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Gene AKR18A1 relevant to detoxification of fusarium toxin and toxic aldehydes and applications of gene AKR18A1

A fusarium and gene technology, applied in the direction of DNA / RNA fragments, applications, genetic engineering, etc., to achieve the effects of easy expression, simple cloning operation, and specific products

Active Publication Date: 2019-01-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether members of the AKR superfamily have detoxification activity against Fusarium toxins has not been reported

Method used

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  • Gene AKR18A1 relevant to detoxification of fusarium toxin and toxic aldehydes and applications of gene AKR18A1
  • Gene AKR18A1 relevant to detoxification of fusarium toxin and toxic aldehydes and applications of gene AKR18A1
  • Gene AKR18A1 relevant to detoxification of fusarium toxin and toxic aldehydes and applications of gene AKR18A1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Cloning of target gene AKR18A1 and construction and transformation of prokaryotic expression vector

[0037] Target sequence cloning: Using the genomic DNA of Sphingomonas sp. degrading bacteria S3-4 (provided by the research group laboratory) as a template, the high-fidelity enzyme KOD plus (purchased from Toyobo, Japan) was amplified Target gene fragment. EcoR I and Hind III restriction sites were added to both ends of the primer, forward primer: 5′-GGAATTCGATGCGCTACAACCGGCTCGGCCG-3′, reverse primer: 5′-CCCAAGCTTGCGCCGC GGCGACGGGCCG-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 μL of betaine, supplemented with ddH 2 O to a total volume of 50 μL. The reaction program was: 95°C pre-denaturation for 5 minutes; 95°C for 30s, 68°C for 2min, 35 cycles; 68°C extension for 10min. After sequencing the target fragment, the sequence ...

Embodiment 2

[0040] Example 2: Expression and purification of AKR18A1 protein

[0041] Induced expression of protein: culture Escherichia coli BL21 strain containing pET-22b-AKR18A1 vector in LB liquid medium, and shake it 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.

[0042] 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-NTA matrix. Add...

Embodiment 3

[0043] Example 3: Catalysis of purified AKR18A1 protein on DON

[0044] The purified AKR18A1 protein was reacted with DON in the buffer solution, and the catalytic activity of the purified protein on DON was calculated by the change of DON content in the reaction system. In 50μL reaction system, including 6μg purified protein, 100μM DON, 2mM NADP + , add buffer to a total volume of 50 μL. After the reaction is completed, add an equal volume of methanol to end the reaction, and use high-performance liquid chromatography (HPLC) to detect the DON content in the solution. The HPLC analysis system consists of: Agilent 1200 semi-preparative high-performance liquid chromatography. Main components: Quaternary pump (1260Quat Pump VL) , autosampler (1260ALS), column oven (1260FCC), UV detector (1260VWD), automatic fraction collector (1260FC-AS), analytical column (Eclipse XDB-C18, 4.6×150mm, 5μm), half Preparative chromatographic column (Eclipse XDB-C18, 9.4×250 mm, 5 μm), operating s...

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Abstract

The invention discloses a gene AKR18A1 relevant to detoxification of fusarium toxin and toxic aldehyde compounds and applications of the gene AKR18A1. The gene AKR18A1 has the sequence as shown in SEQID NO.1, amino acid coded by the gene AKR18A1 comprises a conservative feature structure element of aldo-keto reductase, and the sequence of the amino acid is as shown in SEQ ID NO.2. Purified protein is obtained through prokaryotic expression of the gene AKR18A1, and the in-vitro experiment proves that the purified protein can oxidize deoxynivalenol, so that 3-keto-deoxynivalenol is formed; andmeanwhile, the protein can act on zearalenone and derivatives of zearalenone, including alpha-zearalenol and beta-zearalenol, and also can effectively degrade the toxic aldehyde compounds, namely, glyoxal and methylglyoxal.

Description

technical field [0001] The invention belongs to the field of detoxification of mycotoxins and toxic aldehyde compounds, and in particular relates to an isolated and cloned AKR18A1 gene, the protein encoded by the gene has been confirmed to be able to act on deoxynivalenol (deoxynivalenol, DON). Oxidation to form 3-keto-deoxynivalenol (3-oxo-DON), while reducing zearalenone (ZEN) to produce α-zearalenol (α-zearalenol, α -ZOL) and β-zearalenol (β-zearalenol, β-ZOL), in addition, α-ZOL and β-ZOL can be oxidized to form ZEN. The protein encoded by this gene also has the function of degrading toxic aldehydes glyoxal and methylglyoxal, and the Escherichia coli containing the gene AKR18A1 has significantly improved tolerance to the two aldehydes. 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...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/11
CPCA23K20/189A23L5/25C12N9/0008
Inventor 廖玉才何伟杰李和平袁青松郭茂伟易沭远
Owner HUAZHONG AGRI UNIV
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