Method for improving high-seepage property, oxidative stress resistance and toxicity of beauveria bassiana by utilizing genetic engineering

A technology of Beauveria bassiana and genetic engineering, applied in the field of improving fungal traits, can solve the problems of enhancing stress resistance and virulence of strains

Inactive Publication Date: 2013-06-26
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on enhancing the stress resistance and virulence of strains by destroying the gene encoding the penetrasome protein or changing the expression mode of the penetrame protein gene

Method used

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  • Method for improving high-seepage property, oxidative stress resistance and toxicity of beauveria bassiana by utilizing genetic engineering
  • Method for improving high-seepage property, oxidative stress resistance and toxicity of beauveria bassiana by utilizing genetic engineering
  • Method for improving high-seepage property, oxidative stress resistance and toxicity of beauveria bassiana by utilizing genetic engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Bbtmp1 gene cloning

[0038] Utilize seven kinds of endonucleases EcoR I, Xba I, Nco I, Bgl II, Xho I, Sca I and EcoR V to digest the Beauveria bassiana genome DNA, and link the corresponding linker sequences (with reference to Xiao Yuehua et al., 2002, Acta Genetica Sinica, 29(1):62-66). Then, primers were designed according to the EST sequence (SEQ ID NO.36) obtained by the inventors, and the upstream and downstream sequences of the EST were extended sequentially by using the YADE (Y-shaped adapter extension) method.

[0039] The primers for the extended part of the upstream sequence are:

[0040] First time YADE:

[0041] P1: 5'-AGACTGTGCTCAACGCCATG-3' (SEQ ID NO.1)

[0042] P2: 5'-AGGACCTGGGCATGATTCTC-3' (SEQ ID NO.2)

[0043] Second YADE:

[0044] P3: 5'-ATGGCGTTGAGCACAGTCTT-3' (SEQ ID NO.3)

[0045] P4: 5'-ATGAACCGGTGCGACTGCTC-3' (SEQ ID NO.4)

[0046] Third YADE:

[0047] P5: 5'-GCTGGAGTTGCCAAGTCTAT-3' (SEQ ID NO.5)

[0048] P6: 5'-AGCCCTATGCACACACTCT...

Embodiment 2

[0068] 1. Destruction of the Bbtmp1 gene of Beauveria bassiana by homologous recombination

[0069] The strategy for constructing the Bbtmp1 homologous recombination expression vector is as follows: the partial coding region of the Bbtmp1 gene is replaced by the expression element of the bar gene (SEQ ID NO.37). That is, the flanking sequences of Bbtmp1 are connected at both ends of the bar expression element to form a homologous recombination expression vector, which is introduced into Beauveria bassiana through genetic transformation, and the flanking sequences of Bbtmp1 connected on both sides of the vector and the homologous sequence in the genome of Beauveria bassiana Perform double exchange to replace part of the coding region (273bp) of Bbtmp1 to achieve the purpose of destroying the target gene (such as Figure 5 Shown in A), wherein, the gene sequence after the bar expression element sequence replaces the partial sequence of the Bbtmp1 gene coding region of Beauveria ...

Embodiment 3

[0102] 1. The effect of Bbtmp1 on hyperosmotic and oxidative stress tolerance of Beauveria bassiana

[0103] Collect fresh conidia cultured on SADY at 26°C for 12 days, and use sterilized 0.05% Tween-80 to prepare a concentration of 1×10 7 / ml of conidia suspension. Take 2.0 μl of conidia suspension and inoculate it in a solution containing 1.0M sorbitol, 0.8M NaCl and different concentrations of oxidant H 2 o 2 and vitamin K13 (menadione) Czapek-Dox agar plate (diameter is 90mm) center, in 26 ℃ constant temperature culture for 7 days, analyze Bbtmp1 destruction mutant (Δtmp1), Bbtmp1 reversion complementary transformant (Δtmp1: :tmp1), overexpression of Bbtmp1 transformants (over-Δtmp1), homologous recombination element random integration transformant control (control) and wild strain (WT) sensitivity to hyperosmotic and different oxidants. It was found that compared with the wild strain (WT), the Bbtmp1 disrupted mutant (Δtmp1) was less sensitive to hyperosmotic and oxida...

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Abstract

The invention provides a method for improving high-seepage property, oxidative stress resistance and toxicity of beauveria bassiana by utilizing genetic engineering. The method comprises the steps of: replacing partial sequences of Bbtmp1 gene coding regions of the beauveria bassiana by utilizing bar expression element sequences, and breaking Bbtmp1 genes of the beauveria bassiana, so as to obtain a mutant strain of the beauveria bassiana with high seepage property and the improved oxidative stress resistance and toxicity. The invention further provides a fungus pesticide containing the mutant strain of the beauveria bassiana and an application of the mutant strain of the beauveria bassiana in preparation of the fungus pesticide.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to the improvement of fungal traits by means of genetic engineering. Background technique [0002] Entomopathogenic fungi are a class of important entomopathogenic microorganisms, and are an important natural control factor for controlling insect populations in nature (Clarkson et al., 1996, Trends Microbiol., 4:197-203; Feng et al., 1994, Biocontrol Sci. Technol., 4: 3-34; Roberts et al., 2004, Adv. Appl. Microbiol., 54: 1-70). Among them, Beauveria (Beauveria), Metarhizium (Metarhizium), Isaria (Isaria), Verticillium (Verticillium) and other hyphosporium fungi are widely used in agricultural production due to their significant epidemic potential and production convenience. , forest and health pest biological control. Moreover, unlike bacterial and viral entomopathogenic microorganisms that infect insects through the digestive tract, entomopathogenic fungi are the only microorgani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/80C12N1/15A01N63/04A01P7/04C12R1/645
Inventor 张永军何张江罗志兵裴炎范艳华
Owner SOUTHWEST UNIV
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