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A transgenic strain of Metarhizium anisopliae and its preparation method and application

A technology of Metarhizium anisopliae and transgenic technology is applied in the field of Metarhizium anisopliae and its preparation, which can solve the problems of effective control of the difficult German cockroach population, weak virulence, slow killing effect of entomopathogenic fungi and insecticides, and the like. Improved biosafety, strong reproducibility, and improved insecticidal virulence

Inactive Publication Date: 2017-05-24
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the process of biological control of the German cockroach, the cockroach killing box (Biopath poison bait) with the entomopathogenic fungus Metarhizium anisopliae ESC-1 as the main component was relatively successful and achieved certain results in practical applications, but the entomopathogenic fungus The killing effect of insecticides is usually slow and the toxicity is weak, so it is difficult to achieve effective control of the German cockroach population in a short period of time

Method used

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  • A transgenic strain of Metarhizium anisopliae and its preparation method and application
  • A transgenic strain of Metarhizium anisopliae and its preparation method and application
  • A transgenic strain of Metarhizium anisopliae and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A preparation method of the transgenic bacterial strain of Metarhizium anisopliae:

[0040] First, according to the sequence of the subtilisin-like Pr1a gene whose accession number is EU526905 on GenBank, PCR primers are designed, and the primer sequences are as follows:

[0041] 5'-aacatatgcatctgtctgctcttct-3', as shown in SEQ.ID.NO 1 in the sequence listing;

[0042] 5'-ggcctagttaggcaccgttgtaggcaa-3', as shown in SEQ.ID.NO 2 in the sequence listing.

[0043] According to the above primer sequences, using the genomic DNA of Metarhizium anisopliae strain as a template, the Pr1a gene fragment was amplified, the fragment length was 1378bp, and the sequence fragment was shown in SEQ.ID.NO 3 in the sequence table:

[0044]Secondly, using Pgpd as a strong promoter, TTrpC as a terminator, and Basta as a screening marker, the fusion PCR method is used to construct an overexpression cassette. The structural diagram of the constructed overexpression cassette is as follows figu...

Embodiment 2

[0083] Determination of prla protease activity:

[0084]With reference to the method (1987) of people such as St.Leger, utilize specificity short peptide substrate SUC-Ala-Ala-Pro-Phe-pNA (Sigma) to carry out Prl protease activity assay, take starting strain Metarhizium anisopliae as contrast, The recombinant strain Tp1 expressing prla protease prepared in Example 1 was used as a sample, and its prla protease activity in the cicada slough-inducing medium and non-inducing medium (LB medium) for 50 hours was measured respectively.

[0085] Cicada slough medium formula (g / L): cicada slough powder 2.0g, KH 2 PO 4 0.2g, MgSO 4 0.2g, agar powder 2g, pH8.0;

[0086] LB medium (g / L): peptone 10g, yeast extract 5g, NaCl 10g, agar powder 1.5g, pH 7.2.

[0087] The results of the enzyme activity test showed that: under non-inducing conditions, the protease activity of the starting strain was not detected, and the specific enzyme activity of the recombinant strain was about 18U / mg; un...

Embodiment 3

[0089] Virulence assay of strains:

[0090] The recombinant strain Tp1 of Metarhizium anisopliae prepared in Example 1 was dispersed in commercially available cottonseed oil, and the final spore concentration was prepared to be 1×10 8 pc / mL oil suspension, the starting strain of Metarhizium anisopliae was used as the control. Select male cockroach adults with the same weight after 2-day eclosion as test insects from the culture tank, use a micropipette to take 1 μL of oil suspension to inoculate the cockroach under the pronotum, put it into the culture tank, and adjust the temperature to 26-28 °C , relative humidity 50-60%, photoperiod 16hL / 8hD. Set cottonseed oil blank control. 50 worms were treated each time, and repeated three times. Fresh mouse food was replaced every day, and the number of dead insects in each treatment was regularly observed and recorded every day. Lightly touching the insect body with tweezers, and the feet and wings of the insect did not move were c...

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Abstract

The invention discloses a metarhizium anisopliae transgenic strain as well as a preparation method and an application thereof. The strain is a recombinant engineering bacterium constructed by fusion of metarhizium anisopliae genes and exogenous Prla (subtilisin-like protease A) genes. The metarhizium anisopliae transgenic strain takes metarhizium anisopliae as the basis, takes Pgpd as a strong promoter, takes TTrpC as a terminator and takes Basta as a selection marker, a Prla overexpression box is constructed with a PCR fusion method, then a fungal protoplast is prepared, genetic engineering transformation is conducted with a CaCl2-PEG method, and the overexpression box is inserted in a metarhizium anisopliae gene group randomly for overexpression. The metarhizium anisopliae transgenic strain is good in German cockroach killing effect, higher in toxicity and capable of effectively controlling German cockroach populations in a short time.

Description

technical field [0001] The invention relates to a transgenic strain of Metarhizium anisopliae, a preparation method and application thereof, in particular to a Metarhizium anisopliae which contains an exogenous subtilisin-like protease gene obtained through gene recombination, a preparation method and application thereof. Background technique [0002] German cockroach, commonly known as cockroach, is one of the common worldwide sanitary pests. It can carry a variety of bacteria, viruses and parasite eggs, leading to the spread of diseases. Its secretions or dead worms can also cause severe body allergies. Since it was introduced into China, because of its strong adaptability to habitats, rapid reproduction, and easy resistance to chemical insecticides, its damage to our country has become increasingly serious. [0003] At present, the control of German cockroaches is still dominated by chemical control, but the long-term and large-scale use of highly toxic and high-residue c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12N1/15A01N63/04A01P7/04C12R1/645
Inventor 张凡廖慧萍张洁刘新祝金宝卢敏
Owner SHANDONG NORMAL UNIV
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