Peanut AhFAD2-2A gene promoter and application of peanut AhFAD2-2A gene promoter

A fatty acid dehydrogenase and promoter technology, applied in the preparation of AhFAD2-2A gene promoter, peanut Δ12 fatty acid dehydrogenase AhFAD2-2A gene promoter field, can solve the problem of increasing plant metabolic burden, material and energy waste, plant Morphological changes and other issues can be achieved to improve root development, avoid waste, and improve nutritional quality

Active Publication Date: 2015-11-25
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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  • Peanut AhFAD2-2A gene promoter and application of peanut AhFAD2-2A gene promoter
  • Peanut AhFAD2-2A gene promoter and application of peanut AhFAD2-2A gene promoter
  • Peanut AhFAD2-2A gene promoter and application of peanut AhFAD2-2A gene promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: An isolated peanut Δ 12 fatty acid dehydrogenase AhFAD2-2A gene promoter P AhFAD2-2A and its preparation method

[0050] 1. Peanuts AhFAD2-2A Determination of gene transcription start site:

[0051] According to the RACE kit (SMART TM RACEcDNA Amplification Kit, purchased from Clontech, product number: 634941) operating instructions, the primers used are shown in Table 1, and the first-strand (first strand) cDNA was synthesized using peanut leaf RNA as a template, and the steps were as follows:

[0052] Take 1.0-2.75μL RNA, 1.0μL 5ˊCDSPrimerA, add ddH 2 Make up to 3.75 μL with O; incubate at 72°C for 3 minutes, cool at 42°C for 2 minutes, add 1 μL of SMARTerⅡAoligo, add 5.25 μL of mixed solution (containing 5×first-strand (first strand) buffer 2.0 μL, 1.0 μL DTT, 1.0 μL dNTPsmixture, 0.25 μL RNase Inhibitor and 1.0 μL SMARTScribeReverseTranscriptase), incubate at 42°C for 90 minutes, and stop the reaction at 72°C for 10 minutes to obtain the storage ...

Embodiment 2

[0071] Example 2: Peanuts P AhFAD2-2A Construction of plant expression vector and transformation of Agrobacterium tumefaciens strain EHA105 (purchased from Shanghai Hushang Biotechnology Co., Ltd.)

[0072] 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 sequence was cloned using P AhFAD2-2A Fragment replacement obtained.

[0073] To accomplish this, first use the Sal I / Xba Ⅰ Double digestion cloning vector pMD18- P AhFAD2-2A , 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.

[0074] The cloning vector pMD18- P AhFAD2-2A The about 3Kb fragment and pBI- P ...

Embodiment 3

[0078] Example 3: P AhFAD2-2A Plant expression vector pBI- P AhFAD2-2A Genetic Transformation in Arabidopsis and Screening of Transgenic Plants

[0079] 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 AhFAD2-2A Agrobacterium tumefaciens EHA105 strain, the day before transforming Arabidopsis, pBI- P AhFAD2-2AEHA105 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 below...

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Abstract

The invention discloses an AhFAD2-2A gene promoter (P< AhFAD2-2A >) and an application of the AhFAD2-2A gene promoter, and belongs to the technical field of biology. A nucleotide sequence of the promoter is shown as SEQ ID NO.1. The AhFAD2-2A gene promoter is cloned from peanuts, and is used for building a recombinant expression vector; then, the recombinant expression vector is transformed into arabidopsis thaliana by an agrobacterium-mediated transformation method; a downstream recombinant gene is started to be expressed in seeds, roots and anthers, and is not expressed in other tissues; and the promoter belongs to a tissue specificity promoter, so that the promoter can be applied to transgenic engineering; a target gene expression product is accumulated in a certain organ or tissue; the expression amount in the tissue is increased; a good effect is achieved; meanwhile, the self energy waste of plants is avoided; and important application values are realized in the genetic engineering breeding and transgenic research process.

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 AhFAD2-2A gene promoters, and also involves AhFAD2-2A 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 perspecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82
Inventor 张新友石磊苗利娟黄冰艳齐飞艳秦利刘华董文召汤丰收高伟藏秀旺张忠信
Owner HENAN ACAD OF AGRI SCI
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