A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

Abstract

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified. However, molecular mechanisms of most of these mutants remain to be explored. Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing (mw) mutation which exhibits exceedingly small wings during pupal and adult stages. Compared with the wild type silkworm, relative messenger RNA expression of Bm-app is significantly decreased in the u11 mutant strain which shows mw phenotype. A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity. Furthermore, clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant, demonstrating that Bm-app controls wing development in B. mori. Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins, indicating that it is required for cell mitosis and growth during wing development. We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B. mori.

Keywords: Bombyx mori; CRISPR/Cas9; minute wing; palmitoyltransferase; promoter.

Figure 1

Phenotypes of wild‐type (p50) and mw mutant (u11) strains. Pupal and moth stages of the wild‐type (A) and mw mutant (B) are shown. Red arrows indicate smaller and slightly curled wings in u11 mutant moths.

Figure 2

Bm‐mw mapping on Bombyx mori chromosome 22 and identification of candidate genes. (A) A linkage map based on BC1M genotyping was constructed for group 22. Five sequence‐tagged site (STS) markers and the Bm‐mw locus are shown above and distances between loci (cM) are shown below. (B) Scaffold map of chromosome 22, based on data from the SilkDB database. Orange boxes represent the assembled scaffolds, the names of which are given beneath. The STS markers used for genotyping are localized on the scaffold positions, as indicated by the dotted lines. The green box shows the candidate region linked to the mw locus. The enlarged candidate region is shown beneath along with the position on nscaf3056 of T2272 and terminal, which fix the candidate region. (C) Above is shown the coordinates of nscaf3056. The gray area represents the candidate region, which includes 15 predicted genes according to SilkDB. The directions of the transcription of these genes are represented by arrowheads. For convenience, they are named C17 to C27, C51 to C54 based on their positions. Detailed information for these 15 genes is given in Table S2 in the supplementary material.

Figure 3

Quantitative real‐time polymerase chain reaction analysis of the minute wing candidate genes. (A) Expression patterns of 15 candidate genes in the wing disc of wandering stage larvae. (B) Expression of seven genes between p50 and u11 strains. The asterisks (^***) indicate significant differences (P < 0.0001) compared with the relevant control with a two‐tailed t‐test. Error bars depict ± SEM. ND, not determined. NS, no significance.

Figure 4

Schematic diagram of single‐guide RNA (sgRNA) targeting sites. (A) Schematic diagram of sgRNA‐targeting sites. The boxes indicate the two exons of Bm‐app, and the black line represents the gene locus. The sgRNA targeting sites, S1 and S2, are located on the sense strand of exon‐1 and the sense strand of intron, respectively. The protospacer adjacent motif (PAM) sequence is shown in red. (B) Various types of mutagenesis induced by Cas9/sgRNA injection. The numbers in brackets in the middle of each sequence refer to the 1486 bp‐long interspace fragment that was found between the S1 and S2 sites. The red sequence indicates the PAM sequence. (C) Genomic polymerase chain reaction analyses revealed deletion mutation events in the mutants. The red arrowhead indicates the deleted region. (D) The transcript level of Bm‐app is down‐regulated significantly in Δapp animals. The asterisks (^***) indicate the significant differences (P < 0.0001) compared with the relevant control with a two‐tailed t‐test. Error bars depict ± SEM.

Figure 5

Mutants with abnormal wings induced by Cas9/single‐guide RNA injection. The mutants showed small and curly wing discs during the larval stage (A) and small wings during pupal (B) and adult stages (C, D) The red arrows indicated abnormal position.

Figure 6

Promoter verification in BmN and HK293T cell lines. (A) Putative 2 kb promoter sequences of Bm‐app in p50 and u11 strains were sub‐cloned into PXL‐p50‐EGFP or PXL‐u11‐EGFP plasmid separately to perform cell transfection by using the BmN cell lines. PXL‐IE1Dsred2‐EGFP was used as a control. All three transfected plasmids could drive DsRed2 expression (a–c) while only PXL‐p50‐EGFP could drive enhanced green fluorescent protein (EGFP) expression (a′–c′). (B) Bm‐app promoter activity was analyzed in p50 and u11stains by measuring the Firefly/Renilla ratio. Blank, non‐transfected cells; negative, pGL3‐Basic‐transfected cells; tested, cells transfected with Bm‐app promoter‐2.0 kb (2.0 kb), Bm‐app promoter‐1.5kb (1.5 kb), Bm‐app promoter‐1.0kb (1.0 kb) and Bm‐app promoter‐0.5kb (0.5 kb) in p50 and u11 stains. All data are representative of three independent experiments and expressed as the mean ± SEM. The asterisks (^**) indicate significant differences (^** P < 0.01) compared with the p50 stain.

Figure 7

Bm‐app mutagenesis affected genes expression of the Hippo pathway in Bombyx mori. Relative messenger RNA expressions of genes in the Hippo signalling pathway were determined by quantitative real‐time polymerase chain reaction in wild‐type (WT) and Δapp silkworms. The asterisks (^* or ^** or ^***) indicate significant differences (^* P < 0.05; ^** P < 0.01; ^*** P < 0.001) compared with the WT. Error bars depict ± SEM.