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Promoter of peanut δ12 fatty acid dehydrogenase ahfad2-1b-m gene and its preparation method and application

A fatty acid dehydrogenase and promoter technology, applied in the field of preparation of peanut Δ12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter, AhFAD2-1B-m gene promoter, can solve the problem of increasing plant metabolic burden, material and Energy waste, changes in plant morphology, etc., to achieve the effect of improving seed quality, avoiding waste, and improving fatty acid components

Active Publication Date: 2018-05-25
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, constitutive promoters are widely used in plant genetic engineering. They drive the expression of target genes in various tissues and throughout the developmental stages of plants, which causes waste of materials and energy, increases the metabolic burden of plants, and sometimes affects the growth and development of plants. development, and even cause changes in plant morphology

Method used

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  • Promoter of peanut δ12 fatty acid dehydrogenase ahfad2-1b-m gene and its preparation method and application
  • Promoter of peanut δ12 fatty acid dehydrogenase ahfad2-1b-m gene and its preparation method and application
  • Promoter of peanut δ12 fatty acid dehydrogenase ahfad2-1b-m gene and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: A kind of isolated peanut Δ 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter P AhFAD2-1B-m , whose sequence is the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing;

[0052] a kind of peanut P AhFAD2-1B-m A method for preparing a promoter, comprising the following steps:

[0053] (1) First use the SDS lysis method to extract genomic DNA

[0054] The peanut used in the present invention ( Arachis hypogeae L.) The test materials were sown in the field, and the field was managed normally. Genomic DNA was extracted by SDS lysis method (J. Sambrook. D.W. Russell, translated by Huang Peitang et al., Molecular Cloning Experiment Guide (Third Edition) Science Press).

[0055] The primers used are P AhFAD2-1B-m S:

[0056] 5'- ACGCGTCGACACCAAGTAGCTTCTCAATGGCTCAGATTCG-3'

[0057] P AhFAD2-1B-m A:

[0058] 5'-GACATCTAGATGTTGTGTTGTTAAAGTCCTGTTACCAATG-3';

[0059] (2) Use genomic DNA as a template for PCR amplification.

[0060...

Embodiment 2

[0065] Example 2: P AhFAD2-1B-m Construction of plant expression vector and transformation of Agrobacterium tumefaciens strain EHA105 (purchased from Shanghai Hushang Biotechnology Co., Ltd.)

[0066] To construct the recombinant vector, the plasmid pBI- P AhSAD -GUS (plasmid pBI- P AhSAD -GUS, preserved by the Economic Crops Research Institute of Henan Academy of Agricultural Sciences, the same below) P AhSAD The promoter was cloned P AhFAD2-1B-m Fragment, obtained by substitution.

[0067] To accomplish this, first use the Sal I / Xba Ⅰ Double digestion cloning vector pMD18- P AhFAD2-1B-m , while using Sal I / Xba Ⅰ Double digestion of pBI- P AhSAD -GUS plasmid; the enzyme digestion reaction was carried out in a 37°C incubator, and after about 4 to 6 hours of reaction, it was detected by electrophoresis on a 1% (mass volume ratio, the same below) agarose gel.

[0068] The cloning vector pMD18- P AhFAD2-1B-m The about 3 Kb fragment and pBI- P AhSAD -A ...

Embodiment 3

[0072] Example 3: P AhFAD2-1B-m Plant expression vector pBI- P AhFAD2-1B-m Genetic Transformation in Arabidopsis and Screening of Transgenic Plants

[0073] Arabidopsis transformation was carried out according to the method in the literature (Zhang X.R, et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature, 2006, 1: 1-6). Preparation of plant expression vector pBI- P AhFAD2-1B-m Agrobacterium tumefaciens EHA105 strain, the day before transforming Arabidopsis, pBI- P AhFAD2-1B-m EHA105 was transferred to 200 mL of LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin, and cultured overnight at 28°C and 220 r / min. The next day, use a UV spectrophotometer (SPEKOL 1300) to detect the absorbance of the bacterial solution at a wavelength of 276 nm, and take it out when the absorbance of the bacterial solution is between 1.6 and 2.0. Centrifuge at room temperature (20-25°C, the same below) at 4000 g for 10 mi...

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Abstract

The invention discloses a Δ12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter, a preparation method and an application thereof. The nucleotide sequence of the promoter is shown in SEQ ID NO.1. The present invention clones the peanut Δ12 fatty acid dehydrogenase AhFAD2‑1B‑m gene promoter from peanuts, uses the promoter provided by the present invention to construct a recombinant expression vector, and then transforms the expression vector into Arabidopsis by the method mediated by Agrobacterium Mustard, it can promote the expression of downstream recombinant genes in seeds, cotyledons, and hypocotyls, but not in other tissues. Accumulate in organs or tissues, increase the expression level in tissues, exert better effects, and avoid the waste of energy of the plant itself, so it has important application value in genetic engineering breeding and transgenic research.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology, in particular to a peanut Δ 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoters, and also involves AhFAD2-1B-m Preparation method and application of gene promoter. Background technique [0002] Peanut is one of the most important economic crops in the world, and occupies a pivotal position in the world's edible oil consumption and recreational food. The oil content of seeds is generally 46%-57%, of which oleic acid and linoleic acid are the most important components, and the sum of the two contents generally accounts for more than 80%. Both are unsaturated fatty acids, containing 1 and 1 respectively 2 unsaturated bonds. The content of oleic acid or the ratio of oleic acid / linoleic acid (O / L) is an important biochemical index to measure the storability and quality of peanuts. The larger the value, the better the storability and quality of peanuts. From the persp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/84C12N15/66A01H5/00A01H6/20
Inventor 张新友石磊齐飞艳苗利娟黄冰艳藏秀旺秦利董文召汤丰收张忠信高伟
Owner HENAN ACAD OF AGRI SCI
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