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Angular dioxygenase gene dpea1a2 and its application

A dpea1a2, dioxygenase technology, applied in the field of environmental microorganisms and agriculture, can solve the problem that the microorganism of 2-PBA has not been reported.

Inactive Publication Date: 2019-08-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, three genes or gene clusters that degrade diphenyl ether compounds have been reported at home and abroad, but no microorganisms that can degrade 2-PBA have been reported.

Method used

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  • Angular dioxygenase gene dpea1a2 and its application
  • Angular dioxygenase gene dpea1a2 and its application
  • Angular dioxygenase gene dpea1a2 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Cloning of angle dioxygenase gene dpeA1A2

[0048] 1.1 Extraction of bacterial genome total DNA

[0049] After mass culture of SC-3 (CCTCC NO: M2015417), the total genomic DNA of SC-3 with high purity and large fragments was extracted by CTAB method, dissolved in TE buffer (pH8.0), and stored at -20°C. For specific methods, refer to the "Refined Molecular Biology Experiment Guide" edited by F. Osper et al.

[0050] 1.2 Construction of recombinant plasmids

[0051] The fragments containing the suspected 2-PBA (diphenyl ether) degradation gene cluster were respectively amplified by PCR, and enzyme-linked to the broad host plasmid pBBR1MCS-5 to construct a recombinant plasmid. Through genome analysis and sequence comparison, three suspected target dioxygenase genes were screened out for functional verification experiments, and finally one of the genes was found to be responsible for the degradation of 2-PBA and diphenyl ether.

[0052] Using SC_3 genomic DNA ...

Embodiment 2

[0062] Example 2. High expression of angle dioxygenase gene dpeA1A2 in BL21(pET-29a(+))

[0063] Since the E. coli recombinant strain E. coli DH5α (dpeA1A2) introduced with the dpeA1A2 gene cannot degrade 2-PBA and diphenyl ether, we suspect that E. coli lacks the supporting electron transport chain component, iron, which is required for the catalysis of this dioxygenase. Redoxin and ferredoxin reductases render it incapable of degrading the substrate of interest. In addition, gene alignment did not find related electron transport chain genes around the dpeA1A2 gene. Therefore, we found related electron transport chain genes by gene alignment and expressed them together with dpeA1A2 in BL21(pET-29a(+)) to verify their functions.

[0064] 2.1 Amplification of dpeA1A2 and electron transport chain genes

[0065]Using the genome of strain SC_3 as a template, dpeA1A2 and possible ferredoxin dpeB and reductase gene dpeC sequences were amplified by high-fidelity enzymes with corres...

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Abstract

The invention discloses an angle dioxygenase gene dpeA1A2 and its application. An angle dioxygenase gene dpeA1A2, which is composed of the angle dioxygenase large subunit encoding gene dpeA1 whose size is 1383bp shown in SEQ ID NO.1, and the angle dioxygenase gene dpeA1 whose size is 561bp shown in SEQ ID NO.2 Oxygenase small subunit coding gene dpeA2 and the connecting sequence GATG between the two components. The DpeA1A2BC holoenzyme provided by the present invention can completely degrade 100 mg / L of diphenyl ether within 72 hr; in addition, the DpeA1A2BC holoenzyme can also completely degrade 100 mg / L of 2-phenoxybenzoic acid (2-PBA) within 72 hr. Therefore, the angle dioxygenase gene dpeA1A2 is used in the construction of transgenic crops for degrading diphenyl ether or 2-phenoxybenzoic acid. DpeA1A2BC holoenzyme is used in the degradation of diphenyl ether and 2‑PBA.

Description

technical field [0001] The invention belongs to the fields of applied environmental microorganisms and agriculture, and relates to angle dioxygenase gene dpeA1A2 and its application. Background technique [0002] Chemical pesticides are one of the important means to ensure a bumper harvest in my country, and play a very important role in agricultural production. Pesticide refers to the general term of chemical reagents used to control pests, weeds and other harmful organisms that harm crops and to regulate plant growth. However, due to the extensive use of highly toxic, high-residue, and refractory pesticides for a long time, we are facing environmental problems of increasing soil, groundwater, and atmospheric pesticide pollution. Many pesticides are synthesized from intermediates, so the degradation of pesticide intermediates has wide applicability and high efficiency. Pesticide pollution degradation technology can be divided into thermal degradation, photodegradation, ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/53C12N9/02B09C1/10C02F3/34A62D3/02C12R1/01A62D101/04A62D101/28
CPCA62D3/02C12N1/205C12R2001/01
Inventor 何健蔡舒蔡天明陈立伟施超
Owner NANJING AGRICULTURAL UNIVERSITY
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