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Engineering bacteria for efficiently degrading two fungal toxins and application

A technology of mycotoxins and engineering bacteria, which is applied in the direction of fungi, bacteria, and microorganism-based methods, and can solve problems such as secondary pollution

Active Publication Date: 2012-11-28
ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current adsorbents have basically no effect on these two mycotoxins, and inorganic adsorbents also absorb a large amount of trace nutrients in feed and food. DON adsorbed by clay cannot be decomposed, which will cause secondary pollution

Method used

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  • Engineering bacteria for efficiently degrading two fungal toxins and application
  • Engineering bacteria for efficiently degrading two fungal toxins and application
  • Engineering bacteria for efficiently degrading two fungal toxins and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning of mycotoxin DON degrading enzyme gene DONase

[0032] Construction of Plasmid Library of Deoxynivalenol Degrading Enolase Gene

[0033] (1) The standard strain Fusarium oxysporum (Fusarium oxysporum) ACCC No.36245 (purchased from China Agricultural Microorganism Culture Collection Management Center) was activated and inserted into PDA liquid medium (potato 200g / L, glucose 20g / L, agar 20g / L, PH natural), and then the suspension of the strain was added to 100ml of the same medium, and the shaker condition was 220 rpm, 30°C, 72 hours. After the cultivation was completed, the bacteria were collected by centrifugation at 12000 rpm. RNA extraction (Trizol method);

[0034] (2) Using RNA as a template to reverse transcribe and synthesize a cDNA sequence;

[0035] 2. Acquisition of deoxynivalenol degrading enzyme gene

[0036] Using the cDNA sequence obtained in step 1 as a template, a PCR reaction was performed under the guidance of primers 1 and 2 to a...

Embodiment 2

[0040] Embodiment 2: Cloning of mycotoxin ZEN degrading enzyme gene ZDase

[0041] PCR amplification was carried out using the standard strain G. pink ACCC No. 31535 genomic DNA as a template. Design the following primers to introduce EcoRI and NotI recognition sites (underlined) respectively

[0042] Upstream primer P3: 5'-CCG GAA TTC ATG CGC ACT CGC AGC ACAAT-3'

[0043] Downstream primer P4: 5'-ATAAGAAT GCG GCC G C AAGA TAC TTC TGC GTAAAT T-3'

[0044] The PCR reaction contains 25 μL KOD enzyme Mix in 50 μL PCR reaction mixture, 2 μL each of 12.5 μmoL / L primers, 10 μL template, and 11 μL sterile water to adjust to 50 μL. Conditions are 94 ° C, denaturation 5 min, cycle parameters: 94 ° C denaturation 40 s; Anneal at 54°C for 1 min; extend at 68°C for 1 min; 30 cycles; extend at 68°C for 10 min to complete the extension of the product. The obtained PCR products were subjected to 1.0% agarose gel electrophoresis, and the electrophoresis results were as follows: figu...

Embodiment 3

[0045] Example 3: Construction and verification of recombinant expression vectors

[0046] 1. Construction and verification of recombinant expression vector with exocrine ability

[0047] According to the reported sequence of the exocrine protein α mating factor coding gene on the exocrine expression vector of Pichia pastoris (GenBank Accession No. HQ398363.1 of the SCW gene sequence), the following primers were designed to introduce BamHI and XbaI recognition sites (underlined respectively) out)

[0048] Upstream primer P5: 5'-CGC GGATCC ATGAGATTTCCTTCAATTTT-3'

[0049] Downstream primer P6: 5'-GC TCTAGA TTAATTCGCGGCCGCCCTAG-3′.

[0050] The plasmid DNA of Pichia pastoris expression vector pPIC9k (purchased from Invitrogen) was used as a template for PCR amplification. For PCR reaction, 50 μL of PCR reaction mixture contains 25 μL of KOD enzyme Mix, 2 μL of 12.5 μmoL / L primers, 10 μL of template, and 11 μL of sterile water to adjust to 50 μL. Conditions are 94°C, denat...

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Abstract

The invention discloses engineering bacteria for efficiently degrading two fungal toxins and application. Coding gene of a high-efficiency degradation enzyme of two fungal toxins, i.e., deoxynivalenol (DON) and zearalenone (ZEN), is introduced into the engineering bacteria, so that the engineering bacteria with capacity of efficiently degrading the two toxins, i.e., the DON and the ZEN, is obtained. The engineering bacteria can be directly and effectively applied to diminishing the fungal toxins.

Description

technical field [0001] The invention belongs to the field of fermentation and biotechnology, and specifically relates to the construction and application of two kinds of mycotoxin efficient degradation engineering bacteria. Background technique [0002] Mycotoxins are a class of secondary metabolites produced by toxin-producing fungi during the damage process, mainly including aflatoxins, deoxynivalenol (DON), zearalenone (ZEN) and fumonisins (FUM ) and ochratoxin, etc. In recent years, due to the frequent occurrence of extreme climates in my country, the head blight mainly harms wheat and corn. Wheat and corn continue to increase. In order to ensure the safety of people's consumption and animal husbandry in our country, the State Grain Administration and relevant departments have been infected with severe head blight during the wheat harvest season in the Huanghuaihai region of my country in 2010, resulting in deoxynivalenol ( DON) and zearalenone (ZEN) a large area of ​​whea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/19A62D3/02C12R1/07C12R1/125C12R1/225C12R1/84C12R1/865A62D101/28
Inventor 吴子丹孙长坡任保中伍松陵
Owner ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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