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Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis.

Ou J, Deng HM, Zheng SC, Huang LH, Feng QL, Liu L - BMC Genomics (2014)

Bottom Line: Seventeen transcription factors were significantly up-regulated.Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated.These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Developmental Entomology, Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China. qlfeng@scnu.edu.cn.

ABSTRACT

Background: Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed.

Results: In total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated.

Conclusions: Extensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.

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Related in: MedlinePlus

qRT-PCR results of expression of the chitin degradation related genes. The expression patterns of two chitin degradation genes in the wing disc at six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3) The relative expression levels were normalized to the Bmβ-actin levels. (A) Bm_nscaf2993_229 (chitin deacetylase 4). (B) Bm_nscaf3031_201 (β-N-acetylglucosaminidase 1 precursor). The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR. Different letters indicate statistical significance (p < 0.05).
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Fig6: qRT-PCR results of expression of the chitin degradation related genes. The expression patterns of two chitin degradation genes in the wing disc at six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3) The relative expression levels were normalized to the Bmβ-actin levels. (A) Bm_nscaf2993_229 (chitin deacetylase 4). (B) Bm_nscaf3031_201 (β-N-acetylglucosaminidase 1 precursor). The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR. Different letters indicate statistical significance (p < 0.05).

Mentions: To study the dynamic of chitin in wing disc during the pupal wing transition, the genes that are involved in chitin degradation and synthesis pathways were investigated. Most of the genes that are related to chitin degradation were significantly up-regulated at the PP stage and the genes that are related to chitin synthesis were also up-regulated at the PP or P0 stage (Tables 5 and6). Two critical chitin degradation related genes [Bm_nscaf2993_299 (chitin deacetylase 4) and Bm_nscaf3031_201 (β-N-acetylglucosaminidase 1 precursor)] were significantly up-regulated at the PP stage, then down-regulated at the P0 stage (Figure 6A, B), which were consistent with the results of RNA-Seq (Table 5). Chitin synthesis related genes including Bm_nscaf3027_063 (hexokinase), Bm_nscaf2887_059 (glutamine:F-6-P aminotransferase), Bm_nscaf2823_132 (glucosamine-6-P-N-acetyltransferase), Bm_nscaf2589_266 (chitin synthase A) were significantly up-regulated at the PP or P0 stage, and Bm_nscaf2589_269 (chitin synthase B) was significantly down-regulated from L5D3 to P3 stage (Figure 7A-E), which were generally consistent with the results of RNA-Seq (Table 6). The assembled nucleotide sequences of transcripts (RNA-seq ID) in Tables 5 and6 were provided in Additional files7 and8, respectively. Primers for qRT-PCR were provided in Additional file6.Table 5


Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis.

Ou J, Deng HM, Zheng SC, Huang LH, Feng QL, Liu L - BMC Genomics (2014)

qRT-PCR results of expression of the chitin degradation related genes. The expression patterns of two chitin degradation genes in the wing disc at six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3) The relative expression levels were normalized to the Bmβ-actin levels. (A) Bm_nscaf2993_229 (chitin deacetylase 4). (B) Bm_nscaf3031_201 (β-N-acetylglucosaminidase 1 precursor). The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR. Different letters indicate statistical significance (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196006&req=5

Fig6: qRT-PCR results of expression of the chitin degradation related genes. The expression patterns of two chitin degradation genes in the wing disc at six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3) The relative expression levels were normalized to the Bmβ-actin levels. (A) Bm_nscaf2993_229 (chitin deacetylase 4). (B) Bm_nscaf3031_201 (β-N-acetylglucosaminidase 1 precursor). The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR. Different letters indicate statistical significance (p < 0.05).
Mentions: To study the dynamic of chitin in wing disc during the pupal wing transition, the genes that are involved in chitin degradation and synthesis pathways were investigated. Most of the genes that are related to chitin degradation were significantly up-regulated at the PP stage and the genes that are related to chitin synthesis were also up-regulated at the PP or P0 stage (Tables 5 and6). Two critical chitin degradation related genes [Bm_nscaf2993_299 (chitin deacetylase 4) and Bm_nscaf3031_201 (β-N-acetylglucosaminidase 1 precursor)] were significantly up-regulated at the PP stage, then down-regulated at the P0 stage (Figure 6A, B), which were consistent with the results of RNA-Seq (Table 5). Chitin synthesis related genes including Bm_nscaf3027_063 (hexokinase), Bm_nscaf2887_059 (glutamine:F-6-P aminotransferase), Bm_nscaf2823_132 (glucosamine-6-P-N-acetyltransferase), Bm_nscaf2589_266 (chitin synthase A) were significantly up-regulated at the PP or P0 stage, and Bm_nscaf2589_269 (chitin synthase B) was significantly down-regulated from L5D3 to P3 stage (Figure 7A-E), which were generally consistent with the results of RNA-Seq (Table 6). The assembled nucleotide sequences of transcripts (RNA-seq ID) in Tables 5 and6 were provided in Additional files7 and8, respectively. Primers for qRT-PCR were provided in Additional file6.Table 5

Bottom Line: Seventeen transcription factors were significantly up-regulated.Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated.These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Developmental Entomology, Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China. qlfeng@scnu.edu.cn.

ABSTRACT

Background: Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed.

Results: In total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated.

Conclusions: Extensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.

Show MeSH
Related in: MedlinePlus