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A method for screening transgenic peanuts by constructing an expression vector containing Δ12-fatty acid dehydrogenase gene

A technology of fatty acid dehydrogenase and expression vector, applied in the field of genetic engineering, can solve problems such as environmental and human health adverse effects and damages, and achieve high conversion efficiency and simple operation

Active Publication Date: 2019-05-31
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the process of peanut genetic transformation, using antibiotic genes and herbicide resistance genes as selection marker genes for screening may have adverse effects and damages on the environment and human health

Method used

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  • A method for screening transgenic peanuts by constructing an expression vector containing Δ12-fatty acid dehydrogenase gene
  • A method for screening transgenic peanuts by constructing an expression vector containing Δ12-fatty acid dehydrogenase gene
  • A method for screening transgenic peanuts by constructing an expression vector containing Δ12-fatty acid dehydrogenase gene

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Construction of a plant expression vector containing Δ12-fatty acid dehydrogenase gene

[0038] (1) Design primers according to the AhFAD2B gene sequence (SEQ ID NO.1), and perform PCR amplification using the genomic DNA of Fenghua No. 1 peanut variety as a template, wherein the primer sequences are as follows:

[0039] Upstream primer FAD2B-F: 5'-ATGGGAGCTGGAGGGCGTGT-3' (SEQ ID NO.2),

[0040] Downstream primer FAD2B-R: 5'-TCAGAACTTGTTCTTGTACCAATAA-3' (SEQ ID NO.3);

[0041] The reaction system for PCR amplification is: I-5Mix 25 μl, upstream primer FAD2B-F and downstream primer FAD2B-R 1 μl each, DNA template 1 μl, H 2 O 22 μl; PCR reaction program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 20 s, annealing at 59°C for 20 s, extension at 72°C for 20 s, 30 cycles; extension at 72°C for 2 min.

[0042] (2) connecting the AhFAD2B gene amplified in step (1) to the pMD18-T vector to obtain the AhFAD2B-pMD18-T recombinant plasmid;

[0043] Amo...

Embodiment 2

[0053] Example 2 Construction of the plant expression vector containing the Δ12-fatty acid dehydrogenase gene of the target gene AhPIF3

[0054] (1) Chinese patent document CN107056905A discloses peanut phytochrome interaction factor AhPIF3 and its coding gene. In this example, the genomic DNA of peanut variety Luhua No. 14 was used as a template for PCR amplification to obtain the peanut phytochrome interaction factor coding gene AhPIF3 , the nucleotide sequence of the AhPIF3 gene is shown in SEQ ID NO.6, wherein the primer sequences are as follows:

[0055] AhPIF3-F: 5'-CCC GGTACC ATGCCTTTTTATGAGTTATACCG-3' (SEQ ID NO.7, the underline is the KpnⅠ restriction site),

[0056] AhPIF3-R: 5'-CCC TCTAGA TCAATCATCATAACCAGTCACA-3' (SEQ ID NO.8, the underline is the XbaI restriction site);

[0057] The reaction system for PCR amplification is: I-5Mix 25 μl, upstream primer AhPIF3-F and downstream primer AhPIF3-R 1 μl each, template cDNA 1 μl, H 2 O 22 μl; PCR reaction program: ...

Embodiment 3

[0061] Embodiment 3 Transformation of the plant expression vector containing the Δ12-fatty acid dehydrogenase gene of the target gene AhPIF3

[0062] The recombinant agrobacterium prepared in implementing 2 is transformed into peanut, comprising the following steps:

[0063] (1) Select the plump high-oleic peanut variety K17-15 mature seeds for single sowing, with a plant spacing of 20 cm, and select 20 holes with better growth as recipient plants;

[0064] (2) When the flowering period is approaching, observe every morning, manually remove the flowers to prevent the needles from setting fruit, and the work of removing flowers continues until the blooming period comes;

[0065] (3) After reaching the full flowering stage, inject Agrobacterium from 6:00 am to 8:00 am every day. The specific method: inhale an appropriate amount of Agrobacterium suspension with a 1ml disposable syringe, select the flower buds that open that day, and insert the syringe into the upper part of the c...

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Abstract

The invention relates to a method for utilizing an expression vector containing a delta 12-fatty acid dehydrogenase gene to screen out transgenic peanuts and belongs to the field of gene engineering.According to the method, the delta 12-fatty acid dehydrogenase gene AhFAD2B of peanuts with low content of oleic acid is adopted as a marker gene, the plant expression vector containing the delta 12-fatty acid dehydrogenase gene is created, and application of endogenous genes of the peanuts in fast screening of genetic transformation generations is achieved. According to the method, by carrying out near-infrared detection on one peanut seed grain, transformed bodies can be fast and efficiently screened out under the condition of not damaging the seed. The problems, such as false positive, cumbersome procedures and potential safety hazards in utilization of antibiotics for screening are avoided. The conversion efficiency is high, the operation is simple, and the method is a safe and efficient screening method.

Description

technical field [0001] The invention relates to a method for screening transgenic peanuts by constructing an expression vector containing Δ12-fatty acid dehydrogenase gene, which belongs to the field of genetic engineering. Background technique [0002] Peanut is an important oil crop, and oleic acid and linoleic acid are the main components in its oil. Studies have found that the high oleic acid characteristic of peanuts is controlled by two main genes. Jung et al. isolated these two genes from ordinary peanuts and high oleic peanut mutants (obtained by crossing SunOleic95R as the male parent): AhFAD2A and AhFAD2B (Jung et al. S, Swift D, Sengoku E, Patel M, Teule F, Powell G, Moore K, Abbott A. The high oleate trait in the cultivated peanut Arachis hypogaea L.I. Isolation and characterization of twogenes encoding microsomal oleoyl-PC desaturases. Mol Gen Genet, 2000 , 263:796-805.). These two genes both encode △12-fatty acid dehydrogenase, the main function is to catalyz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/53C12N1/21A01H5/10A01H6/54G01N21/3563G01N21/359C12R1/01
CPCC12N9/0083C12N15/8247C12Y114/99033G01N21/3563G01N21/359
Inventor 赵术珍王兴军石素华厉广辉孙金波李膨呈赵传志夏晗侯蕾
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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