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Bacterial enzyme composition for preventing and treating root-knot nematode and preparation method of bacterial enzyme composition

An enzyme composition and a technology for root-knot nematodes, which are applied in the field of biomedicine, can solve the problems of high cost, limited application, residue of allyl chloride, etc., and can avoid the reduction of product activity, prevent and control root-knot nematodes quickly and long-term, and realize quick-acting killing. The effect of the bug effect

Pending Publication Date: 2022-07-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most common method is chemical synthesis, but the cost is high and there are allyl chloride residues, or the bulbs of Liliaceae plants are broken and enzymatically hydrolyzed and then extracted, but the operation is complicated and the cost is high, which greatly limits its application in agriculture.

Method used

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  • Bacterial enzyme composition for preventing and treating root-knot nematode and preparation method of bacterial enzyme composition
  • Bacterial enzyme composition for preventing and treating root-knot nematode and preparation method of bacterial enzyme composition
  • Bacterial enzyme composition for preventing and treating root-knot nematode and preparation method of bacterial enzyme composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Construction of Bacillus subtilis strains overexpressing S-alkyl-L-cysteine ​​sulfoxide lyase LCC1

[0058] (1) Design primers according to the S-alkyl-L-cysteine ​​sulfoxide lyase LCC1 gene sequence and carry out PCR amplification to obtain the S-alkyl-L-cysteine ​​sulfoxide lyase LCC1 gene fragment; The pHT43 expression vector plasmid was extracted and double digested and recovered by gel.

[0059] (2) The recovered S-alkyl-L-cysteine ​​sulfoxide lyase LCC1 gene fragment and pHT43 plasmid fragment were ligated. Ligation product transforms competent cells E. coli Transformants were picked by DH5α for full-length sequencing of gene fragments.

[0060] (3) The recombinant plasmid was transferred into Bacillus subtilis WB800N competent cells, recovered, and coated on LB plate (100 μg / ml Amp). The transformants were picked and cultured in LB medium (100 μg / ml Amp) at 37 °C for 14 h with shaking. PCR and electrophoresis to verify the results are as follows figure 1 ...

Embodiment 2

[0063] Construction of Bacillus subtilis strain LCC1 by recombinant integration of S-alkyl-L-cysteine ​​sulfoxide lyase

[0064] The recombinant plasmid pIEFBPR-S-alkyl-L-cysteine ​​sulfoxide lyase LCC1 gene was constructed, and the bacterial-derived S-alkyl-L-cysteine ​​sulfoxide lyase LCC1 gene sequence was cloned into the integration vector pIEFBPR. At the multiple cloning site between the homology arm bpr front and the DR sequence, the recombinant plasmid transforms the host strain WB800N and integrates into the protease-encoding gene on the chromosome of WB800N bpr middle of the sequence. First-crossover transformants were selected with spectinomycin (100 μg / ml). Transformants were cultured in anti-LBG-free (LB+1% glucose) medium for 24 h, and the second exchange occurred spontaneously. The culture liquid is transferred to LBG medium, induced by IPTG, cultured (or directly coated on LBG plate containing IPTG), induced by IPTG mazF Lethal gene expression, if no second ...

Embodiment 3

[0066] Top-pot fermentation of Bacillus subtilis overexpressing S-alkyl-L-cysteine ​​sulfoxide lyase LCC1 and recombinantly integrated Bacillus subtilis expressing S-alkyl-L-cysteine ​​sulfoxide lyase LCC1

[0067] (1) Bacillus subtilis WB800N fermentation overexpressing S-alkyl-L-cysteine ​​sulfoxide lyase LCC1:

[0068] The fermentation medium was prepared as follows:

[0069] The formula of the fermentation medium (g / l) according to the mass-volume ratio (g / l) is: soybean meal powder 20, glucose 20, cornmeal 20, manganese sulfate 0.2, yeast powder 5, corn steep liquor 4, sodium chloride 2, phosphate diphosphate Potassium hydrogen 3, magnesium sulfate 0.5, ferric chloride 0.3, pH 7.2, put into a 5 L fermenter, and sterilized at 121 °C for 20 min.

[0070] Primary seed culture: Pick a single colony from the plate and inoculate it into 5 ml of liquid LB seed medium, chloramphenicol (5 μg / ml), 37°C, 220 rpm, overnight culture.

[0071] Secondary seed culture: insert the above...

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Abstract

The invention relates to the technical field of biological medicines, in particular to a bacterial enzyme composition for preventing and treating root-knot nematode and a preparation method thereof. The method comprises the following steps: carrying out overexpression and integrated expression on a bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 in bacillus subtilis; a bacillus subtilis overexpression strain is utilized, high-enzyme-activity protein is obtained through lactose induction in the fermentation process, enzyme is secreted out of cells, and enzyme dry powder is obtained through spray drying; bacillus subtilis is utilized to integrate and express strains, enzyme products which are continuously and stably expressed are obtained through the fermentation process, and microbial inoculum / enzyme dry powder is obtained through spray drying. The substrate and bacterial enzyme dry powder are compounded to form a product, so that in-situ catalysis of enzyme is realized to produce an insecticidal product diallyl thiosulfinate, and rapid killing and long-term control effects of root-knot nematode are realized. A plate inhibition experiment shows that the product has a 100% lethal effect on soil root-knot nematode, and the control effect on root-knot nematode of greenhouse and field crops is obvious.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a mycoenzyme composition for preventing and controlling root-knot nematodes and a preparation method thereof. Background technique [0002] The root-knot nematode seriously restricts the sustainable development of agriculture in my country. The root-knot nematode can infect the roots of crops and form root nodules, resulting in difficulties in absorbing nutrients, withering and death of crops. Nematode disease is widespread, serious and difficult to control. Especially in greenhouse cultivation, root knot nematode has developed into a devastating pest and disease in many old greenhouses due to continuous cropping. Therefore, there is an urgent need for products that can effectively inhibit root-knot nematodes. [0003] At present, chemical control is quick and easy, and can effectively control the damage of root-knot nematodes. Commonly used chemical nematicides include th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/88C12N15/75C12N15/60C12P11/00A01N63/22A01N63/50A01P5/00C12R1/125
CPCC12N1/20C12N9/88C12N15/75C12P11/00A01N63/22A01N63/50
Inventor 杨春玉李春芳刘远翔
Owner SHANDONG UNIV
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