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CRISPR/Cas9‐mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis
Author(s) -
Dong XiaoTong,
Liao Hui,
Zhu GuanHeng,
Khuhro Sajjad Ali,
Ye ZhanFeng,
Yan Qi,
Dong ShuangLin
Publication year - 2019
Publication title -
insect science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 45
eISSN - 1744-7917
pISSN - 1672-9609
DOI - 10.1111/1744-7917.12544
Subject(s) - biology , sex pheromone , cas9 , chilo suppressalis , mutant , crispr , mutagenesis , microbiology and biotechnology , gene , genetics , botany , lepidoptera genitalia
Pheromone‐binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo . In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis , one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP‐single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target‐gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1‐sgRNA injected eggs and 19.5% for PBP3‐sgRNA injected eggs. Second, by an in‐crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA‐injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis , as well as a valuable reference in methodology for gene functional study in other genes and other moth species.