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Promoter BgIosP513 and preparation method and application thereof

A technology for promoters and monocotyledonous plants, which is applied in the field of promoters and can solve problems such as limited regulatory effects

Active Publication Date: 2010-10-20
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Adh-1 promoter is mainly used in monocotyledonous plants, and has limited effect on the regulation of gene expression in most dicotyledonous plants

Method used

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  • Promoter BgIosP513 and preparation method and application thereof
  • Promoter BgIosP513 and preparation method and application thereof
  • Promoter BgIosP513 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: PCR amplification of the P513 promoter fragment and pMD18-T+P513 recombination body construction

[0061] PCR amplification

[0062] Using a plant genome DNA extraction kit (TIANGEN new plant genome DNA extraction kit, catalog number: DP320-02) to extract rice Japanese eyeballs (preserved on December 18, 2009 at the Wuhan University Preservation Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, That is, the Genomic DNA of the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC-P200910), according to the sequence of the promoter in the rice Nippon gDNA, a pair of PCR-specific amplification primers (upstream primers) were designed at the beginning and end respectively. F1, add restriction enzyme site EcoR I and protection base, downstream primer R1, add restriction enzyme site Pst I and protection base). Using the above-mentioned extracted gDNA of rice japonicus as a template, high-fidelity Ex Taq (TaKaRa, DRR10...

Embodiment 2

[0081] Embodiment 2: Construction of carrier-p8+P513 recombinant vector

[0082] According to the operation manual of TIANGEN ordinary plasmid small extraction kit (catalogue number: DP103-03), extract the cloning vector with the P513 promoter sequence of the present invention from the Escherichia coli DH5α-P513 transformed with the promoter P513 constructed in Example 1 pMD18-T+P513; After purification, digest with the corresponding restriction enzymes EcoR I (NEB, B0101S) and Pst I (NEB, R0140T), recover the corresponding promoter insert fragment, and use the same method as the p8 plasmid The large fragments of the vector recovered after digestion with the restriction endonuclease were ligated.

[0083] Transform the resulting ligation product p8+P513 recombinant vector into the competent cell DH5α prepared according to the calcium chloride method shown in the "Molecular Cloning Experiment Guide" (third edition, Science Press), and culture it upside down at 37°C for 16-24 ...

Embodiment 3

[0108] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P513 cells

[0109] The p8+P513 recombinant vector constructed as described in Example 2 and the p8 plasmid as a control were respectively transformed into the root cancer prepared by the calcium chloride method described in the "Molecular Cloning Experiment Guide" (third edition, Science Press). Competent cells of Agrobacterium EHA105 (preserved in China Center for Type Culture Collection (CCTCC) on December 24, 2009, with a preservation number of CCTCC M 209315), the specific method is as follows:

[0110] The Agrobacterium tumefaciens competent cells EHA105 were taken out from the ultra-low temperature refrigerator and thawed on ice. After thawing, add 5 μl of p8+P513 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minutes, freeze in liquid nitrogen for 5 minutes, thaw at 37°C for 5 minutes, add 800 μl of normal temperature LB Liquid culture medium...

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Abstract

The invention relates to a promoter, in particular to a promoter of a monocotyledon such as paddy rice, and a preparation method and application of the promoter. The promoter has a nucleotide sequence shown by SEQ ID NO:1, or has a variant which has a function of the promoter and is selected from the following sequences: 1) a nucleotide sequence in hybridization with the nucleotide sequence shown by the SEQ ID NO:1 under a high-grade stringent condition; 2) a nucleotide sequence for performing substitution, deletion and adding modification on one or more basic groups of the nucleotide sequence shown by the SEQ ID NO:1; and 3) a nucleotide sequence having at least 90 percent of sequence identity with the nucleotide sequence shown by the SEQ ID NO:1. The invention also relates to the preparation method of the promoter and the application of the promoter in adjusting the expression of a target gene in the monocotyledon and adjusting rice breeding.

Description

technical field [0001] The present invention relates to a promoter, especially a promoter of a monocotyledonous plant such as rice, as well as a preparation method and application of the promoter. Background technique [0002] The promoter is a part of the gene, usually located upstream of the 5' end of the structural gene, and is a DNA sequence that RNA polymerase recognizes, binds and initiates transcription. The promoter can guide the holoenzyme to correctly combine with the template, activate RNA polymerase, and initiate gene transcription, thereby controlling the initiation time and degree of gene expression (transcription). In transgenic plants, the promoter is one of the important factors affecting the expression efficiency of the transgene, and the selection of a high-efficiency promoter is the key to high-efficiency expression of foreign genes. [0003] Promoters can be divided into three categories according to their transcriptional patterns: constitutive promoter...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N1/21C12N5/10C12P19/34C12N15/82C12N15/84C12R1/01
Inventor 黄三文李宁束礼平李华
Owner HUADA GENE RES & DEV CENT HANGZHOU
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