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Separation clone and expression mode identification of promotor region of rice endosperm special expression gene

A promoter and rice technology, applied in the field of plant genetic engineering, can solve the problems of enhancing the expression strength of the promoter, and unable to determine the characteristics of the specific expression of the promoter's endosperm, etc.

Active Publication Date: 2010-09-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is also reported in the literature that the GCN4 element only plays a role in enhancing the expression intensity of the promoter in the endosperm, and it does not determine the specific expression characteristics of the promoter in the endosperm (Vickers et al., 2006)

Method used

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  • Separation clone and expression mode identification of promotor region of rice endosperm special expression gene
  • Separation clone and expression mode identification of promotor region of rice endosperm special expression gene
  • Separation clone and expression mode identification of promotor region of rice endosperm special expression gene

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: endosperm specific expression candidate gene ( gb: AK107215.1 ) expression profile validation

[0033] Material preparation: The material used in the present invention is an indica rice variety (Oryza sativa ssp. indica) Minghui 63 (an excellent rice restorer line widely used in my country), and the planting method is hydroponics. The composition of the nutrient solution is as follows: 1.44mM NH 4 NO 3 , 0.3mM NaH 2 PO 4 , 0.5mM K 2 SO 4 , 1.0mM CaCl 2 , 1.6mM MgSO 4 , 0.17mM NaSiO 3 , 50μm Fe-EDTA, 0.06μM (NH 4 ) 6 Mo 7 o 24 , 15 μM H 3 BO 3 , 8 μM nCl 2 , 0.12 μM CuSO 4 , 0.12 μM ZnSO 4 , 29 μM FeCl 3 , 40.5 μM Citric acid, pH 5.5 (Yoshida et al., 1976). Take leaves, leaf sheaths, stems, roots and ears at the booting stage, and take endosperm 14 days after fertilization. RNA was extracted using the Trizol Reagent (Invitrogen, Carisbad, CA, USA) method. The total RNA was detected by 1.4% agarose gel electrophoresis and concentration ...

Embodiment 2

[0035] Example 2: Acquisition of endosperm-specific expression promoter EnP2 candidate fragments and corresponding deletion fragments (refer to "Molecular Cloning Experiment Guide (Second Edition)" (J. Sambrook et al., 1996) for routine operations in molecular biology

[0036] Extraction of Minghui 63 Genomic DNA: Take fresh leaves of Minghui 63 at the peak tillering stage to extract its genomic DNA. The specific method is the CTAB method reported by Murray (Murray and Thompson.1980). After the extracted DNA is completely dissolved Store in -20°C refrigerator.

[0037] Candidate genes were extracted from the bioinformatics website NCBI (http: / / www.ncbi.nlm.nih.gov / ) gb: AK107215.1 The upstream sequence of , specifically the interval from -1085 to +91 (transcription initiation point is +1) totals 1176 bp as a candidate promoter fragment. Name it EnP2. Using the PCR method, using the extracted Minghui 63 genomic DNA as a template, by designing specific primers (EnP2F: AAG...

Embodiment 3

[0041] Example 3: Construction of transformation vectors for endosperm-specific expression of promoter EnP2 candidate fragments and corresponding deletion fragments (refer to "Molecular Cloning Experiment Guide (Second Edition)" (J. Sambrook et al., 1996) for routine operations in molecular biology)

[0042] (1) Digest vector DX2181. The DX2181 vector was transformed on the basis of pCAMBIA1380 (the vector used by the Australian CAMBIA (Center for the Application of Molecular Biology to International Agriculture, CAMBIA) laboratory in the world for public communication), and at the multiple cloning site with the opposite Direction Constructed a GUS gene and EGFP gene respectively, the vector map and multiple cloning sites and other information can be found in image 3 . Digest DX2181 with HindIII and BamHI, recover the digested product with UNIQ-10 Column DNA Gel Recovery Kit (produced by Shanghai Sangon Bioengineering Technology Service Co., Ltd.), detect the integrity of th...

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Abstract

The invention belongs to the field of plant genetic engineering. Seven promotors with different lengths (EnP2, EnP2-967, EnP2-771, EnP2-591, EnP2-281, EnP2-186 and EnP2-155) at the upstream of the same endosperm special expression gene (GeneBank has an accession number gb: AK107215.1) are cloned in a rice gene bank, and nucleotide sequences thereof are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6 and SEQ ID NO: 7. The promotors are expressed in endosperm of a transferred plant, and the expression is reduced with the growth of the endosperm; and the promotors are not expressed in other tissues, such as leaves, leaf sheaths, stems and straws, roots, flowers, glumes and endosperms. The invention also discloses a preparation method of the seven promotors and corresponding expression vectors, and application thereof guided into rice by using a transgenosis method mediated by bacillus.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the isolation and cloning of different length promoter regions of the same endosperm-specific expression gene in rice and the identification of expression patterns. Background technique [0002] The growth and development of higher organisms is a process of orderly expression and synergistic effect of different genes in time and space. In this process, the opening, closing, expression site, and expression level of gene expression will be finely regulated. The regulation of gene expression is a multi-level complex process, which is controlled by different regulatory factors and is also realized at multi-stage levels, namely pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational levels. Although gene expression is a multi-level regulatory system in higher organisms, regulation at the transcriptional level is ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A01H5/00
Inventor 林拥军叶荣建陈浩
Owner HUAZHONG AGRI UNIV
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