Recombinant bacterium targeted to silence plutella xylostella GNBP2 gene and application thereof in pest control

A Plutella xylostella, recombinant bacteria technology, applied in application, bacteria, genetic engineering and other directions, to achieve the effect of improving mortality

Active Publication Date: 2020-01-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] There is currently a lack of a method for controlling insects by silencing Gram-negative bacterial binding proteins

Method used

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  • Recombinant bacterium targeted to silence plutella xylostella GNBP2 gene and application thereof in pest control
  • Recombinant bacterium targeted to silence plutella xylostella GNBP2 gene and application thereof in pest control
  • Recombinant bacterium targeted to silence plutella xylostella GNBP2 gene and application thereof in pest control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Cloning of cDNA sequence of Plutella xylostella gram-negative bacteria binding protein gene GNBP2

[0067] 1. Experimental method

[0068] 1. Unigene amplification of GNBP2 gene

[0069] According to the laboratory determination of the diamondback moth midgut transcriptome database. Using the total RNA of Plutella xylostella as a template, the first cDNA was obtained by reverse transcription; primers (GNBP2F and GNBP2R) were designed with Unigene sequence, the Unigene sequence was amplified by PCR, and the product was ligated with pMD18-T vector and transformed into E. coli competent cell DH5α , Screen the correct streaked single colony, inoculate it into LB liquid medium containing antibiotics, and perform double enzyme digestion with PstI and EcoRI after the plasmid is extracted by the plasmid extraction kit, and send the recombinant plasmid identified by the enzyme digestion to Guangzhou Aiji Bio Technology Co., Ltd. sequencing. The sequencing results were comp...

Embodiment 2

[0075] Example 2 Temporal and spatial expression pattern of GNBP2 gene of Plutella xylostella

[0076] 1. Experimental method

[0077] Select healthy samples of diamondback moths of different developmental stages, including eggs, 1st instar larvae, 2nd instar larvae, 3rd instar larvae, 4th instar larvae, pre-pupa, pupae and adult samples, and dissect the 4th instar diamondback moth on ice. Collect 6 tissue samples of head, hemolymph, midgut, martens tube, fat body, and epidermis, and extract the synthesis of total RNA and cDNA first strand. Based on the full-length cDNA sequence of Plutella xylostella GNBP2 and RPS13 gene sequence, a pair of fluorescent quantitative PCR primers were designed respectively (the primers are: GNBP2-qRT-F and GNBP2-qRT-R, RPS13-qRT-F and RPS13-qRT- R), perform real-time fluorescent quantitative PCR detection.

[0078] 2. Experimental results

[0079] The result is figure 2 As shown, the GNBP2 gene of Plutella xylostella is expressed in all instars of Pl...

Embodiment 3

[0080] Example 3 Sensitivity detection of Plutella xylostella GNBP2 to different microorganisms

[0081] 1. Experimental method

[0082] Inoculate fresh glycerol bacteria of Escherichia coli (E.coli), Staphylococcus aureus (S.aureus), S. marcescens (S. marcescens) and B. thuringiensis (B. thuringiensis) preserved in the laboratory. Cultivate the seed bacteria overnight at 37°C, 240rpm in the LB medium without any resistance, and inoculate the seed bacteria 1:100 into the fresh LB medium without any resistance, 37°C, 240rpm, OD 600 =0.8, 12000g centrifugation for 10 minutes to collect the bacteria, wash the bacteria twice with PBS, count the concentration of the mother liquid bacteria with a hemocytometer, and then dilute to 1.0×10 with PBS 8 CFU / mL is reserved.

[0083] Transferring B. bassiana and I. fumosorosea stored in the laboratory to a PDA plate, and then placing them in an incubator (12L:12D) at 25°C for 7 days. After sporulation, add 20 mL of 0.05% Tween-80 solution to the ...

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Abstract

The invention discloses a recombinant bacterium targeted to silence a plutella xylostella GNBP2 gene and application thereof in pest control. Specifically, the nucleotide sequence of plutella xylostella pattern recognition receptor protein gene GNBP2 is shown in SEQ ID NO:1; the amino acid sequence of the plutella xylostella pattern recognition receptor protein GNBP2 is shown in SEQ ID NO:2. According to the recombinant bacterium, it is found for the first time that a pest immune system is the negative bacteria-binding protein GNBP2 gene in a target system, the expression of the gram-negativebacteria-binding protein GNBP2 and downstream antibacterial peptide gene can be effectively reduced, and the sensibility of the plutella xylostella to the gram-negative bacteria, gram-positive bacteria, and fungi is greatly improved, and the mortality is further increased. According to the recombinant bacterium, a theoretical foundation is laid for the development of human and livestock safe immunosuppressants, a novel theory is provided for the biological control of pests, and novel strategies and novel approaches of pest control are provided for green agricultural production and sustainabledevelopment in China.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more specifically, to a recombinant bacterium targeted to silence the diamondback moth GNBP2 gene and its application in pest control. Background technique [0002] There are many kinds of insects with different shapes. They belong to arthropods among invertebrates. They are the most numerous animal group on earth. They account for more than 50% of all biological species (including bacteria, fungi, and viruses). They are almost everywhere. Every corner of the world. Has a high degree of adaptability and effective defense mechanism. [0003] Insect immune system is different from mammals. It does not have B and T lymphocyte system and therefore does not have the highly specific acquired immune system of higher animals. However, insects have a highly effective innate immune system that produces an immune response to non-specific factors. Studies have shown that the immune response of insects is...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/705C12N15/113C12N15/70C12N1/21A01N57/16A01P7/04C12R1/19
CPCA01N57/16C07K14/705C12N15/1138C12N15/70C12N2310/14
Inventor 许小霞金丰良洪莹莹鞠雯燕曾路张雨欣花艳艳曹苗苗
Owner SOUTH CHINA AGRI UNIV
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