- editing of pigment transporter genes in the swallowtail butterfly Papilio xuthus. - However, little is known about the copy numbers of pigment transporter genes in the butterfly genomes and about the roles of pigment transporters in the development of swallowtail butterflies.. - Results: Here, we have identified 56 ABC transporters and 58 Rab members in the genome of swallowtail butterfly Papilio xuthus. - Our results indicate that the disruption of each gene produced mutated phenotypes in the colors of larvae (cuticle, testis) and/or adult eyes in G0 individuals but have no effect on wing color. - Comparative transcriptomes revealed 606 ~ 772 differentially expressed genes (DEGs) in the mutants of four ABCG transporters and 1443 DEGs in the mutants of lightoid. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Our study provides new insights into the function of ABC transporters and small G-proteins in the morphological development of butterflies.. - For ABC transporters, ATP binding and hydro- lyzing in the nucleotide-binding domains (NBDs) is a necessary process to transport a wide spectrum of sub- strates (e.g., amino acids, sugars, heavy metal ions and conjugates, peptides, lipids, polysaccharides, xenobiotic and chemotherapeutic drugs) via the integral transmem- brane domains (TMDs) across lipid membranes [24, 25].. - melano- gaster, which are involved in the uptake of pigment. - precursors in ommochromes and pteridines pathways in the development of cells of Malpighian tubules and compound eyes [26–29]. - armi- gera) and verified to play an important role in the devel- opment of larval epidermis or/and adult eyes [3, 10].. - Among 33 Rab genes identified in the genome of D. - Here, we systematically identified potential ABC trans- porters and Rab protein family in the genome of P.. - lightoid) in fruit fly, in the development of P. - Combining comparative transcriptomics of mutants and wild-types, we provide new insights into the func- tion of ABC transporters and small G-proteins in the morphological development of swallowtail butterflies.. - We comprehensively identified copy number of ABC gene family in the genome of the swallowtail butterfly P.. - These data suggest ABCG may play a more important role in the evolution of diverse insects. - mori, and the orange boxs show the genes in the genome of D. - Four Px genes highlighted in grey in ABCG subfamily were selected to investigate their function in the development of P. - We identified 58 and 51 Rab members in the genomes of P. - Rab23 is involved in the regulation of the number and planar polarization of the adult cuticular hairs in Drosophila [46]. - mori, and the orange boxs show the gene in the genome of D. - Lightoid, highlighted in red in cluster D, was selected to investigate its function in the development of P. - Mutations in the white gene. - We also observed that the testis of the fifth-instar larval mutants showed part or complete disappearance of white external sheath and red follicular epithelium (Fig. - No changes in shape and color were observed in the pupa and adult wing (Add- itional file 2: Fig. - Mutations in the scarlet gene. - No mor- phological changes were observed in the injected G0 lar- vae, but 36.36% (four individuals: three females and one male) emerged adults of G0 showed abnormal eyes with mosaic stripes of white and black/red-brown (Table 1 and Fig. - Mutations in the brown and ok genes. - We observed that 22.86% of the fifth-instar larvae of G0 showed a translucent cuticle (Table 1 and Fig. - Mutations in the lightoid gene. - disappearance of V-shape white markings in the fourth- instar larvae of G0 (Fig. - All six G0 mutants of white (three 5th-instar larvae and three adults) showed the dis- ruption mutated rate) in all or part of target sites with numerous deletions (1 – 84 bp), inserts (1 – 30 bp) or substitutions in the targeted regions (Additional file 3: Fig. - Four G2 adult mutants of scarlet showed a deletion of 8 – 11 bp in the target site T_684 in all clones (Additional file 3: Fig. - Transcriptome profiling of the mutants. - Specifically, a homo- zygous 8-bp deletion was identified at the region of tar- get site T_684 in the transcripts of four investigated scarlet mutants of G2 (Additional file 1: Table S8. - The epidermal tissues of the fourth instar larvae (L4). - CRISPR/Cas9 gene-editing affect the expression of the genes, the expression level (Fragments per Kilobase Million, FPKM) of the exon involved with target sites were acquired by manually distinguishing the mutated reads and normal reads in the mutant samples (Fig. - b, d, e), suggesting that the normal transcripts were less transcribed after CRISPR/Cas9-induced mutations, thus leading to the down-expression of the five genes. - c Testes of the fifth instar larva. - The area with obviously morphological mutation in mutants and their corresponding part in wild-type were highlighted in red circle in the panels of (a) and (d) and in red square (b). - The epidermis of the fifth-instar larvae among mutants of white, brown, ok, and lightoid and their wild-types have and 1443 DEGs, re- spectively (Fig. - Table S9), suggesting some intersections in the expression profile of these transported-related genes. - We found 10 DEGs shared in the mutants of all four ABC transporter and another 30 DEGs shared among the fifth-instar larval mutants of four gene white, brown, ok and lightoid (Additional file 1: Table S9).. - GO enrichment ana- lysis show most DEGs in mutants of all five genes enriched in the molecular function categories and biological process (Fig. - 5 Brown and ok mutants showed morphological mutation in the fifth instar larva (L5). - DEGs of brown, ok, and white fifth-instar lar- val mutants enriched in the KEGG pathway of trans- porters. - We used CRISPR/Cas9-based mutagenesis to uncover the roles of four ABCGs (white, scarlet, ok, and brown) and one Rab member (lightoid) in the morphological de- velopment of swallowtails butterfly for the first time.. - White play a key role in the morphological development of both larvae (cuticle, testis) and adults (eye color), while other four transporters play an important role in larvae (cuticle: brown, ok, lightoid. - 6 Lightoid mutants showed morphological mutations in the fourth instar larvae (L4), the fifth instar larvae (L5) and the testis of L5. - 7 Expression level of the exons in which the target sites are located. - The brown and white form het- erodimer and transport the pteridines precursor into the pigment granules in the eye development of D. - 8 The number of differentially expressed genes (DEGs) among mutants of the five genes and their Venn diagram. - a The number of DEGs between mutants of the five edited genes and their wild-types. - b Venn diagram of DEGs of mutants of the five edited genes and their wild-types. - Another possibility is that the five edited genes do have result in the reduced transportation of kynurenine but other genes may also transport kynurenine into wings and rescue the low concentration of kynurenine, finally leading to unchanged color in their wings. - 9 The functional enrichment of Gene Ontology (GO) term and KEGG pathway for the differentially expressed genes (DEGs) which were down and up expressed in the mutated groups. - We noticed the mutants of all five genes have eight shared DEGs, sug- gesting some intersections in the expression profiles of these transported-related genes (Additional file 1: Table S9). - In insects, the ommochromes path- way is also the most important route for elimination of tryptophan metabolites, which are toxic in the presence of excessive quantities [63]. - DEGs and some enriched GO items were shared in the mutants of four ABCG members (white, scarlet, brown and ok) suggesting these homologs shared some com- mon molecular basis in different developmental stages of different mutants (Additional file 1: Table S9). - Among four ABC mutants, scarlet is eye mutants of G2 adult and other three (white, brown, ok) are cuticle mutants of the fifth-instar larvae. - on the other hand, similar genes were enriched in the pathway of transporters in the fifth-instar larval mutants of brown, ok, and white. - These results suggest that lightoid plays a role in signal trans- mission mainly through the phosphorylation cascade in the process of pigment transportation. - For the first time, we comprehensively identified copy number of ABC family (56) and Rab family (58) in the genome of the swallowtail butterfly P. - The results indicated that all these five genes play an im- portant role in the morphological development of larvae (cuticle and/or testis) and/or adults’ eye color, but have no effect on wing color. - Genomic DNA extraction and mutagenesis detection Part epidemic tissues of the fifth-instar larval mutants of four genes (white, brown, ok, lightoid) or the thorax and abdomen of adults (white, scarlet) and their correspond- ing wild types were dissected in phosphate buffer saline and then used to extract genomic DNA using TreliefT- MAnimal Genomic DNA Kit (TsingKe, China) following the manufacturer’s protocols. - Except some adult mutants of white gene, part tissues of the same individual for the mutants and wild-types are also used for RNA extraction as described in the following part. - Part epidemic tissues of the fifth-instar larvae for the mutants of four genes (white, brown, ok, lightoid) and wild types or the head tissues of adult for mutant of scarlet gene and wild types were dissected for RNA ex- traction and sequencing. - The quality of the reads was evaluated using FastQC (https://www.bioinformatics.. - Then we manually count the number of the mutated reads which cover in the target sites for each sample, calculate the expression level (FPKM) of mu- tated transcripts and normal transcript in CRISPR-in- dued mutated individuals for exons with target sites.. - Additional file 1: Table S1 Gene numbers in the subfamilies of ABC transporter in the genomes of Papilio xuthus, other 31 insects, other five arthropods and human. - Details of the 56 ABC transporters identified in the genome of Papilio xuthus. - Details of the Rab families identified in the genome of Papilio xuthus and Bombyx mori.. - The target of the CRISPR-edited locus and primer for genotyping sequences. - The mutations in the target regions from transcriptomics data. - Note that in the panels of B, C and E, incomplete shapes of hindwings were produced during flying. - NA, not applicable because mutation type did not appear in the se- quenced clones. - Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm, Bombyx mori. - Eye pigments in wild-type and eye-color mutant strains of the African malaria vector Anopheles gambiae. - Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum. - The ABCs of eye color in Tribolium castaneum: orthologs of the Drosophila white, scarlet, and brown genes. - A single-base deletion in an ABC transporter gene causes white eyes, white eggs, and translucent larval skin in the silkworm w-3(oe) mutant. - Functional analysis of the ABCs of eye color in Helicoverpa armigera with CRISPR/Cas9-induced mutations. - Mutational analysis of the traffic ATPase (ABC) transporters involved in uptake of eye pigment precursors in Drosophila melanogaster. - Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration. - RNA interference-mediated knockdown of eye coloration genes in the western tarnished plant bug (Lygus hesperus knight). - Identification of stage-specific larval camouflage associated genes in the swallowtail butterfly, Papilio xuthus. - Advances and perspectives in the application of CRISPR/Cas9 in insects. - definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. - Immune function of a Rab-related protein by modulating the JAK-STAT signaling pathway in the silkworm, Bombyx mori. - Drosophila Rab23 is involved in the regulation of the number and planar polarization of the adult Cuticular Hairs. - Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti. - Molecular characterization of eye pigmentation-related ABC transporter genes in the ladybird beetle Harmonia axyridis reveals striking gene duplication of the white gene. - Bombyx ortholog of the Drosophila eye color gene brown controls riboflavin transport in Malpighian tubules. - Annotation and expression of carboxylesterases in the silkworm, Bombyx mori. - Systemic organ wasting induced by localized expression of the secreted insulin/IGF antagonist ImpL2. - Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of notch signaling by the JAK/STAT pathway. - Genome editing in the butterfly type-species Papilio machaon. - Genome-wide identification and characterization of ATP-binding cassette transporters in the silkworm, Bombyx mori
Xem thử không khả dụng, vui lòng xem tại trang nguồn hoặc xem
Tóm tắt