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Peanut delta 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter and its preparation method and use

A fatty acid dehydrogenase and promoter technology, applied in biochemical equipment and methods, DNA preparation, botany equipment and methods, etc., can solve the problems of increasing the metabolic burden of plants, waste of materials and energy, and changes in plant morphology, achieving Improve seed quality, avoid waste, improve the effect of fatty acid composition

Active Publication Date: 2016-04-20
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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  • Peanut delta 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter and its preparation method and use
  • Peanut delta 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter and its preparation method and use
  • Peanut delta 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter and its preparation method and use

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 peanuts (Arachishypogeae L.) used in the present invention were sown in the field and managed normally in the field. Genomic DNA was extracted by SDS lysis method (written by J. Sambrook D.W. Russell, translated by Huang Peitang et al., Molecular Cloning Experiment Guide (Third Edition) Science Press).

[0055] The primer used was 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] The reaction system is...

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 Institute of Economic Crops, Henan Academy of Agricultural Sciences, the same below) on P AhSAD The promoter was cloned to obtain P AhFAD2-1B-m Fragment, obtained by substitution.

[0067] To accomplish this purpose, first cut the cloning vector pMD18-P with SalI / XbaI AhFAD2-1B-m , while cutting pBI-P with SalⅠ / XbaⅠ 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 About 3Kb fragment and pBI-PAhSAD -A fragment of about 10 Kb cut by GUS enzyme was rec...

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] Transformation of Arabidopsis thaliana was carried out according to the method in the literature (Zhang X.R, et al. Agrobacterium-mediated transformation of Arabidopsisthaliana using the floraldip method. Nature, 2006, 1:1-6). Preparation of plant expression vector pBI-P AhFAD2-1B-m The Agrobacterium tumefaciens EHA105 strain, the day before transforming Arabidopsis, pBI-P AhFAD2-1B-m EHA105 was transferred into 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 an ultraviolet spectrophotometer (SPEKOL1300) to detect the absorbance value of the bacterial liquid at a wavelength of 276 nm, and take it out when the absorbance value of the bacterial liquid is between 1.6 and 2.0. Centrifuge at room temperature (20~25°C, the same b...

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Abstract

The invention discloses a peanut delta 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter and its preparation method and use. A nucleotide sequence of the promoter is shown in the formula of SEQ ID NO. 1. A peanut delta 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter is cloned, a recombinant expression vector is prepared from the promoter, based on the expression vector, arabidopsis thaliana is transformed through an agrobacterium tumefaciens mediated method, expression of a downstream recombinant gene in seeds, cotyledon and hypocotyls is started, and the gene promoter is not expressed in other tissue and is a tissue-specific promoter so that the gene promoter can be used for transgenic engineering, target gene expression products are accumulated in an organ or tissue, an expression level of the tissue is improved, good effects are produced and plant energy waste is avoided and thus the gene promoter 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 The fatty acid dehydrogenase AhFAD2-1B-m gene promoter also relates to the preparation method and application of the AhFAD2-1B-m 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/84C12N15/66A01H5/00
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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