS and Enh8::GUS) Plant Expression Vectors pRDEnh6G and pRDEnh8G

[0070] The construction process of the plant expression vectors pRDEnh6G and pRDEnh8G driven by inducible and enhanced constitutive promoters of GUS fusion genes (Enh6::GUS and Enh8::GUS) is shown in Figure 4, and the specific methods are as follows:

[0071] After pBSEnh6G and pBSEnh8G were digested with restriction endonucleases Hind III and Bgl II respectively, freeze-thaw method ("Molecular Cloning" third edition) or DNA fragment recovery kit (Easy-NA GelExtraction Kit, Germany Omeg-Bio / TEK product) recover and purify the enhanced constitutive promoter Enh6 and Enh8 fragments (about 1200bp), respectively, and the large fragment (about 13.6 kb) to obtain a recombinant plasmid. The obtained recombinant plasmid was transformed into Escherichia coli strain DH5α by "freezing-thawing method" (Molecular Clonin...

Embodiment 3

[0072] In this step, a chemically inducible promoter PR-N driving the GUS gene (PR-N::GUS) plant expression vector pRDPR-NG without inserting the enhancing element was constructed as a control. Example 3 Transformation of Enh6::GUS, Enh8::GUS fusion gene tobacco acquisition and identification

[0073] 1. Transformation of Agrobacterium and identification of transformants

[0074] with CaCl 2 Competent cells of Agrobacterium tumefaciens strain LBA4404 (product of Life Technology, USA) were prepared by the method (Molecular Cloning, third edition). The plant expression vectors pRDEnh6G and pRDEnh8G containing Enh6::GUS and Enh8::GUS fusion genes were transferred into the prepared LBA4404 competent cells by freeze-thaw method (Molecular Cloning, third edition). Inoculate the transformed LBA4404 cells into YEB solid medium containing streptomycin (Str) 100mg / L and kanamycin (Kan) 50mg / L, culture in the dark at 28°C for 48-72h, pick the plate A single colony was inserted into YE...

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Abstract

The invention discloses a plant-inducible and enhanced super-strong constitutive promoter and its application. The promoter has the following nucleotide sequence: the nucleotide sequence described in sequence 4 or 6 in the sequence listing, and a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent hybridization conditions. The promoter of the present invention can drive the constant expression of genes in plant cells, and at the same time has the ability to respond to chemical inducers. Under the induction of inducers, it can drive the superexpression of genes. Under the condition of no inducer, its driving ability is about It is twice that of the CaMV 35s promoter; after induction by chemical inducers, its driving ability is about 5 times that of the CaMV 35s promoter.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a plant-inducible enhanced constitutive promoter and its application. Background technique [0002] Promoters are generally located in the 5' flanking region of functional genes, and can combine with trans elements such as RNA polymerase (RNApolymerase) and other protein cofactors to control the initiation and speed of gene transcription. Promoter is the most important factor among gene expression regulators, which basically determines whether, when and where a gene is expressed. According to the mode of action and function, promoters can be roughly divided into three categories: specific promoters, inducible promoters and constitutive promoters [Wang Guanlin, Fang Hongyun, 2002, Principles and Technology of Plant Genetic Engineering (Second Edition), Beijing, Science and Technology Press]. [0003] Genes under the regulation of organ-and / or tissue-speci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82
Inventor 肖兴国韩晓红朱建伟刘建兵王伟
Owner CHINA AGRI UNIV
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