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Iprodione-degrading bacteria, degrading enzyme IpaH, encoding gene ipaH of degrading enzyme IpaH and application of iprodione-degrading bacteria, degrading enzyme IpaH and encoding gene ipaH

A technology of iprodione and gene, applied in iprodione-degrading bacteria, degrading enzyme IpaH and its coding gene ipaH and its application field

Active Publication Date: 2018-03-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Several bacterial strains that degrade iprodione have been reported at present, but there are no reports about iprodione-degrading enzymes and genes as of the date of this patent application, which limits people's use of microbial (enzyme) degradation technology to eliminate soil and agricultural products. iprodione residue

Method used

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  • Iprodione-degrading bacteria, degrading enzyme IpaH, encoding gene ipaH of degrading enzyme IpaH and application of iprodione-degrading bacteria, degrading enzyme IpaH and encoding gene ipaH
  • Iprodione-degrading bacteria, degrading enzyme IpaH, encoding gene ipaH of degrading enzyme IpaH and application of iprodione-degrading bacteria, degrading enzyme IpaH and encoding gene ipaH
  • Iprodione-degrading bacteria, degrading enzyme IpaH, encoding gene ipaH of degrading enzyme IpaH and application of iprodione-degrading bacteria, degrading enzyme IpaH and encoding gene ipaH

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1.1 Isolation of iprodione-degrading bacteria YJN-5 (CCTCC NO.M 2017440)

[0036] The enrichment matrix used to enrich the iprodione-degrading strain was taken from the farmland soil of the vineyard in Langfang City, Hebei Province. Take 5.0 g of the farmland soil and add it to 100 ml of basic salt medium, add 50 mg L -1 Iprodione, 30°C, 180r·min -1 Cultivate for 5 days, transfer to the same medium with 5% inoculum size, transfer continuously for 3 times, and dilute the enrichment solution in gradient, take 10 -4 ~10 -7 Each 0.1mL of the diluted enrichment solution was applied to the 100mg·L -1 On the solid medium plate of iprodione, after culturing at 30°C for 3 days, pick the grown single colony and inoculate it in a medium containing 100mg·L -1 In iprodione inorganic salt medium, 30°C, 180r min -1 Cultivate on a shaking table for 3 days, and repeat to verify its degradation effect.

[0037] The basic salt medium formula is (1L): 1.0g NH 4 NO 3 , 1.0g NaCl, 1.5...

Embodiment 2

[0043] The cloning of embodiment 2 gene ipaH (technical route figure 1 )

[0044] The strategy adopted for cloning the gene is the shotgun method. First extract the total DNA of bacterial strain YJN-5, the total DNA is partially digested with Sau3AI and the pUC118BamHI / BAP vector (after pUC118DNA is digested by the restriction enzyme BamHI through the single cutting point on its multiple cloning site, and then sourced from E.coli Alkaline phosphatase (BAP) was dephosphorylated) enzyme-linked, and the enzyme-linked product was transformed into Escherichia coli DH5α competent cells to construct a total DNA library of strain YJN-5, and all clones were coated on a medium containing 100ppm ampicillin-resistant and 500ppm iprodione on the LB plate (iprodione is insoluble in water, and a precipitate will be formed in the LB plate, so the method of transparent hydrolysis circle is used to screen positive clones). Using this cloning method allows high-throughput screening of librarie...

Embodiment 3

[0062] Example 3 High-efficiency expression, purification and functional determination of iprodione hydrolase IpaH in BL21 (pET-29a (+)) (technical route figure 2 )

[0063] (1) PCR amplification of gene ipaH

[0064] Forward primer: 5’-GGAATTC CATATG GGTACTTCACCCCCAG-3' (SEQ ID NO.3) and reverse primer: 5'-CCG CTCGAG ACCAGCGTTGATGAACGG-3' (SEQ ID NO.4) was used as a primer to amplify the ipaH gene fragment from Paenarthrobacterureafaciens.YJN-5 (CCTCC NO.M 2017440) genomic DNA by PCR

[0065] Amplification system:

[0066]

[0067] PCR amplification program:

[0068] a. Denaturation at 98°C for 3 minutes;

[0069] b. Denaturation at 98°C for 0.5min, annealing at 58°C for 0.5min, extension at 72°C for 1.5min, and 30 cycles;

[0070] c. Extend at 72°C for 10 minutes and cool to room temperature.

[0071] (2) The PCR product was double digested with NdeI and XhoI.

[0072] Enzyme cutting system:

[0073]

[0074] In a 37°C water bath, react for 30 minutes. The...

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Abstract

The invention belongs to the field of applied environment microorganisms and agriculture, and relates to iprodione-degrading bacteria, a degrading enzyme IpaH, an encoding gene ipaH of degrading enzyme IpaH and application of the iprodione-degrading bacteria, the degrading enzyme IpaH and the encoding gene ipaH. The full-length of open reading frame of the iprodione-degrading enzyme gene ipaH is 1410bp, and a sequence is shown as SEQ ID NO.1, an encoded product IpaH contains 469 amino acids and a sequence of the encoded product IpaH is SEQ ID NO.2. IpaH can degrade iprodione. The gene ipaH canbe used to construct transgenic engineering bacteria or crops degrading the iprodione, and the degrading enzyme IpaH can prepare enzyme preparations for eliminating iprodione residues in soils, waterbodies and agricultural products.

Description

technical field [0001] The invention belongs to the fields of applied environmental microorganisms and agriculture, and relates to iprodione-degrading bacteria, degrading enzyme IpaH and its coding gene ipaH and applications thereof. Background technique [0002] Fungicides are the most important weapon in the control of crop diseases. Iprodione is a dicarboximide high-efficiency broad-spectrum, contact-killing fungicide with a wide bactericidal spectrum. Its main control targets are various vegetables, fruit trees and fruit storage diseases caused by Botrytis and Bustle. It has a good control effect on fungal diseases such as early leaf defoliation, gray mold, and early blight of crops. Since most fungicides are low-efficiency or low-efficiency pesticides, the obvious control effect can only be seen after a period of time after application, so the dosage is often deliberately increased several times or even dozens of times during use, and the fungicide becomes One of the ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/78C12N15/55C12N15/70C12N1/21C02F3/34B09C1/10A62D3/02C12R1/01C12R1/19C02F101/38A62D101/04A62D101/28
CPCA62D3/02A62D2101/04A62D2101/28B09C1/10C02F3/34C02F2101/38C12N9/78C12N1/205C12R2001/01
Inventor 洪青杨战功邱吉国闫新何健蒋建东黄星陈凯朱建春
Owner NANJING AGRICULTURAL UNIVERSITY
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