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Recombinant broad-spectrum metarhizium as well as preparation method and application thereof

A metarhizium anisopliae, broad-spectrum technology, applied in the field of transgenic strains and its preparation, can solve the problems of unsatisfactory effect, danger, long lethal time, etc., achieve the effect of increasing concentration, good biological safety, and improving insecticidal efficiency

Active Publication Date: 2018-04-17
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the wild-type broad-spectrum Metarhizium anisopliae generally has a long lethal time and the effect is not ideal, while the strains modified by genetic engineering methods are potentially dangerous to the environment or humans

Method used

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  • Recombinant broad-spectrum metarhizium as well as preparation method and application thereof
  • Recombinant broad-spectrum metarhizium as well as preparation method and application thereof
  • Recombinant broad-spectrum metarhizium as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The preparation of the metarhizium anisopliae of embodiment 1 recombination broad-spectrum

[0045] In this example, the knockout of the monoamine oxidase gene (marked as MAA_03753) in the broad-spectrum Metarhizium anisopliae Roberts (marked as MAA) is taken as an example. The gene bank accession number of MAA_03753 is NW_011942171.1, monoamineoxidase[EC:1.4.3.4 ], the specific sequence is shown in SEQ ID NO:1. In this embodiment, Metarhizium anisopliae broad-spectrum Roberts is not limited, and other broad-spectrum Metarhizium anisopliae having monoamine oxidase, such as Metarhizium anisopliae, etc. may be used. In this embodiment, the type of plasmid is not limited, as long as it contains Bar gene and / or Ben gene. For example, pDHt-Bar plasmid can be used, and pDHt-Ben plasmid can also be used. The pDHt-Bar plasmid is taken as an example below for illustration.

[0046] 1. Knockout plasmid construction of MAA_03753

[0047] Primers MAA_03753Fs and MAA_03753Rs, MA...

Embodiment 2

[0079] Determination of tryptamine content in embodiment 2 recombinant broad-spectrum Roberts metarhizium anisopliae

[0080] The wild-type MAA, the recombinant MAA-KO screened in Example 1 and the wild-type obligate bacterium Metarhizium anisopliae (referred to as MAC) were cultured on PDA plates respectively. After culturing for 15 days, the spores of wild-type MAA, recombinant MAA-KO and MAC were inoculated into the L15 medium containing the hemolymph of migratory locusts (preparation of the hemolymph of migratory locusts: add 200 μL of fresh hemolymph was filtered with a 0.22 μm filter. Add 100 μL of the above prepared hemolymph to each ml of L15 medium when cultivating mycelia), and cultured in a light-proof incubator at 28°C for 6 days, and then Mycelia were collected and treated with ddH 2 O washed out the medium twice, and then freeze-dried at -20°C. Weigh 1 mg of dried mycelia, lyse with 100 μL of 0.1 M perchloric acid, grind, centrifuge at 5200 g at 4 °C for 30 min...

Embodiment 3

[0094] Example 3 Tryptamine affects the detection of ROS generation

[0095] The scattered male migratory locusts three days after eclosion were randomly divided into three groups on average, which were marked as the control group Ck-4d, the wild-type group MAA-4d and the recombinant mutant group KO-4d. The control group was not treated, and the wild The type group was infected with wild-type MAA, and the mutant group was infected with the MAA-KO screened in Example 1. After 4 days, the hemolymph of migratory locusts was taken in 500 μL of L15 medium, and rapidly centrifuged at 300 rpm, 4° C., for 10 Pour off the supernatant after 10 minutes, add 500 μL of L15 medium containing 0.1 μM Mitosox Red (red fluorescent probe), incubate at 37°C in the dark for 10 minutes, centrifuge to remove the supernatant, suspend the cells in L15 medium and put them on the machine.

[0096] Detector: Beckman CytoFLEX;

[0097] Detection channel: PE;

[0098] Collect 8000 cells and count the pro...

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Abstract

The invention provides recombinant broad-spectrum metarhizium; the recombinant broad-spectrum metarhizium down-regulates the expression of monoamine oxidase or does not express the monoamine oxidase,or the content of tryptamine in a recombinant broad-spectrum metarhizium body is higher than that of tryptamine in wild type broad-spectrum metarhizium; the recombinant broad-spectrum metarhizium is the strain per se, the progenies of the strain, the conidia produced by the strain, the mycelia produced by the strain, or any combination of the progenies, the conidia and the mycelia. A monoamine oxidase gene in the recombinant broad-spectrum metarhizium is knocked out, so that the concentration of tryptamine in a broad-spectrum metarhizium robertsii is significantly increased, the insecticidal efficiency is further obviously increased, and the half lethal time LT50 of the wild-type broad-spectrum metarhizium is shortened to 6.136+ / -0.488 days from 7.33+ / -0.445 days; furthermore, the recombinant broad-spectrum metarhizium is harmless to the environment, good in biological safety and non-toxic to human beings.

Description

technical field [0001] The invention relates to a transgenic bacterial strain and its preparation method and application, in particular to a recombinant broad-spectrum Metarhizium anisopliae capable of improving the insecticidal efficiency of the broad-spectrum Metarhizium anisopliae and its preparation method and application. Background technique [0002] Entomopathogenic fungi, compared with chemical insecticides, have the advantages of environmental friendliness, strong stress resistance, ability to spread in large quantities, and high selectivity, and are widely used as a class of biological pesticides. However, entomopathogenic fungi still have the shortcoming of long lethal time as insecticides. It is an important direction of current research to improve the effect of fungal insecticides by studying the pathogenic mechanism of fungi and transforming fungi by means of genetic engineering. E.g: [0003] 1. High expression of hydrolase genes secreted by fungi. Overexpr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80A01N63/04A01P7/04C12R1/645A01N63/30
CPCC12N9/0022C12N15/80C12Y104/03004A01N63/30
Inventor 王云丹童希文康乐
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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