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Recombined insect molting hormone inactivation gene Bbsp::egt and insecticidal fungistatic agent thereof

A technology of ecdysone and gene, applied in the field of recombinant insect ecdysone inactivated gene Bbsp::egt and its insecticide fungicide, can solve the problem of interfering with host insect-related hormone levels, low infection efficiency of entomopathogenic fungi, and reducing insect immunity Resist ability and other issues, to achieve the effect of reducing immune resistance, enhancing insecticidal effect, and improving control effect

Active Publication Date: 2016-03-30
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention also provides that the recombinant insect ecdysone inactivation gene Bbsp:egt is applied to insecticide fungicides, interferes with the relevant hormone levels of host insects, and reduces the immune resistance of insects, thereby improving the insecticidal fungus Beauveria bassiana. Insecticidal efficacy, solving the problem of low infection efficiency of entomopathogenic fungi

Method used

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  • Recombined insect molting hormone inactivation gene Bbsp::egt and insecticidal fungistatic agent thereof
  • Recombined insect molting hormone inactivation gene Bbsp::egt and insecticidal fungistatic agent thereof
  • Recombined insect molting hormone inactivation gene Bbsp::egt and insecticidal fungistatic agent thereof

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Experimental program
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Embodiment 1

[0043] 1. Cloning and synthesis of Bbsp::egt gene

[0044] A signal peptide sequence of Beauveria bassiana chitinase was added to the front end of the LdMNPV virus egt gene, and XbaI restriction sites were added at both ends, and then artificially synthesized, and Bbsp::egt was cloned into the puc57-simple vector , obtained the correct cloning vector and named it puc57-simple-Bbsp::egt.

[0045] 2. Construction of overexpression vector

[0046] The Bbsp::egt fragment was constructed on the overexpression vector puc-bar-Pgpda by conventional molecular biology methods. First, XbaI single-digested puc57-simple-Bbsp::egt to obtain about 1.8kb Bbsp::egt target fragment, which was cloned into the linearized puc-bar-Pgpda vector (about 5.5 kb). The recombinant vector was transformed into Escherichia coli DH5α, and the orientation was screened with P1 / P2 primers (the P1 primer is located in the Pgpda region of the promoter, and the P2 primer is located inside the Bbsp::egt, so the ...

Embodiment 2

[0062] In order to elucidate the effect of high-level expression of Bbsp::egt gene on the virulence of Beauveria bassiana strain, the larvae of Mellonella mellonella was used as test insects for bioassay. Transformants F1, F2 and wild type (wt) were inoculated on PDA solid medium, cultured at a constant temperature of 26°C for 20 days, and the conidia were scraped and dispersed in 0.05% Tween 80 solution to obtain a concentration of 2×10 7 Spores / ml of spore suspension. Melonella mellonella was divided into 30 per plate, and each set of three plates was used as a parallel experiment. The 0.05% Tween 80 solution without spores was used as a negative control. The fungi were inoculated by body wall infection, that is, every 30 test insects were immersed in 30ml of spore suspension, and after 20s, the water was taken out for 6-7s. The survival rate of the test insects was counted every 12 hours after the infection.

[0063] After determination, it was found that the virulence of...

Embodiment 3

[0066] Data and observations from previous experiments indicated that Bbsp::egt was involved in the immune regulation of insects. In order to clarify the changes in the resistance of insects to pathogenic bacteria after inoculation with transformed strains, seven insect antibacterial-related genes were selected for detection of expression levels, namely: Ferritin, Gallerimycin, Cecropin, Gloverin, GST, PGRP-A, PGRP-B, β- actin is an internal reference gene. Among them, PGRP-A and PGRP-B encode peptidoglycan recognition proteins, which can be used as pattern recognition molecules to specifically recognize the conserved structure of invading pathogens. After pattern recognition, the immune system of insects will be activated; Gallerimycin, Cecropin and Gloverin encode three different antimicrobial peptides in insects; Ferritin and GST encode ferritin and glutathione sulfhydryl transferase, respectively. The former is a protein that stores and transports iron ions, and participat...

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Abstract

The invention relates to a recombined insect molting hormone inactivation gene Bbsp::egt and an insecticidal fungistatic agent thereof. The recombined insect molting hormone inactivation gene Bbsp::egt contains coding beauveria bassiana chitinase signal peptide sequences (Bbsp) and coding lymantria dispar nuclepolyhedrovirus (hereinafter referred to as LdMNPV) ecdysteroid uridine 5minute-diphosphonic acid-glucosyltransferase gene (egt), and has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. The molecular biological technique is adopted to shift into beauveria bassiana to obtain correct genetic transformation bacterial strains. The recombined Bbsp::egt bacterial strain obtained by the invention can disturb the normal operation of a hormone system in vivo of insects, and is beneficial to reducing immune resistance of insects on insecticidal fungi beauveria bassiana and accelerating knockout of fungus on hosts, so that the insecticidal fungi virulence is improved.

Description

technical field [0001] The present invention relates to a novel recombinant ecdysteroid uridine 5'-diphosphate-glucosyltransferase encoding gene Bbsp::egt, specifically containing a signal peptide encoding Beauveriabassiana chitinase (Bbsp) and a recombinant sequence encoding the gypsy moth nuclear polyhedrosis virus (LdMNPV) decidoid uridine 5'-diphosphate-glucosyltransferase gene (egt). [0002] The present invention relates to a fungal expression vector expressing the above-mentioned recombinant sequence. [0003] The present invention also relates to an insecticide and fungicide expressing the above-mentioned recombinant vector. Background technique [0004] Most of the chemical pesticides such as DDT and pyrethrins frequently used in traditional agricultural production will seriously change the structure of the ecosystem and be harmful to the human body, and even concentrate in the food chain, posing a potential threat to human health. Therefore, under this backgrou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/80C12N15/66A01N63/04A01P7/04
CPCA01N63/30C07K2319/02C12N9/1051C12N15/66C12N15/80C12Y204/01
Inventor 范艳华冯雪瑶裴炎金丹
Owner SOUTHWEST UNIV
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