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Peanut diacylglycerol acyltransferase AhDGAT3 promoter as well as preparation method and application thereof

A diacylglycerol acyl and promoter technology, applied in the field of plant genetic engineering, to achieve the effects of inhibiting diffusion, preventing gene drift, and enriching germplasm resources

Active Publication Date: 2015-10-14
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The increasing maturity of transgenic technology provides a new way to improve the yield and quality of peanuts, but it faces the safety risk of transgenic

Method used

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  • Peanut diacylglycerol acyltransferase AhDGAT3 promoter as well as preparation method and application thereof
  • Peanut diacylglycerol acyltransferase AhDGAT3 promoter as well as preparation method and application thereof
  • Peanut diacylglycerol acyltransferase AhDGAT3 promoter as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: a kind of peanut P AhDGAT3 A method for preparing a promoter, the method comprising the following steps:

[0054] 1. Peanuts P AhDGAT3 Preparation of the promoter:

[0055] Use the sodium dodecyl sulfate (SDS) lysis method to extract the DNA of peanuts (J. Sambrook. D.W. Russell, translated by Huang Peitang et al. Molecular Cloning Test Guide (Third Edition) Science Press), according to the genome walk Kit (Genome Walker TM Universal kit, produced by Clontech, product number: 638904) operating procedures, genome walking, walking for two rounds of PCR amplification, cloning to obtain peanuts P AhDGAT3 Promoter (2181 bp).

[0056] The primers used are listed in Table 1.

[0057] Table 1 Genome walking clone P AhDGAT3 Primers used

[0058]

[0059] peanut P AhDGAT3 The specific steps of preparation are as follows:

[0060] According to the genome walking kit (Genome Walker TM Universal kit, produced by CLONTECH Company, product number: 638...

Embodiment 2

[0075] Example 2: P AhDGAT3 Plant expression vector pBI- P AhDGAT3 The genetic transformation in Arabidopsis thaliana and the selection of transgenic plants were carried out by referring to the method in the literature (ZhangX.R, et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature, 2006, 1: 1 -6), the operation steps are as follows: after bolting of wild-type Arabidopsis thaliana, cut off the developed siliques with clean scissors one day before transformation, and amplify 200 mL of Agrobacterium to be transformed. The next day, centrifuge the prepared Agrobacterium at room temperature at 5000 rpm, collect the bacterial liquid, resuspend the Agrobacterium with 5% sucrose, and add Silwet L-77 at a final concentration of 0.02%. Then immerse the inflorescences in the Agrobacterium solution for 15 seconds, take them out and wrap them in a black plastic bag to keep them moist, remove the plastic bag the next day, put the trans...

Embodiment 3

[0082] Embodiment 3: peanut P AhDGAT3 Functional Analysis and Application of Promoter

[0083] The present invention is cloned for the first time P AhDGAT3 sequence and its functional analysis. From the T1 generation of positive seedlings screened in the steps of Arabidopsis thaliana transformation and PCR detection in Example 2, the seeds (ie T2 generation) were harvested by selfing. The tissues of 10 lines of T2 generation were taken at different stages for GUS staining.

[0084] PCR verification: Seeds of positive T1 generation transgenic plants were sown on MS (mentioned above) medium, placed at 4°C for 3-5 days, and then germinated in a plant growth chamber. Seedlings and roots, stems, leaves, flowers, siliques, and immature embryos at 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days after flowering were subjected to histochemical staining.

[0085] The staining process of the T2 generation is as follows: Soak the sample in the GUS staining solution for...

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Abstract

The invention discloses a peanut diacylglycerol acyltransferase AhDGAT3 promoter (P<AhDGAT3>) as well as a preparation method and application thereof. The nucleotide sequence of the promoter is shown in SEQ ID No.1. A (P<AhDGAT3>) sequence is cloned through a genome walking method, genome DNA is extracted through an SDS cracking process, endonuclease is used for enzyme digestion of DNA in different systems, after DNA is connected with a joint fragment, first PCR amplification is carried out by taking GSP1 and AP1 as primers, and secondary PCR amplification is carried out by taking GSP2 and AP2 as primers, so that a DNA sequence containing (P<AhDGAT3>) is obtained. The promoter has the function of driving exogenous gene expression, the expression part of the promoter is in an embryo of a young and tender seed, the promoter is a tissue specific expression promoter, can drive a target gene to express and accumulate in the embryo of the young and tender seed when being applied to transgenic engineering, and avoids unnecessary waste in a plant metabolic process, therefore, the promoter has a good application prospect in the plant transgenic engineering.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a peanut diacylglycerol acyltransferase gene AhDGAT3 Promoter (named P AhDGAT3 ), preparation method and application in plant genetic engineering. Background technique [0002] The advantage of breeding crop varieties by means of transgenics is that transgenics are not limited by the kinship of organisms. In theory, the genes used for transformation can come from any species or artificially synthesized genes. In 2014, the global planting area of ​​transgenic plants reached 180 million hectares, with an annual growth rate between 3% and 4%. At present, transgenic technology has become an important means of improving plant varieties. [0003] There are two essential elements in plant transgenesis, namely promoter and gene. Promoter is a segment of DNA in organisms. It is a cis-regulatory sequence located upstream of the gene transcription initiation site. It b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/84C12N1/21A01H5/10
Inventor 张新友石磊齐飞艳苗利娟张忠信黄冰艳董文召汤丰收高伟藏秀旺秦利
Owner HENAN ACAD OF AGRI SCI
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