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Promoter BgIosP587, 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: 2010-09-22
深圳华大基因农业控股有限公司
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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 BgIosP587, and preparation method and application thereof
  • Promoter BgIosP587, and preparation method and application thereof
  • Promoter BgIosP587, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1P587

[0059] The PCR amplification of embodiment 1P587 promoter fragment and the construction of pMD18-T+P587 recombinant vector

[0060] PCR amplification

[0061] Using a plant genomic DNA extraction kit (TIANGEN new plant genomic DNA extraction kit, catalog number: DP320-02) to extract rice Nipponbare (preserved in Wuhan University Preservation Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province on December 18, 2009, namely China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC-P200910) Genomic DNA, according to the sequence of the promoter in the rice Nipponbare gDNA, a pair of PCR-specific amplification primers (upstream primer F1, upstream primer F1, Add restriction enzyme site EcoR I and protection base, downstream primer R1, add restriction enzyme site Sbf I and protection base). Using the rice Nipponbare gDNA extracted above as a template, high-fidelity Ex Taq TM (TaKaRa, DRR100B) polymerase for PCR amplification. As shown in Table 1....

Embodiment 2p8

[0082] Construction of embodiment 2p8+P587 recombinant vector

[0083] According to the operating manual of the TIANGEN ordinary plasmid small extraction kit (catalogue number: DP103-03), extract the cloning vector with the P587 promoter sequence of the present invention from the Escherichia coli DH5α-P587 transformed with the promoter P587 constructed in Example 1 pMD18-T+P587; after purification, digest with the corresponding restriction enzymes EcoR I (NEB) and Sbf I (NEB), recover the corresponding promoter insert, and use the same restriction endonucleases as the p8 plasmid The large vector fragments recovered after digestion with Dicer enzyme were ligated.

[0084] Transform the resulting ligation product p8+P587 recombinant vector into competent cells 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 hours. After the colonies grow out, sin...

Embodiment 3

[0110] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P587 cells

[0111] The p8+P587 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 on December 24, 2009 at the Wuhan University Collection Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, that is, the China Type Culture Collection Center (CCTCC), the preservation number is CCTCC M 209315), and the specific method as follows:

[0112] 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+P587 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minutes, freeze in liquid nitrogen f...

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Abstract

The invention relates to a promoter, in particular to a promoter for monocotyledons such as rice, and a preparation method and application thereof. The promoter of the invention has a nucleotide sequence shown in SEQ ID NO: 1, or has a variant which has the functions of the promoter and is selected from the following sequences: 1) a nucleotide sequence hybridized with the nucleotide sequence shown in SEQ ID NO: 1 under the condition of advanced tightness; 2) a nucleotide sequence carrying out substitution, loss, modification and addition of one or more basic groups on the nucleotide sequence shown in SEQ ID NO: 1; and 3) a nucleotide sequence having at least 90 percent of sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In addition, the invention also relates to a method for preparing the promoter and the application of the promoter in adjusting and controlling the target gene expression in the monocotyledons.

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): C12N15/113C12N15/63C12N1/21C12N5/10C12N15/10A01H5/00
Inventor 张耕耘李宁全志武彭小华
Owner 深圳华大基因农业控股有限公司
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