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Peanut δ9-stearyl-acp dehydrogenase ahsad promoter and its preparation method and application

A promoter and stearyl technology, applied in the field of plant genetic engineering and biology, can solve the problem that the supply cannot meet the requirements of peanut oil, and achieve the effects of improving crop quality, photosynthesis, and disease resistance

Active Publication Date: 2018-02-16
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, my country's peanut production has made great progress and has become the world's largest peanut producer. However, the current supply of China's peanut oil market cannot meet the demand for peanut oil. Therefore, research on peanut breeding should be strengthened to increase peanut production to the country's edible oil supply. Safety is of great practical significance

Method used

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  • Peanut δ9-stearyl-acp dehydrogenase ahsad promoter and its preparation method and application
  • Peanut δ9-stearyl-acp dehydrogenase ahsad promoter and its preparation method and application
  • Peanut δ9-stearyl-acp dehydrogenase ahsad promoter and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: a kind of peanut P AhSAD The preparation method of promoter

[0048] The peanuts used in the present invention ( Arachis hypogeae L.), the test material was sown in the field, and the field was managed normally.

[0049] 1. Peanuts P AhSAD Preparation of promoter:

[0050] Peanut DNA was extracted by sodium dodecyl sulfate (SDS) lysis method (J. Sambrook. D.W. Russell, translated by Huang Peitang et al. Molecular Cloning Test Guide (Third Edition) Science Press), followed by genome walking Kit (GenomeWalker TM Universal kit, produced by Clontech, Cat. No. 638904) operating procedures, perform genome walking, perform two rounds of PCR amplification by walking, and clone to obtain peanuts P AhSAD Promoter (718bp). The primers used for walking are shown in Table 1.

[0051] Table 1 Genome walking clones P AhSAD Primers used

[0052]

[0053] peanut P AhSAD The specific steps for the preparation of the promoter are as follows:

[0054] Accor...

Embodiment 2

[0063] Example 2: Peanuts P AhSAD Construction of plant expression vector and transformation of Agrobacterium tumefaciens strain GV3101 (purchased from Shanghai Hushang Biotechnology Co., Ltd., the same below):

[0064] The constructed recombinant vector is obtained by cloning the 35S promoter on the plasmid pBI121 (purchased from TaKaRa company). P AhSAD Fragment replacement. To accomplish this, first use Kpn Ⅰ / Bam HI double digestion cloning vector pMD18- P AhSAD , while using Kpn Ⅰ / Bam The pBI121 plasmid was digested with HI.

[0065] The digestion reaction was carried out in a 37°C incubator, and after about 4-6 hours, it was detected by 1% (mass volume ratio) agarose gel electrophoresis. The cloning vector pMD18- P AhSAD The small fragment digested by the enzyme and the large fragment digested by the pBI121 (described above) were recovered using a DNA gel recovery kit.

[0066] Press pMD18- P AhSAD The ratio of the small fragments cut by the enzyme an...

Embodiment 3

[0069] Example 3: P AhSAD Plant expression vector pBI- P AhSAD Genetic Transformation and Screening of Transgenic Plants in Arabidopsis

[0070] Arabidopsis thaliana was transformed according 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 the wild-type Arabidopsis is bolted, cut off the developed siliques with clean scissors the day before transformation, and amplify 200 mL of Agrobacterium to be transformed. The next day, centrifuge the prepared Agrobacterium at 5000 rpm at room temperature, collect the bacterial solution, resuspend the Agrobacterium with 5% sucrose, and add Silwet L-77 with a final concentration of 0.02%; then immerse the inflorescence in the Agrobacterium solution for 15 After taking it out, wrap it in a black plastic bag to keep it moisturizing. After the second day, remove the plastic bag and pu...

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Abstract

The invention discloses a peanut Δ9-stearyl-ACP dehydrogenase gene AhSAD promoter (PAhSAD), a preparation method and an application. The promoter has the nucleotide sequence shown in SEQ ID No.1. The PAhSAD sequence is cloned by using the genome walking method, the genomic DNA is extracted by the SDS cleavage method, and the DNA is digested with blunt-end endonuclease in different systems respectively. After ligation with the adapter fragment, the first round of PCR amplification was carried out with GSP1 and AP1 as primers, and the second round of amplification was carried out with GSP2 and AP2 as primers to obtain the DNA sequence containing PAhSAD. The promoter PAhSAD of the present invention has the function of driving the expression of exogenous genes, and its expression sites are in the roots, stems, leaves, fruit needles and seeds of plants. This promoter with tissue-specific expression has a role in plant genetic engineering. important application value.

Description

technical field [0001] The invention relates to the field of plant genetic engineering and biotechnology, in particular to a peanut AhSAD promoter (named P AhSAD ) and preparation methods and applications. Background technique [0002] Plant promoters play a key role in the regulation of gene expression. The regulation of gene expression is the result of the combined action of many factors. Generally, according to the sequence of events, gene regulation is divided into transcriptional level regulation, translational level regulation and protein processing level regulation. The proteins and RNAs encoded by genes and their secondary metabolites are extremely important for maintaining the whole life activities of organisms. Any error in the regulation of gene expression can have serious consequences for life. Therefore, the mechanism study of gene expression regulation has always been a hot spot in molecular biology research. The regulation at the transcriptional level i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/84A01H5/00A01H6/20
Inventor 张新友石磊苗利娟黄冰艳齐飞艳张忠信汤丰收高伟藏秀旺刘华董文召
Owner HENAN ACAD OF AGRI SCI
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