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
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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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