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Fusarium toxin removal path related genes ADH, AKR6D1 and AKR13B2 and their application

A fusarium and gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of unknown genes related to metabolic pathways, and achieve the effects of product specificity, huge development and application potential, and simple cloning operation.

Active Publication Date: 2018-04-17
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the metabolic pathway of DON degradation into 3-epi-DON and the related genes involved are still unknown

Method used

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  • Fusarium toxin removal path related genes ADH, AKR6D1 and AKR13B2 and their application
  • Fusarium toxin removal path related genes ADH, AKR6D1 and AKR13B2 and their application
  • Fusarium toxin removal path related genes ADH, AKR6D1 and AKR13B2 and their application

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Cloning of target genes ADH, AKR6D1, AKR13B2 and construction and transformation of prokaryotic expression vectors

[0029] Target sequence cloning: Using the genomic DNA of Devosia sp. degrading bacteria D6-9 (provided by the research group laboratory) as a template, the high-fidelity enzyme KOD plus (purchased from Toyobo, Japan) was used to amplify the target sequence Gene fragment. Amplification primer of gene ADH, forward primer: 5′-CATGCTGACGGGGCCGCGG-3′, reverse primer is 5′-CTTGGCTTCGGGCAGGGCG-3′; amplification primer of gene AKR6D1, forward primer is 5′-ATGGAATATCGTCGTCTGGG-3′, The reverse primer is 5′-CTAGAACCGCTGCGGGCCGG-3′; the amplification primer of the gene AKR13B2, the forward primer is 5′-ATGTCAGAACCCAATGCAGC-3′, and the reverse primer is 5′-TCACGAAGCCCCTTTCCAGCTC-3′. 50μL reaction system: 5μL of 10×KOD buffer, 25mmol / L MgSO 4 2 μL, 5 μL of 2 mmol / L dNTPs, 1.5 μL of each 10 μmol / L primer, 1 μL of KOD plus (1U / μL), 1 μL of template cDNA, 5 μ...

Embodiment 2

[0032] Example 2: Expression and purification of ADH, AKR6D1, AKR13B2 proteins

[0033] Induced expression of protein: culture the Escherichia coli BL21 strain containing the expression vector in LB liquid medium, in a shaker at 37°C (200r / min) to OD 600 It reaches about 0.6; add IPTG (purchased from Sigma, USA) at a final concentration of 0.4 mM, place in a shaker at 16°C (140r / min) for induction and culture for 12h.

[0034] Purification of protein: collect the cells by centrifugation, and resuspend the cells with an appropriate amount of lysis buffer (according to the ratio of 1:20-1:40). Vortex vigorously to fully dissolve the cells. Before cell disruption, 100 μM protease inhibitor PMSF was added at a ratio of 1:100 (v:v), and E. coli cells were disrupted with a high-pressure cell disruptor. The bacterial cell disruption solution was centrifuged at 16000r / min for 30min, the supernatant was collected, and all passed through the protein purification column filled with Ni-...

Embodiment 3

[0035] Embodiment 3: the catalysis of purified ADH protein to DON

[0036] The purified ADH protein was reacted with DON in buffer, in a 50 μL reaction system, including 6 μg purified protein, 100 μM DON, 100 μM PQQ, 100 μM Ca + , add Tris-HCL buffer solution (pH 7.5) to a total volume of 50 μL, and react at 30°C. After the reaction is completed, add an equal volume of methanol to end the reaction. Use high-performance liquid chromatography (HPLC) to detect the DON content in the solution, and analyze the composition of the system by HPLC : Agilent 1200 semi-preparative HPLC main components: quaternary pump (1260QuatPump VL), autosampler (1260ALS), column thermostat (1260FCC), UV detector (1260VWD), automatic fraction collector (1260FC-AS ), analytical chromatographic column (Eclipse XDB-C18, 4.6×150mm, 5μm), semi-preparative chromatographic column (EclipseXDB-C18, 9.4×250mm, 5μm), operating system (Agilent ChemStation, B.04.03). HPLC analysis conditions: injection volume 10μ...

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Abstract

The invention discloses Fusarium toxin removal path related genes ADH, AKR6D1 and AKR13B2 and their application. Purified proteins are acquired by prokaryotic expression of the genes ADH, AKR6D1 and AKR13B2; in-vitro experiments prove that the three purified proteins can jointly catalyze DON (deoxynivalenol) to form 3-isomer-deoxynivalenol (3-eip-DON), wherein the protein ADH can oxidize DON to form 3-oxo-deoxynivalenol (3-oxo-DON), and the proteins AKR6D1 and AKR13B2 can further reduce 3-oxo-DON to form 3-epi-DON having lowest toxicity presently known.

Description

technical field [0001] The invention belongs to the field of mycotoxin detoxification in food safety, and specifically relates to genes ADH, AKR6D1 and AKR13B related to the detoxification pathway of fusarium toxins and applications thereof. The protein encoded by the ADH gene has been confirmed to act on deoxynivalenol (deoxynivalenol, DON) and oxidize it to form 3-keto-deoxynivalenol (3-oxo-DON), while AKR6D1, AKR13B2 protein can reduce 3-oxo-DON to form 3-isomer-deoxynivalenol (3-epi-DON), which is extremely weak in toxicity. Background technique [0002] Deoxynivalenol (DON) is a widely distributed mycotoxin mainly produced by fungi of the genus Fusarium. These toxin-producing fungi infect the floret tissues of cereal crops such as wheat, barley, and corn in the field, and colonize the developing kernels. Thus, the toxins produced by these fungi accumulate directly in the ripening grain and thus enter downstream products (Bai, G., and Shaner, G. Management and resistan...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/11C12P17/18
CPCC12N9/0006C12P17/181C12Y101/01001
Inventor 廖玉才何伟杰李和平易沭远张静柏黄涛
Owner HUAZHONG AGRI UNIV
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