Vitelline Membrane Protein 26 Mutagenesis, Using CRISPR/Cas9, Results in Egg Collapse in Plutella xylostella

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 the VMP26 gene and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a worldwide migratory pest of cruciferous plants. The PxVMP26 gene 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 that PxVMP26 was specifically expressed in female adults and was significantly highly expressed in the ovary. Further anatomical analysis indicated that the expression level of PxVMP26 in the ovarian tube with an incomplete yolk was significantly higher than that in the ovarian tube with a complete yolk. CRISPR/Cas9-induced PxVMP26 knockout successfully created two homozygous strains with 8- and 46-bp frameshift mutations. The expression deficiency of the PxVMP26 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 the PxVMP26 gene 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 the VMP gene in the oocyte formation and embryonic development of P. xylostella, using CRISPR/Cas9 technology, provides a basis for screening new genetic control targets of P. xylostella.

Keywords: diamondback moth; egg formation; embryonic development; gene expression; mutation.

Figure 1

Phylogenetic relationship of PxVMP26 and the small molecular VMPs of other Lepidoptera species. The phylogenetic tree was constructed by MEGA 7 using the neighbor joining method with 1000 bootstrap replicates. The VMP26 in P. xylostella is indicated by the red star.

Figure 2

The developmental and tissue-specific expression profiles of the PxVMP26 in P. xylostella. (A–C) The expression profiles of PxVMP26 transcripts were analyzed by qRT-PCR; the mRNA level was normalized to ribosomal protein genes L32 (RPL32), L8 (RPL8), and elongation factor 1 alpha (EF-1α). Data shown as mean ± SE representing three biological replicates. Different letters on the bars indicate significant differences (p < 0.05) using LSD’s multiple range test. (D) The expression profiles of PxVMP26 protein were analyzed by western blot. The proteins from different stage (30 ug) or tissue (20 ug) was separated using 15% SDS-PAGE, and tubulin was used as the internal reference. L1–4, 1–4 instar larvae; PF/PM-1/2/3, male and female pupae 1/2/3 d; F/M-1/2/3, male and female adults 1/2/3 d; no full, ovarian tube with incomplete yolk deposition; full, ovarian tube with complete yolk deposition. The VMP26 protein is indicated by the red arrows.

Figure 3

Sequencing and identification of the mutant genotypes of PxVMP26 based on CRISPR/Cas9. (A) Representative sequencing maps of PCR products from wild type WT and G₀ adults with mutation at the target site, which are highlighted in the red boxes. (B) The mutant types of PxVMP26 gene in G₁ generation. The mutant sites are indicated by the red boxes. The deleted bases are displayed with dashed lines, and the inserted bases are shown in the red boxes. The numbers of mutant bases are demonstrated at the right of each allele (–, deletion; +, insertion). Eight bp mutation, 5-base insertion and 3-base deletion; 40 bp mutation, 40-base deletion; 46 bp mutation, 1-base insertion and 45-base deletion.

Figure 4

Homozygous mutation types of the PxVMP26 gene. (A) Eight bp mutation (5-base insertion and 3-base deletion) and (B) 46 bp mutation (1-base insertion and 45-base deletion) are highlighted with red box.

Figure 5

The mutation efficiency of PxVMP26 induced by CRISPR/Cas9. (A) The transcription levels of PxVMP26 were analyzed by qRT-PCR; the mRNA level was normalized to ribosomal protein L32 (RIBP). Data shown as mean ± SE represented with three biological replicates. Different letters on the bars indicate significant differences (p < 0.05) using LSD’s multiple range test. (B) The expression profiles of PxVMP26 protein were analyzed by western blot; 30 ug of protein was separated using 15% SDS-PAGE, and tubulin was used as the internal reference. The VMP26 protein is indicated by the red arrows. (C) The freshly dissected ovaries from newly emerged Mut and WT females were treated with the PxVMP26 polyclonal antibody and Alex Fluor Plus 594-conjugated secondary antibody (goat anti-rabbit) (red) and stained with DAPI Fluoromout-G^TM for DNA (blue); bar = 50 um.

Figure 6

The fecundity and egg size of P. xylostella after PxVMP26 knockout. (A) The total number of laid egg. (B) Hatching rate. (C) Egg length. (D) Egg width. Data shown as mean ± SE. Different letters on the bars indicated significant differences (p < 0.05) using LSD’s multiple range test. WT × WT, WT^♀ mated with WT^♂; Mut-8 × 8, Mut-8^♀ mated with Mut-8^♂; Mut-46 × 46, Mut-46^♀ mated with Mut-46^♂; Mut-8 × WT, Mut-8^♀ mated with WT^♂; Mut-46 × WT, Mut-46^♀ mated with WT^♂.

Figure 7

(A) The vitelline deposition of P. xylostella eggs after PxVMP26 knockout. WT × WT, WT^♀ mated with WT^♂; Mut-8 × 8, Mut-8^♀ mated with Mut-8^♂; Mut-46 × 46, Mut-46^♀ mated with Mut-46^♂. (B) Morphologies of eggs were observed by digital microscope VHX-2000C (KEYENCE, Japan), bar = 100 μm in WT and bar = 200 μm in Mut. (C) Rate of abnormality of the eggs. Data shown as mean ± SE. NS indicates no significant difference (p > 0.05), and different letters on the bars indicate significant differences (p < 0.05), using LSD’s multiple range test.

Figure 8

Microscopic structure of the P. xylostella vitelline membrane after PxVMP26 knockout. (A) WT. (B) Mut-8. (C) Mut-46. The ultrastructure of vitelline membrane was observed by transmission electron microscope (TME) (H-7650, HITACHI), bar = 500 nm. Oo, oocyte; FC, follicular cell; VM, vitelline membrane (electron-dense region); ex, exochorion; ie, inner-endochorion; oe, outer-endochorion; c, columnar layer.