Recombinant fungus targeting expression of silent pest-pattern recognition protein GNBP3 gene and application of recombinant fungus in pest control

A technology of pattern recognition receptors and recombinant bacteria, applied in applications, fungi, genetic engineering, etc., can solve problems such as the identification of escaping and invading microorganisms, shorten the death time, improve the use efficiency and range of use, and increase the mortality rate Effect

Active Publication Date: 2020-02-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no microbial pesticides that can escape their body's recognition of invading microorganisms

Method used

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  • Recombinant fungus targeting expression of silent pest-pattern recognition protein GNBP3 gene and application of recombinant fungus in pest control
  • Recombinant fungus targeting expression of silent pest-pattern recognition protein GNBP3 gene and application of recombinant fungus in pest control
  • Recombinant fungus targeting expression of silent pest-pattern recognition protein GNBP3 gene and application of recombinant fungus in pest control

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Cloning of Example 1 Plutella xylostella gram-negative bacteria binding protein gene GNBP3 cDNA sequence

[0058] 1. Experimental method

[0059] 1. Unigene amplification of GNBP3 gene

[0060] According to the Plutella xylostella transcriptome database determined in the laboratory, the GNBP3 Unigene sequence was obtained through bioinformatics analysis and screening. In order to ensure the correctness of the sequence GNBP3, a pair of Unigene primers (GNBP3FUni and GNBP3RUni) were designed. The primer sequences are shown in Table 1.

[0061] Using Plutella xylostella cDNA as a template, the Unigene sequence was amplified by PCR. After the product was recovered, T cloned, and after the recombinant plasmid was extracted, pMD18-T-GNBP3 was digested with PstI and EcoR I to identify the recombinant plasmid pMD18-T-GNBP3. The correct recombinant plasmid pMD18-T-GNBP3 was sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with...

Embodiment 2

[0067] The spatio-temporal expression pattern of embodiment 2 diamondback moth GNBP3 gene

[0068] 1. Experimental method

[0069] Samples of different developmental stages of healthy Plutella xylostella were selected, including eggs, larvae, prepupa, pupae, and adults. Healthy 4th instar Plutella xylostella larvae were selected and dissected on ice to collect six tissue samples including head, hemolymph, midgut, Malpighian tubules, fat body and epidermis.

[0070] Extract total RNA and synthesize first-strand cDNA from all samples. According to the full-length cDNA sequence of Plutella xylostella GNBP3 and the cDNA sequence of RPS13 gene of Plutella xylostella, two pairs of fluorescent quantitative PCR primers were designed respectively. GNBP3 quantitative primers are GNBP3 primers GNBP3-qF and GNBP3-qR, Rps13 primers are Rps13-qF and Rps13-qR, the primer sequences are shown in Table 1, RT-qPCR reaction system is 25 μL: ddH2O 8.5 μL, SYBR Premix ExTaqⅡ 12.5 μL, upstream Pr...

Embodiment 3

[0073] Example 3 Sensitivity Detection of Plutella xylostella GNBP3 Gene to Fungi

[0074] 1. Experimental method

[0075]Three kinds of entomogenous fungi preserved in the laboratory, Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea were transferred to PDA plates, and then placed in a 25°C incubator ( 12L:12D) for 7 days. After sporulation, add 20mL 0.05% Tween-80 solution on the plate, gently scrape the spores on the plate with an inoculation loop, pour the prepared solution into a beaker, and stir with a magnetic stirrer for 30 minutes. After the spores are completely dispersed, filter the bacterial liquid with double layers of medical gauze to obtain a spore suspension. Use a hemocytometer to count the concentration of spores in the mother liquor, and use 0.05% Tween-80 solution to make 1.0×10 8 CFU / mL of spore suspension for later use.

[0076] The larvae of Plutella xylostella xylostella on the first day of the 4th instar were selected and treated w...

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Abstract

The invention discloses a recombinant fungus targeting expression of silent pest-pattern recognition protein GNBP3 gene and application of the recombinant fungus in pest control. The invention constructs recombinant beauveria bassiana capable of inhibiting or blocking GNBP3 gene transcription. Therefore, when the recombinant beauveria bassiana infects pests, the expression of antibacterial peptidein the pests is remarkably reduced, the signal transduction of pest recognition immune entomogenous fungi is weakened or interrupted, and the pests are more easily infected with pathogenic microorganisms such as the entomogenous fungi to die. The recombinant beauveria bassiana significantly improves the death rate of the pests, shortens the death time, and has concentration dependence. The recombinant beauveria bassiana is combined with a microbial insecticide bacillus thuringiensis Bt or serratia marcescens so as to significantly improve the death rate and death time of pests, it is indicated that the recombinant beauveria bassiana is a very good synergist of the microbial insecticide, does not improve the killing effect, widens the insecticidal spectrum, reduces the drug resistance of the microbial insecticide, and is an environment-friendly microbial insecticide.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a recombinant fungus targeting the expression of the GNBP3 gene for silencing the pest pattern recognition protein and its application in pest control. Background technique [0002] There are about 1.5 million species of insects that have been scientifically recorded in the world, accounting for about half of the biological species on the earth, and about 45% of them use the leaves of green plants as food to harm plants. The insects that eat crops will seriously damage hard crops. Variety yield, which in turn affects farmers' income. [0003] At present, the control of agricultural pests mainly relies on chemical pesticides, but the extensive use of chemical pesticides has caused shocking environmental pollution, which has caused serious harm to human health, and at the same time increased the resistance of insects. Therefore, it is necessary to choose effective and non-pollutin...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/80C12N1/15C12Q1/6888A01N63/30A01P7/04C12R1/645
CPCC07K14/43563C12N15/80C12Q1/6888C12Q2600/136C12Q2600/158
Inventor 许小霞金丰良洪莹莹曹苗苗李树忠曾路张雨欣花艳艳
Owner SOUTH CHINA AGRI UNIV
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