Abstract
Molting is an important physiological process in the larval stage of Bombyx mori and is controlled by various hormones and peptides. The silkworm mutant that exhibits the phenotype of non-molting in the 2nd instar (nm2) is incapable of molting in the 2nd instar and dies after seven or more days. The ecdysone titer in the nm2 mutant is lower than that in the wildtype, and the mutant can be rescued by feeding with 20E and cholesterol. The results of positional cloning indicated that structural alteration of BmCPG10 is responsible for the phenotype of the nm2 mutant. To explore the possible relationship between BmCPG10 and the ecdysone titer as well as the genes affected by BmCPG10, digital gene expression (DGE) profile analysis was conducted in the nm2 mutant, with the wildtype strain C603 serving as the control. The results revealed 1727 differentially expressed genes, among which 651 genes were upregulated and 1076 were downregulated in nm2. BLASTGO analysis showed that these differentially expressed genes were involved in various biological processes, cellular components and molecular functions. KEGG analysis indicated an enrichment of these differentially expressed genes in 240 pathways, including metabolic pathways, pancreatic secretion, protein digestion and absorption, fat digestion and absorption and glycerolipid metabolism. To verify the accuracy of the DGE results, quantitative reverse transcription PCR (qRT-PCR) was performed, focusing on key genes in several related pathways, and the results were highly consistent with the DGE results. Our findings indicated significant differences in cuticular protein genes, ecdysone biosynthesis genes and ecdysone-related nuclear receptors genes, but no significant difference in juvenile hormone and chitin biosynthesis genes was detected. Our research findings lay the foundation for further research on the formation mechanism of the nm2 mutant.
Highlights
The silkworm, Bombyx mori, a holometabolous lepidopteran, is an experimental model for molecular entomology in the fields of genetics, physiology and biochemistry [1]
Early-late genes are activated by the early genes, followed by a series of late genes activated by the early-late genes, such as eclosion hormone biosynthesis genes, neuropeptide genes, cuticular protein genes and hydrolase genes, and the activating signals are amplified by the key nuclear receptor βFTZ-F1 [2]
From the digital gene expression (DGE) libraries, we acquired 12,912,619 and 12,900,347 clean reads from the 13,129,213 and 13,129,324 raw reads after sifting of 216,594 and 216,594 adapter reads, 21,750 and 23,019 reads in which unknown bases were more than 10% and some low quality reads that the percentage of low quality bases was over 50% in a read in the wildtype C603 and the nm2 mutant, respectively
Summary
The silkworm, Bombyx mori, a holometabolous lepidopteran, is an experimental model for molecular entomology in the fields of genetics, physiology and biochemistry [1]. Molting is an important physiological process in the silkworm and is controlled by prothoracicotropic hormone (PTTH), 20-hydroxyecdysone (20E), juvenile hormone (JH) and neuropeptides. When it becomes necessary to molt, ecdysone is synthesized in the prothoracic gland promoted by PTTH, after which activated 20E is combined with the heterodimeric nuclear receptor EcR-USP to promote the expression of early genes. When the 20E titer decreases in the late phase of molting, numerous pigmentation-related enzymes are expressed and transported to the epidermis. The resulting pigments participate in the formation of a new epidermis [3, 4]. The pupating process requires the disappearance of juvenile hormone, and ecdysone plays a dominant role [5, 6]
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