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Rice root tip specific expression promoter Pro-Os04g24469 and application thereof

A pro-os04g24469, promoter technology, applied in the fields of molecular biology and genetic engineering

Inactive Publication Date: 2014-04-02
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no related reports on the rice Os04g24469 promoter regulating the root-specific expression and application of exogenous genes

Method used

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  • Rice root tip specific expression promoter Pro-Os04g24469 and application thereof
  • Rice root tip specific expression promoter Pro-Os04g24469 and application thereof
  • Rice root tip specific expression promoter Pro-Os04g24469 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Expression Analysis of Os04g24469 Gene in Different Rice Tissues

[0018] 1.1 Rice total RNA sample preparation

[0019] The roots and leaves of rice seedlings grown for 15 days were taken respectively, and stored in liquid nitrogen for quick-freezing; the young ears of rice with a length of about 2 cm when heading began to be taken, and quickly stored in liquid nitrogen. After the rice tissue samples were thoroughly ground with liquid nitrogen, the total RNA of the samples was extracted using a plant RNA extraction kit (QIAGEN). The concentration and quality of RNA samples were measured with a NanoDrop ND-1000 nucleic acid detector.

[0020] 1.2 Synthesis of first-strand cDNA

[0021] Take 2.5 μg of each of the above RNA samples, according to the method provided by Invitrogen The first strand synthesis system first added 1 μl 10mM dNTP and 1 μl Oligo(dT)20, added water to make up the volume to 10 μl, incubated at 65°C for 5 minutes, and immediately place...

Embodiment 2

[0025] Example 2: Cloning of rice root tip-specific expression promoter Pro-Os04g24469

[0026] 2.1 Biomaterials

[0027] 2.1.1 Plant material

[0028] The rice transgenic receptor material is japonica Nipponbare

[0029] 2.1.2 Strains and vectors

[0030] The Agrobacterium strain used was EHA105, and the vector was pCAMBIA1300.

[0031] 2.2 Method

[0032] 2.2.1 Cloning of rice root tip-specific expression promoter Pro-Os04g24469

[0033]Rice Nipponbare leaves were used as materials, and the total DNA of rice was extracted with a plant genome extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.). According to the genome sequence of rice Os04g24469 published on the rice database website (http: / / rice.plantbiology.msu.edu / index.shtml), the PCR amplification primers for the upstream promoter sequence were designed, and the 5' ends of the two primers were respectively Add restriction site sequences and protection bases. The primer sequences were: pro-F: 5'-TGCTCTAGATT...

Embodiment 3

[0044] Example 3: The rice root tip-specific expression promoter Pro-Os04g24469 regulates the expression of the reporter gene GUS

[0045] GUS histochemical staining and microscopic observation

[0046] The root and leaf materials of the transgenic rice grown for 7 days were taken, soaked in the staining solution containing the GUS display substrate x-gluc, kept at 37°C for 2-4 hours in the dark, and then photographed. The result is as image 3 As shown, specific staining occurs only at the root tip of the main root and the root tip of the lateral root of rice, while other parts of the root and leaves are not stained, indicating that the Pro-Os04g24469 promoter can regulate the expression of the target gene only in the root tip of rice, and the specificity is very high. powerful. The formula of GUS staining solution is: 100mmol / L phosphate buffer (pH7.0), 5mmol / L EDTA, 0.5mmol / L potassium ferricyanide, 0.5mmol / L potassium ferrocyanide, 0.5%Triton X-100, 0.2 mg / L X-gluc in w...

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Abstract

The invention relates to a rice root tip specific expression promoter Pro-Os04g24469 and application thereof. The promoter has a nucleotide sequence shown as SEQ ID NO:1 or a nucleotide sequence which is derived by replacing or deleting one or more nucleotide from the nucleotide sequence and / or adding one or more nucleotide into the nucleotide sequence and has the same function as the nucleotide sequence shown as SEQ ID NO:1. The invention also provides a carrier, a transgenic cell line and engineering bacteria which contain the promoter and provides a transgenic line with the promoter. The invention provides the application of the rice root tip specific expression promoter Pro-Os04g24469 in regulating and controlling downstream genetic expression. The rice root tip specific expression promoter Pro-Os04g24469 has the beneficial effects that the new rice root tip specific expression promoter is provided for the first time, a foundation is laid for further analysis on cis acting element of root specific expression in following steps, and a new promoter element can also be provided for plant root system development and transformation and reconstruction of root configuration as well as plant stress resistance gene engineering and study on genetic improvement.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and mainly relates to a rice root tip-specific expression promoter Pro-Os04g24469 and an application thereof. Background technique [0002] A promoter is a DNA sequence involved in the transcription of a particular gene and its regulation. It determines whether, when and where a gene is expressed. According to the mode of action and function, promoters can be divided into three categories: constitutive promoters, specific promoters and inducible promoters. In plant genetic engineering, promoters with different expression regulation levels are used to promote the expression of target genes. For example, the Ubiqutin promoter from corn and the Actin promoter from rice can regulate the strong expression of target genes in roots and leaves, and are widely used promoters in plant transgenics. Although constitutive promoters can drive the non-specific high-efficien...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N5/10C12N1/21A01H5/00
Inventor 王芳邓敏娟徐磊赵红玉易可可
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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