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Recombinant metarhizium anisopliae and its use for promoting plant root growth

A technology of plant root system and metarhizium anisopliae, applied in the field of recombinant fungi and its preparation, can solve the problems of unreported plant root system growth, etc., and achieve the effect of promoting plant root system growth and good biological safety

Active Publication Date: 2021-06-15
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its use for promoting plant root growth has not been reported in the art

Method used

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  • Recombinant metarhizium anisopliae and its use for promoting plant root growth
  • Recombinant metarhizium anisopliae and its use for promoting plant root growth
  • Recombinant metarhizium anisopliae and its use for promoting plant root growth

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The preparation of the metarhizium anisopliae of embodiment 1 recombination broad-spectrum

[0024] 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.

[0025] 1. Knockout plasmid construction of MAA_03753

[0026] Primers MAA_03753Fs and MAA_03753Rs, MA...

Embodiment 2

[0058] Determination of tryptamine content in embodiment 2 recombinant broad-spectrum Roberts metarhizium anisopliae

[0059] 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 in a dark incubator at 28°C for 6 days, the mycelia were collected and washed 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, take the supernatant, and wash with Na 2 CO 3 Neutralize to make the pH value around 6, take the supernatant and filter it with a 0.22 μm microporous membrane, and set aside. The content of tryptamine in the supernatant was detected by HPLC.

[0060] HPLC detection:

[0061] Agilent 1100, G1315A fluorescence detector (FLD), chromatographic column is C18 column;

[0062] Mobile phas...

Embodiment 3

[0073] Embodiment 3 Recombinant broad-spectrum Roberts metarhizium anisopliae promotes plant root growth

[0074] Inoculate the wild-type MAA and the recombinant MAA-KO prepared in the above examples on PDA medium for culture, scrape the spores of MAA and MAA-KO respectively, add an appropriate amount of peanut oil to suspend the spores, vortex, and use a glass The spores were collected after filtering with silk floss and resuspended with peanut oil. Count using a cell counting plate under a microscope, and make a final concentration of 1 × 10 by resuspending and counting several times. 6 Spores / ml.

[0075] Arabidopsis thaliana was purified at 4 degrees and put on the medium (0.5×MSP21) after 4 days. The MSP21 medium does not contain nitrogen, and then put it into the climate box, keep the temperature at 21 degrees, the day-night ratio is 16 hours: 8 hours, and the culture plate is 30 degrees place. When the plant grows two leaves, add 2uL 5ug / uL locust protein extract to ...

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Abstract

The invention relates to a recombinant broad-spectrum metarhizium anisopliae and its use for promoting the growth of plant roots, belonging to the field of agricultural biotechnology. The recombinant broad-spectrum Metarhizium anisopliae expresses down-regulated monoamine oxidase or does not express monoamine oxidase, which can promote plant root growth.

Description

technical field [0001] The invention relates to a recombinant fungus and its preparation method and use, in particular to a recombinant broad-spectrum metarhizium anisopliae capable of increasing tryptamine production and its use for promoting plant root growth. Background technique [0002] Extensive use of chemical fertilizers in farmland can quickly provide the nutrients needed by crops at the initial stage, but it will lead to soil acidification, uneven fertility and groundwater pollution. Microbial fertilizers can replace part of chemical fertilizers to solve the problem of soil degradation caused by long-term application of chemical fertilizers. Microbial fertilizers can be divided into five categories according to the types of microorganisms: ① bacterial fertilizers (such as rhizobia fertilizers, nitrogen-fixing bacterial fertilizers, phosphorus-dissolving bacterial fertilizers, potassium-dissolving bacterial fertilizers, and photosynthetic bacterial fertilizers); ② a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C05F11/08C12R1/645
CPCC05F11/08C12N15/80C12N1/145C12R2001/645
Inventor 王云丹童希文康乐
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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