Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant

Wu, Fan; Wang, Pingyang; Zhao, Qiaoling; Kang, Lequn; Xia, Dajing; Qiu, Zhiyong; Tang, Shengnan; Li, Muwang; Shen, Xingjia; Zhang, Guozheng

doi:10.1371/journal.pone.0153549

Cited by 11 publications

(18 citation statements)

References 32 publications

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“…BmCPG10 [15] and BmCP-like [21] were upregulated in the nm2 mutant, with the fold-changes of 2.37 and 86.16, respectively. These results were consistent with previous studies, which indicated that the DGE data were accurate and reliable.…”

Section: Resultsmentioning

confidence: 99%

Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm, Bombyx mori

Wang

et al. 2017

PLoS ONE

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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.

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Section: Resultsmentioning

confidence: 99%

Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm, Bombyx mori

Wang

et al. 2017

PLoS ONE

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“…BmCPV‐miR‐1 mimics and NCs were dissolved in DEPC water and injected into the haemocoel of silkworm larvae of 5th instars through the intersegmental membrane with a microsyringe following the method previously described (Terenius et al ., ; Wu et al ., ). One microgram (as a low dose) or 2 μg (as a high dose) mimics were injected into each larva, 1 μg NC injected as NC group and 1 μg DEPC water as control group.…”

Section: Methodsmentioning

confidence: 97%

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

et al. 2019

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Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus-encoded microRNAs (miRNAs) have been proven to play important roles in host-pathogen interactions. In this study we identified a BmCPV-derived miRNA-like 21 nt small RNA, BmCPV-miR-1, from the small RNA deep sequencing of BmCPV-infected silkworm larvae by stem-loop quantitative real-time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV-miR-1 at the 5 untranslated region. It was found that the expression of BmCPV-miR-1 and its target gene BmIAP were both up-regulated in BmCPV-infected larvae. At the same time, it was confirmed that BmCPV-miR-1 could up-regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV-miR-1 mimics could up-regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV-infected larvae, BmCPV-miR-1 mimics could be further up-regulated and inhibitors could lower the virus-mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV-miR-1 mimics could up-regulate and inhibitors down-regulate their replication in the infected silkworm. These results implied that BmCPV-miR-1 could inhibit cell apoptosis in the infected silkworm through up-regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.

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“…The soaking method especially suits RNAi analysis in insect cells and tissues, as well as in insects of specific life stages, such as eggs and neonate larvae [10]. The soaking method has also been used in moth cells [18,33], eggs [50], and larvae [19]. …”

Section: Methods To Introduce Exogenous Dsrna Into the Mothsmentioning

confidence: 99%

RNA Interference in Moths: Mechanisms, Applications, and Progress

Wang

Chen

et al. 2016

Genes

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The vast majority of lepidopterans, about 90%, are moths. Some moths, particularly their caterpillars, are major agricultural and forestry pests in many parts of the world. However, some other members of moths, such as the silkworm Bombyx mori, are famous for their economic value. Fire et al. in 1998 initially found that exogenous double-stranded RNA (dsRNA) can silence the homolog endogenous mRNA in organisms, which is called RNA interference (RNAi). Soon after, the RNAi technique proved to be very promising not only in gene function determination but also in pest control. However, later studies demonstrate that performing RNAi in moths is not as straightforward as shown in other insect taxa. Nevertheless, since 2007, especially after 2010, an increasing number of reports have been published that describe successful RNAi experiments in different moth species either on gene function analysis or on pest management exploration. So far, more than 100 peer-reviewed papers have reported successful RNAi experiments in moths, covering 10 families and 25 species. By using classic and novel dsRNA delivery methods, these studies effectively silence the expression of various target genes and determine their function in larval development, reproduction, immunology, resistance against chemicals, and other biological processes. In addition, a number of laboratory and field trials have demonstrated that RNAi is also a potential strategy for moth pest management. In this review, therefore, we summarize and discuss the mechanisms and applications of the RNAi technique in moths by focusing on recent progresses.

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Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant

Cited by 11 publications

References 32 publications

Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm, Bombyx mori

Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm, Bombyx mori

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

RNA Interference in Moths: Mechanisms, Applications, and Progress

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