Gentisic acid dioxygenase as well as coding gene and application thereof

Abstract

The invention belongs to the field of application environment microorganism and agriculture, and discloses gentisic acid dioxygenase as well as a coding gene and application thereof. A gentisic acid dioxygenase gene dsmD has a nucleotide sequence shown in SEQ ID NO.1, the total length of the dsmD is 1053bp, 350 amino acids are coded, and the amino acid sequence of the dsmD is shown in SEQ ID NO.2.The gentisic acid dioxygenase gene is the first gene disclosed to degrade 3-chlorogentic acid, an intermediate metabolite of dicamba, and a protein coded by the gene is capable of carrying out ring-opening degradation on gentisic acid and 3-chlorogentic acid. The gentisic acid dioxygenase provided by the invention can degrade gentisic acid and 3-chlorogentisic acid of 100mg / l within 30min. Therefore, the gentisic acid dioxygenase gene dsmD has a great potential in constructing transgenic crops for degrading dicamba, and the gentisic acid dioxygenase protein DsmD has a good application prospect in degrading dicamba and benzene ring substances.

Description

technical field

[0001] The invention belongs to the fields of environmental microorganisms and agriculture, and relates to a gentisate dioxygenase and its coding gene and application, in particular to a gentisate dioxygenase and the application of its coding gene participating in the microbial degradation process of dicamba. Background technique

[0002] Chemical pesticides are an important means to ensure modern agricultural productivity. Rational use of pesticides can effectively prevent and control crop diseases and increase crop yields. However, improper use of pesticides will cause environmental damage to soil, water bodies, etc. The use of herbicides can effectively reduce agricultural labor intensity and increase crop yields, but with the extensive use of herbicides, the damage caused by herbicide residues to the soil is becoming more and more serious. Microbial remediation technology is an in-situ bioremediation technology with good effect, low cost and no secondary ...

Examples

Embodiment 1

[0029] Embodiment 1. Cloning of gentisate dioxygenase gene dsmD

[0030] 1.1 Search for gentisate dioxygenase gene

[0031] 1.1.1 Substrate spectrum experiment of Rhizorhabdus dicambivorans Ndbn-20, an efficient dicamba-degrading bacterium

[0032] The research material of this experiment is Rhizorhabdus dicambivorans Ndbn-20, a high-efficiency dicamba-degrading bacterium isolated by members of our laboratory. The date is November 5, 2014. The substrate spectrum experiment of Ndbn-20 is to add appropriate concentration of 3,6-DCSA analogue to the inorganic salt medium, regularly sample and use the method of ultraviolet scanning to detect the degradation status, the degradation status is shown in Table 1. Through the substrate spectrum experiment, it was found that Ndbn-20 can degrade gentisic acid but not catechol, so it is speculated that the ring-opening substrate of 3,6-DCSA degradation reaction may be gentisic acid or gentisic acid analogue3 ,6-Dichlorogentisic acid.

...

Embodiment 2

[0044] Example 2 High expression of gentisate dioxygenase gene in BL21 (pET-24b (+))

[0045] 2.1 PCR amplification of gentisate dioxygenase gene

[0046] Forward primer: 5'-CGGGAATTC CATATG ACTGCTACGTCGATCAAACAC-3' (SEQ ID NO.3) and reverse primer: 5'-CCG CTCGAG Using CGCGCTACGCCAAAGGCCCAG-3' (SEQ ID NO.4) as a primer, a gentisate dioxygenase gene fragment was amplified from Ndbn-20 genomic DNA by PCR.

[0047] Amplification system:

[0048]

[0049] PCR amplification program:

[0050] a. Denaturation at 98°C for 3 minutes;

[0051] b. Denaturation at 98°C for 0.5min, annealing at 58°C for 0.5min, extension at 72°C for 1.0min, and 30 cycles;

[0052] c. Extend at 72°C for 10 minutes and cool to room temperature.

[0053] 2.2 Double digestion of PCR product and plasmid, product purification and enzyme connection

[0054] The PCR product was purified using a gel purification recovery kit. For specific methods, refer to the kit instructions. Purified PCR products an...

technical field

[0001] The invention belongs to the fields of environmental microorganisms and agriculture, and relates to a gentisate dioxygenase and its coding gene and application, in particular to a gentisate dioxygenase and the application of its coding gene participating in the microbial degradation process of dicamba. Background technique

[0002] Chemical pesticides are an important means to ensure modern agricultural productivity. Rational use of pesticides can effectively prevent and control crop diseases and increase crop yields. However, improper use of pesticides will cause environmental damage to soil, water bodies, etc. The use of herbicides can effectively reduce agricultural labor intensity and increase crop yields, but with the extensive use of herbicides, the damage caused by herbicide residues to the soil is becoming more and more serious. Microbial remediation technology is an in-situ bioremediation technology with good effect, low cost and no secondary ...

Examples

Embodiment 1

[0029] Embodiment 1. Cloning of gentisate dioxygenase gene dsmD

[0030] 1.1 Search for gentisate dioxygenase gene

[0031] 1.1.1 Substrate spectrum experiment of Rhizorhabdus dicambivorans Ndbn-20, an efficient dicamba-degrading bacterium

[0032] The research material of this experiment is Rhizorhabdus dicambivorans Ndbn-20, a high-efficiency dicamba-degrading bacterium isolated by members of our laboratory. The date is November 5, 2014. The substrate spectrum experiment of Ndbn-20 is to add appropriate concentration of 3,6-DCSA analogue to the inorganic salt medium, regularly sample and use the method of ultraviolet scanning to detect the degradation status, the degradation status is shown in Table 1. Through the substrate spectrum experiment, it was found that Ndbn-20 can degrade gentisic acid but not catechol, so it is speculated that the ring-opening substrate of 3,6-DCSA degradation reaction may be gentisic acid or gentisic acid analogue3 ,6-Dichlorogentisic acid.

...

Embodiment 2

[0044] Example 2 High expression of gentisate dioxygenase gene in BL21 (pET-24b (+))

[0045] 2.1 PCR amplification of gentisate dioxygenase gene

[0046] Forward primer: 5'-CGGGAATTC CATATG ACTGCTACGTCGATCAAACAC-3' (SEQ ID NO.3) and reverse primer: 5'-CCG CTCGAG Using CGCGCTACGCCAAAGGCCCAG-3' (SEQ ID NO.4) as a primer, a gentisate dioxygenase gene fragment was amplified from Ndbn-20 genomic DNA by PCR.

[0047] Amplification system:

[0048]

[0049] PCR amplification program:

[0050] a. Denaturation at 98°C for 3 minutes;

[0051] b. Denaturation at 98°C for 0.5min, annealing at 58°C for 0.5min, extension at 72°C for 1.0min, and 30 cycles;

[0052] c. Extend at 72°C for 10 minutes and cool to room temperature.

[0053] 2.2 Double digestion of PCR product and plasmid, product purification and enzyme connection

[0054] The PCR product was purified using a gel purification recovery kit. For specific methods, refer to the kit instructions. Purified PCR products an...

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