Application of dicamba intermediate product 3,6-dichlorogentisate dechlorinase dsmh2 and its coding gene

A technology of dichlorogentisic acid dechlorination enzyme and dichlorogentisic acid, which is applied in the field of environmental microorganisms and agriculture, and can solve the problems that restrict the research of dicamba's environmental behavior and ecological safety.

Active Publication Date: 2019-12-24
NANJING AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dicamba is stable in the soil and can generally be maintained for more than 40 days. The main force of its degradation in the environment is microorganisms. At present, a variety of dicamba degrading strains have been screened, and multiple dicamba degradation genes have been cloned. The further degradation mechanism, degradation gene and enzyme of the microbial degradation intermediate product 3,6-dichlorogentisic acid of dicamba have not been reported yet, which seriously restricts the research on the environmental behavior and ecological safety of dicamba

Method used

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  • Application of dicamba intermediate product 3,6-dichlorogentisate dechlorinase dsmh2 and its coding gene
  • Application of dicamba intermediate product 3,6-dichlorogentisate dechlorinase dsmh2 and its coding gene
  • Application of dicamba intermediate product 3,6-dichlorogentisate dechlorinase dsmh2 and its coding gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cloning of embodiment 1.3,6-dichlorogentisate dechlorase gene dsmH1, dsmH2

[0033] 1.1 Metabolite detection

[0034] 1.1.1 Detection of crude enzyme 3,6-DCGA dechlorase activity of dicamba-degrading strain Ndbn-20

[0035] The strain used in this experiment is Rhizorhabdus dicambivorans Ndbn-20 (formerly named Sphingomonas sp.Ndbn-20), an efficient dicamba-degrading bacterium isolated by members of our laboratory. Ndbn-20 was cultured in 100ml 1 / 5LB liquid medium To the logarithmic phase, the cells were collected by centrifugation, washed twice with PBS buffer, resuspended in 10ml PBS buffer, ultrasonically disrupted (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 5-10 minutes, and centrifuged at 12000rpm for 40min , collect the supernatant, which is the crude enzyme solution.

[0036]Enzyme activity reaction system (1ml): add PBS (50mM, pH 7.0), 0.1mM 3,6-dichlorogentisic acid, 100μl of crude enzyme solution, add different cofactors NADH, NADPH, GSH,...

Embodiment 2

[0039] High expression of embodiment 2 dechlorinase gene in BL21 (pET-24b (+))

[0040] 2.1 PCR amplification of dechlorinase gene

[0041] Forward primer: 5'-TAAGAAGGAGATATACATATGACCCATCTGGACCTGTACAATTAC-3' (SEQ ID NO.3), reverse primer: 5'-TGGTGGTGGTGCTCGAGGATCCCACCCTTCCAATTGGGCATC-3' (SEQ ID NO.4). dsmH2 was amplified from Ndbn-20 genomic DNA by PCR.

[0042] Amplification system:

[0043]

[0044] PCR amplification program:

[0045] a. Denaturation at 98°C for 3 minutes;

[0046] 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;

[0047] c. Extend at 72°C for 10 minutes and cool to room temperature.

[0048] 2.2 Double digestion of PCR product and plasmid, product purification and enzyme connection

[0049] The PCR product was purified using a gel purification recovery kit. For specific methods, refer to the kit instructions. Purified PCR products and plasmids were digested with corresponding enzymes re...

Embodiment 3

[0071] Verification of the physiological function of embodiment 3 dechlorinase in bacterial strain Ndbn-20

[0072] In order to verify the physiological function of the dechlorinase in the strain Ndbn-20, the present invention constructed the dsmH2 insertion mutation vector, which is to insert the about 509bp dsmH2' fragment in the middle of the reductive dechlorinase gene into the PstI and BamHI restriction sites of pJQ200SK between points.

[0073] With forward primer: 5'-CTTGATATCGAATTCCTGCAGTTGATGAAGCTGGAGCATACGCGGC-3' (SEQ ID NO.5), reverse primer: 5'-GCTCTAGAACTAGTGGATCCGTCGTCCCATAGCCGGGCAAAATTC-3' (SEQ ID NO.6), dsmH2' was amplified.

[0074] The insertion mutant vector pJQ200SK was double-digested with PstI and BamHI, and the method for constructing the vector is shown in Example 2 for the construction of an expression vector. Transform the constructed vector into DH5α.

[0075] 3.2 Insertion mutation

[0076] Insert donor bacteria DH5α(pJQdsmH2), acceptor bacteria ...

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Abstract

The invention discloses a dicamba intermediate product 3,6-dichloro gentisic acid dechlorination enzyme DsmH2 and application of an encoding gene thereof. The nucleotide sequence of the DsmH2 is SEQ ID No. 1 and has a total length of 741bp; 246 amino acids are encoded, and the amino acid sequence of the DsmH2 is SEQ ID No. 2. The DsmH2 is firstly found to be a glutathione S-transferase type of reductive dechlorination enzyme participating in the downstream metabolic pathway of dicamba, and can degrade 100 mg / l of 3,6-dichloro gentisic acid within 30 minutes. Therefore, a 3,6-dichloro gentisicacid dechlorination enzyme gene dsmH2 has a huge application potential in the construction of degraded dicamba transgenic crops.

Description

technical field [0001] The invention belongs to the fields of environmental microbes and agriculture, and relates to the application of two dechlorinases and their coding genes involved in the microbial degradation process of the herbicide dicamba. Background technique [0002] Pesticide refers to the general term for chemical reagents used to prevent and control pests, weeds and other harmful organisms that harm crops, as well as to regulate plant growth. It is an important means to ensure modern agricultural productivity. Rational use of pesticides can effectively prevent and control crop diseases and increase crop yield. The use of herbicides can effectively reduce agricultural labor intensity and increase crop yields. However, with the large-scale and long-term use of herbicides, the harm caused by herbicide residues to the atmosphere, soil and water is becoming more and more serious, and it also poses a threat to human health. Pesticide pollution remediation technology ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/14C12N15/70C12N1/21C12N15/82B09C1/10C02F3/34C12R1/19C02F101/36
CPCB09C1/10B09C2101/00C02F3/342C02F2101/306C02F2101/36C12N9/14C12N15/70C12N15/8274C12Y308/01
Inventor 何健李娜姚利丁德荣陶青陈乐刘斌彭乾
Owner NANJING AGRICULTURAL UNIVERSITY
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