Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori
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ABSTRACT: A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-inding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.
Project description:Insect cuticle plays essential roles in multiple physiological functions. During molting and metamorphosis, tremendous changes occur in silkworm cuticles. Silkworm is a model of Lepidoptera insects; however, little is known about the stage expression profiles of genes in cuticles of silkworm. In the present study, we selected 16 developmental stages, ranging from day 1 of the first instar larvae to day 8 of pupae, to perform microarray-based expression profiles. The data told us that various functions and physiological pathways were activated in the cuticle. Moreover, the expression profiles of cuticular protein genes, as the important components of cuticle, were investigated. The current study provides important insights for the functional study of insect cuticle and the regulation of insect cuticular protein genes.
Project description:Insect cuticle plays essential roles in multiple physiological functions. During molting and metamorphosis, tremendous changes occur in silkworm cuticles. Silkworm is a model of Lepidoptera insects; however, little is known about the stage expression profiles of genes in cuticles of silkworm. In the present study, we selected 16 developmental stages, ranging from day 1 of the first instar larvae to day 8 of pupae, to perform microarray-based expression profiles. The data told us that various functions and physiological pathways were activated in the cuticle. Moreover, the expression profiles of cuticular protein genes, as the important components of cuticle, were investigated. The current study provides important insights for the functional study of insect cuticle and the regulation of insect cuticular protein genes. Transcription profiling experiments, 16 developmental stages (samples) were analyzed. Dual-channel experiments, with test samples labeled by Cy5 and common reference samples labeled by Cy3. Common reference sample was used for data normalization. One biological replicate. No dye-swaps.
Project description:The Dominant obese translucent (Obs) mutant of the silkworm results in a short and stout larval body, translucent phenotype, and abnormal pigmentation in the integument. The Obs mutant also displays difficulties in ecdysis and metamorphosis. In the present study, to gain an understanding of multiple Obs phenotypes, we investigated the phenotypes of Obs and performed a comparative analysis of the larval integument proteomes of Obs and normal silkworms. The phenotypic analysis revealed that the Obs larvae are indeed short and fat, and that chitin and uric acid content were lower but melanin content was higher in the Obs mutant. Proteomic analysis revealed that 244 proteins were significantly differentially expressed between Obs and normal silkworms, some of which are involved in uric acid metabolism and melanin pigmentation. Twenty-six proteins were annotated as cuticular proteins, including RR motif-rich cuticular proteins (CPR), glycine-rich cuticular protein (CPG), hypothetical cuticular protein (CPH), cuticular protein analogous to peritrophins (CPAP), and the chitin_bind_3 motif proteins, and accounted for over 84% of the abundance of the total significantly differentially-expressed proteins identified. Moreover, 22 of the 26 cuticular proteins were down-regulated in the Obs mutant. Comparative proteomic analysis suggested that the multiple phenotypes of the Obs mutant are related to changes in the expression of proteins that participate in cuticular formation, uric acid metabolism, and melanin pigmentation. These results also provide a reference for studying the gene responsible for the Obs mutant.
Project description:To investigate the function of small RNAs in cuticle biosynthesis in B. napus, we constructed four small RNA libraries from stem epidermis of wax-deficient and wild-type materials for sequencing. Subsequently, a total of 43,840,451 clean reads were generated and 24nt sequences represented the dominant percentage. Totally, 300 unique conserved miRNAs were identified and 8 of them showed differentially expressed. In addition, the expression of six novel miRNAs were also changed. After target prediction and function annotation, three miRNAs (aly-miR165a-5p, aly-miR408-5p, and novel-mir2) might influence cuticle morphogenesis not only by repressing the biosynthesis of cuticular substrates and components, but also blocking the transmembrane transport processes. The expression level of aly-miR408-5p and aly-miR165a-5p were increased in glossy materials examined by stem-loop qRT-PCR, while their predicted target genes (BnaC04g29170D, BnaA01g25630D, BnaA06g40560D) were down-regulated. These results suggested that miRNAs might influence cuticle biosynthesis by repressing cuticle-related genes expression in B. napus. Both the biosynthesis and secretion processes of cuticlular components might be influenced by miRNAs. These results will promote the study of post-transcriptional regulation mechanisms of cuticle biosynthesis in B. napus and provide new direction for further research.
2018-11-30 | GSE115073 | GEO
Project description:Analysis of silkworm larvae cuticular microbiomes
Project description:The epidermis of plants forms a protective barrier against biotic and abiotic stress. Little is known about how breaches in the epidermis are repaired, especially those of mature leaves. Here, we investigated wound healing in the mature leaves of Arabidopsis. We discover a novel wound protection mechanism comprising a multi-layered ligno-suberized barrier covered with cuticular wax. This barrier is formed by mesophyll cells that adopt an epidermal fate. This cell fate transition is regulated in two steps by ATML1, a key transcription factor in epidermal specification. First, mesophyll cells of protective layer 1 (P1), just beneath the wound, transition into epidermal cells, sealing the wound by depositing cuticle, a mechanism that involves signaling through ethylene and reactive oxygen species produced by RBOHE. This signaling also promotes P1 cell death, ensuring wax accumulation on the surface. Second, the underlying protective layer 2 (P2) undergoes ligno-suberization, driven by jasmonic acid and RBOHD, forming a cork-like layer on the leaf surface. ATML1 regulates this process in P2 as well. Thus, wound healing in mature leaves involves integration of ethylene and jasmonic acid signaling with ATML1-mediated epidermal cell specification to coordinate cell layer-specific functions, including cuticular wax formation and ligno-suberization. This novel protective mechanism also occurs in the leaves of tobacco and Capsella, suggesting it is widespread.
Project description:The present study reports the genetic and biochemical characterization of a dominant glossy mutant allele (BnaA. GL) in B. napus that results in a glossy phenotype. Results from transmission electron microscopy and scanning electron microscopy revealed the GL mutant exhibits reduced deposition of the cuticle layer, which was confirmed by a cuticular wax analysis. The wax compositional analysis revealed an increase in aldehydes but a severe decrease in alkanes, ketones and secondary alcohols. Genetic mapping narrowed the BnaA. GL gene to the end of A9 chromosome, where a gene homologous to ECERIFERUM1 (CER1) in Arabidopsis locates.<br><br>Then, we conducted a microarray analysis to find the differentially expressed genes between normal phenotype and glossy plants. Two comparisons were performed: wild type parent VS. the GL parent, and the bulked normal phenotype DH lines VS. the bulked glossy DH lines. The DH lines are generated from F1 plants of two parents, and RNA samples from three DH lines were combined to make a bulked sample for each phenotype. <br><br>Although no discernible mutation was apparent in the B. napus gene, this cDNA microarray chip assay revealed coordinated down regulation of genes encoding enzymes of the cuticular wax biosynthetic in the glossy mutant with BnCER1 being one of the most severely suppressed genes.
Project description:Verson’s glands are segmental pairs of dermal glands attached to the epidermis in lepidopteran larvae. They produce macromolecules during intermolt period and empty them during each molt. Morphological, histochemical, developmental, and protein analysis studies have been conducted to determine the functions of Verson’s glands. However, the exact role of Verson’s glands remains unclear. In our previous study, a strain of transgenic fall armyworm, Spdoptera frugiperda expressing green fluorescence protein (GFP) and Systemic RNA interference defective protein 1 (SID-1) from Caenorhabditis elegans was established to improve RNA interference (RNAi) efficiency. Unexpectedly, we found that GFP fluorescence was significantly brighter in Verson’s glands than in other tissues. Also, RNAi efficiency improved more in Verson’s glands than in other tissues. We took advantage of improved RNAi efficiency to explore the function of Verson’s glands. RNA-seq analysis revealed that genes highly expressed in Verson’s glands code for cuticular proteins, molting fluid proteins, hemolymph proteins, and antimicrobial peptides. Injection of dsRNA targeting essential genes interfered with Verson’s glands growth. These studies revealed that Verson’s glands contribute to hemolymph, cuticle, molting fluid, and immune response during molting. These studies also provide useful tools for future research in identifying the physiological role of Verson’s glands in lepidopteran insects.
Project description:The cuticles of arthropods, including aquatic crustaceans like Daphnia, provide an interface between the organism and its environment. Thus, the cuticle’s structure influences how the organism responds to and interacts with its surroundings. Here, we used label-free quantification proteomics to provide a proteome of the molted cuticle of Daphnia magna, which has long been a prominent subject of studies on ecology, evolution, and developmental biology, anddetected 278 high confidence proteins. Using protein sequence domain and functional enrichment analyses, we identified chitin-binding structural proteins and chitin modifying enzymes as most abundant protein groups in the cuticle proteome.Structural cuticular protein families showed a similar distribution to those found in other arthropods and indicated proteins responsible for the soft and flexible structure of the Daphnia cuticle . Finally, cuticle protein genes were clustered as tandem gene arrays in the Daphnia genome, indicating their importance for adaptation to environmental change. The cuticle proteome presented here will be a valuable resource to the Daphnia research community, informing investigations on diverse topics such as the genetic basis of interactions with predators and parasites.