Abstract

Vitelline membrane proteins (VMPs) are the main proteins that form the inner shell (vitelline membrane layer) of insect eggs and are an integral part of egg formation and embryo development. Here, we characterized the molecular structure and expression patterns of theVMP26gene and analyzed its reproductive functions in diamondback moth,Plutella xylostella(L.), a worldwide migratory pest of cruciferous plants. ThePxVMP26gene was shown to be a single exon gene that contained an open reading frame of 852 base pairs (bp) encoding 283 amino acids. Both qPCR and western blot analyses showed thatPxVMP26 was specifically expressed in female adults and was significantly highly expressed in the ovary. Further anatomical analysis indicated that the expression level ofPxVMP26in the ovarian tube with an incomplete yolk was significantly higher than that in the ovarian tube with a complete yolk. CRISPR/Cas9-inducedPxVMP26knockout successfully created two homozygous strains with 8- and 46-bp frameshift mutations. The expression deficiency of thePxVMP26 protein was detected in the mutant strains using immunofluorescence and western blot. No significant difference was found in the number of eggs laid within three days between wild and mutant individuals, but there was a lower egg hatchability. The loss of thePxVMP26gene changed the mean egg size, damaged the structure of the vitelline membrane, and increased the proportion of abnormal eggs due to water loss, resulting in egg collapse. This first analysis of the roles of theVMPgene in the oocyte formation and embryonic development ofP. xylostella, using CRISPR/Cas9 technology, provides a basis for screening new genetic control targets ofP. xylostella.Keywords:diamondback moth;egg formation;mutation;gene expression;embryonic development