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Seed-specific promoter and application thereof

A specific, grain-based technology, applied in applications, angiosperms/flowering plants, introduction of foreign genetic material using vectors, etc., can solve the problems of gene silencing, difference in expression activity, and inability of expression activity to meet genetic engineering, etc. Effects of highly tissue-specific expression

Inactive Publication Date: 2012-07-04
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are differences in the expression activities of these promoters to promote the expression of exogenous genes, and their expression activities cannot meet the needs of genetic engineering. Moreover, when multiple genes are required to be connected in series, if the same promoter is used to drive the expression of different genes, it will easily lead to gene silencing.

Method used

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  • Seed-specific promoter and application thereof
  • Seed-specific promoter and application thereof
  • Seed-specific promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0031] Experimental example 1. Discovery and sequence analysis of a specific promoter

[0032] 1. Discovery of the promoter sequence

[0033] 1. DNA extraction kit (Tiangen) was used to extract genomic DNA from seedlings of oat variety Jizhangyan No. 1 germinated in the dark.

[0034] 2. Design a pair of primers according to the conserved region of the existing promoter (GENBANKACCESSION NO.AY795082, see sequence 2 in the sequence listing) as follows:

[0035]F1 (upstream primer): 5'-cg aagctt tggaaagtcattttgcctc-3';

[0036] R1 (downstream primer): 5'-gc ccatgg gagattgtagaaggtggattgg-3'.

[0037] 3. Using the genomic DNA in step 1 as a template, perform PCR amplification with the primer pair designed in step 2 to obtain a PCR amplification product.

[0038] PCR system (20μl): containing 50ng genomic DNA, 10pmol upstream primers, 10pmol downstream primers, 250μM each of dNTPs, 2μl 10×buffer (50mM KCl, 10mM Tris-HCl, 1.5mM MgCl 2 , pH 8.3) and 2.0U pfu polymerase (Dalia...

experiment example 2

[0044] Experimental example 2, construction of recombinant plasmid

[0045] 1. Construction of recombinant plasmid A

[0046] (1) Using the recombinant plasmid pTglo as a template, PCR amplification is performed with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0047] (2) Restriction endonucleases HindIII and NcoI double digest the PCR amplified product of step (1), and reclaim the digested product.

[0048] (3) Plasmid pCAMBIA3301 was double digested with restriction endonucleases HindIII and NcoI, and the vector backbone (about 11076bp) was recovered.

[0049] (4) Ligate the digestion product of step (2) with the carrier backbone of step (3) to obtain a ligation product.

[0050] (5) The ligation product of step (4) is subjected to PCR identification (using a primer pair composed of F1 and R1), enzyme digestion identification (using HindIII and NcoI double enzyme digestion) and sequencing identification in sequence to obtain recombinant plas...

Embodiment 3

[0062] Embodiment 3, the acquisition of transgenic plants

[0063] Prepare transgenic plants with recombinant plasmid A, recombinant plasmid B and recombinant plasmid C respectively (each plasmid transforms 2000 scutellaria):

[0064] 1. Separation and pretreatment of wheat scutellum

[0065] Collect immature wheat K35 seeds (scutellum size 1mm) pollinated 14 days, process 30 seconds with 75% (volume ratio) ethanol aqueous solution, 0.1% (g / 100mL) mercury chloride (HgCl 2 ) aqueous solution for 10 minutes, the immature embryos were removed with a dissecting needle in a sterile operating table, and the embryos were inoculated in dedifferentiation medium with the hypocotyl facing down. On the next day, the hypocotyl was excised from the contact point between the hypocotyl and the scutellum of the seed with a No. 15 blade under a microscope to obtain the scutellum. Put the scutellums from which the hypocotyls were removed on the dedifferentiation medium respectively, keep the s...

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Abstract

The invention discloses a seed-specific promoter and application thereof. The promoter is a DNA molecule shown as a sequence 1 in a sequence table. The invention also provides a method for specifically expressing genes in plant seeds. The method comprises the following steps of: transferring a recombinant expression vector containing the DNA molecule into a plant, and promoting downstream genes to be specifically expressed in the plant seeds by using the DNA molecule. The promoter has high-efficiency promoting activity and high tissue-specific expression, and provides a good base material forimproving traits of wheat seeds by using a transgenic technology.

Description

technical field [0001] The invention relates to a grain-specific promoter and its application. Background technique [0002] A promoter is a DNA sequence that RNA polymerase can recognize and bind to, thereby initiating gene transcription, usually located upstream of the gene. A typical promoter includes CAAT-box and TATA-box, which are the recognition and binding sites of RNA polymerase, respectively, and are generally located dozens of bases upstream of the transcription start site. There are usually some special DNA sequences upstream of the core promoter, namely cis-acting elements, to which transcription factors bind to activate or repress gene transcription. Once the RNA polymerase is located and bound to the promoter, gene transcription can be initiated, so the promoter is an important element in the regulation of gene expression, and its interaction with trans-acting factors such as RNA polymerase and other protein cofactors is the promoter regulation The essence o...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 夏先春李根英何中虎李玉莲高洁宋国琦宋华东黄承彦
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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