Promoter BgIosP529, and preparation method and use thereof

A technology of promoter and expression method, which can be applied in the fields of botanical equipment and methods, biochemical equipment and methods, horticultural methods, etc., and can solve the problems of limited regulation effect and the like

Active Publication Date: 2013-08-21
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 BgIosP529, and preparation method and use thereof
  • Promoter BgIosP529, and preparation method and use thereof
  • Promoter BgIosP529, and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: PCR amplification of P529 promoter fragment and construction of pMD18-T+P529 recombinant vector PCR amplification

[0081] Use the plant genomic DNA extraction kit (TIANGEN new plant genomic DNA extraction kit, catalog number: DP320-02) to extract rice Nipponbare (the application number is 200910238992.0, the invention name is "a kind of promoter BgIosP587, its preparation method and use) According to the sequence of the promoter in the rice Nipponbare gDNA, a pair of PCR-specific amplification was designed at the beginning and the end respectively. Primers (upstream primer F1, plus restriction site KpnI and protective bases, downstream primer R1, plus restriction site SbfI and protective bases). Using the gDNA of rice Nipponbare extracted above as a template, high-fidelity Ex Taq (TaKaRa, DRR100B) polymerase was used for PCR amplification. As shown in Table 1.

[0082] Table 1 PCR system for gene promoter amplification

[0083]

[0084]

[0085]...

Embodiment 2

[0100] Embodiment 2: Construction of carrier-p8+P529 recombinant vector

[0101] According to the operating manual of the TIANGEN ordinary plasmid small extraction kit (catalogue number: DP103-03), extract the cloning vector with the P529 promoter sequence of the present invention from the Escherichia coli DH5α-P529 transformed with the promoter P529 constructed in Example 1 pMD18-T+P529; After purification, digest with the corresponding restriction endonucleases KpnI and SbfI, recover the corresponding promoter insert fragment, and use the same restriction enzymes as the p8 plasmid to recover the vector Large fragments are concatenated.

[0102] The resulting ligation product p8+P529 recombinant vector was transformed into competent cells DH5α prepared according to the calcium chloride method shown in "Molecular Cloning Experiment Guide" (third edition, Science Press), and cultured upside down at 37°C for 16-24 hours. After the transformants grow into colonies, single clon...

Embodiment 3

[0128] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P529 cells

[0129] The p8+P529 recombinant vector constructed as described in Example 2 and the p8 plasmid as a control were respectively transformed into root cancer prepared by the calcium chloride method described in the "Molecular Cloning Experiment Guide" (third edition, Science Press). Agrobacterium EHA105 (preserved in the invention application with the application number 200910238992.0 and the invention title "A promoter BgIosP587, its preparation method and use", and published on September 22, 2010, with the preservation number CCTCC M 209315) Competent cells, the specific method is as follows:

[0130]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+P529 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minutes, freeze in liquid nit...

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Abstract

The invention relates to a promoter BgIosP529, and a preparation method and use thereof. The promoter has a nucleotide sequence which is selected from any of the following groups and has a prompter function: a, a nucleotide sequence represented by SEQ ID No.1; b, a nucleotide sequence complementary to the SEQ ID No.1; c, a nucleotide sequence which can be crossed with the nucleotide sequence a orthe nucleotide sequence b under a highly strict condition; d, a nucleotide sequence obtained by modifying the nucleotide sequence a or the nucleotide sequence b by substituting, losing and increasingone or more bases; and e, a nucleotide sequence having an identity of at least 90 percent with the nucleotide sequence a or the nucleotide sequence b. The invention also relates to the use of the promoter in the control over the expression of a target gene in monocotyledons or dicotyledons and the use of the promoter in seed breeding.

Description

technical field [0001] The present invention relates to the field of gene regulation. Specifically, the present invention relates to a promoter, especially the rice promoter BgIosP529, and a method for preparing the promoter. The present invention also relates to the use of the promoter in regulating the expression of target genes in monocotyledonous plants or dicotyledonous plants. 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21C12N5/10C12N15/11C12N15/10A01H4/00A01H5/00C12R1/19C12R1/01
Inventor 张耕耘全志武李宁倪雪梅
Owner 深圳华大基因农业控股有限公司
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