Comparative transcriptomic analysis of the l-4i silkworm (Lepidoptera: Bombyx mori) mutants and its wild-type strain P33 by RNA-Seq
Introduction
The domesticated silkworm (Bombyx mori) is a domesticated holometabolous insect that has been used for silk production for approximately 5000 years and is still of great economic importance in developing countries, especially in China (International Silkworm Genome, 2008). Moreover, as being first sequenced lepidopteran (Mita et al., 2004; Xia et al., 2004), it is also a model insect used in various fields of biology, including physiology, biochemistry, developmental biology, neurobiology, screening of antimicrobial drugs, and environmental monitoring (Meng et al., 2017; Kawamoto et al., 2019). Furthermore, silkworm has also been used as a bioreactor expressing recombinant protein production (Tomita et al., 2003; Hino et al., 2006; Iizuka et al., 2009) and producing novel silk biomaterials (Teulé et al., 2012; Xu et al., 2018). Significant progress has been achieved in silkworm research since the genome was sequenced (Xia et al., 2014), such as constructing a fine genome sequence (Kawamoto et al., 2019) and a genetic variation map (Xia et al., 2009), DNA methylation (Xiang et al., 2010), and the SilkDB database (Wang et al., 2005; Duan et al., 2010; Lu et al., 2020).
An advantage of working with domesticated silkworms over other lepidopteran species is the availability of mutations and inbred lines. More than 400 Mendelian mutations have been identified, and most mutants have been found in China and Japan (Goldsmith et al., 2005). Understanding the mechanism behind these silkworm mutants is important for investigating the development of silkworms, and lethal genes could be used for pest control. During the production and preservation of the bivoltine silkworm variety P33, a lethal mutant in fourth instar larvae (l-4i) was discovered. They feed slowly and stop development after the fourth instar and finally die on day 3 or day 4 of the fourth instar (Kang et al., 2015b). It has been found that the gene expression of β-glucosidase was significantly lower in larvae of l-4i than P33 (Kang et al., 2015a). In this study, comparative transcriptomic analysis of the l-4i mutant and P33 was performed using RNA-Seq. Some important biological processes and pathways were significantly changed in the l-4i mutant, and a large number of differential RNA alternative splicing events were found between l-4i and P33.
Section snippets
Silkworm preparation
The wild silkworm strain P33 and l-4i mutant heterozygotes were maintained in our lab (Sericulture Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu Province, China). Their larvae were fed fresh mulberry leaves under standard conditions with alternating 12 h of light and 12 h of darkness at 25 ± 2 °C and relative humidity 65 ± 5%. Three silkworm larvae were collected for each sample on day 2 of the 4th instar. After removing the mulberry leaves in the midgut, the
RNA-Seq analysis
RNA-Seq generated 25.4–30.3 million reads for l-4i samples and 18.5–27.7 million reads for P33 samples (Table S2). These raw sequencing data have been submitted to SRA in NCBI (BioProject ID: PRJNA681471).
Compared to the gene expression in P33, 2013 genes were significantly downregulated (fold change <0.5 and p value <0.01), and 2405 genes were significantly upregulated (fold change >2 and p value <0.01) in the l-4i mutant (Fig. 1, Fig. 2, Fig. S1, Fig. S2, Fig. S3, Fig. S4, Fig. S5, Tables S2,
Discussion
In 2015, our laboratory reported the l-4i mutant (Kang et al., 2015b), but the molecular mechanism remains unclear. In this study, the whole bodies of the l-4i mutant and its original strain P33 were used to perform RNA-Seq, and the results revealed that 2013 genes were significantly downregulated. Twenty biological processes, including spliceosomal snRNP assembly, protein folding and protein catabolic processes, were significantly enriched in these downregulated genes, and 2405 genes were
Conclusion
In this study, a total of 4418 differentially expressed genes were identified in the l-4i mutant; compared to P33, 2405 genes were upregulated and 2013 genes were downregulated in the l-4i mutant. Twenty biological processes, including spliceosomal snRNP assembly, protein folding and protein catabolic processes, were significantly enriched in these downregulated genes, and 20 biological processes, including purine nucleobase metabolic process, nucleoside metabolic process and de novo IMP
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (grant number 31972616, Qiaoling Zhao).
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