Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 38 (2021) 100800

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Comparative Biochemistry and Physiology - Part D:

Genomics and Proteomics
journal homepage: www.elsevier.com/locate/cbpd

Comparative transcriptomic analysis of the l-4i silkworm (Lepidoptera:

Bombyx mori) mutants and its wild-type strain P33 by RNA-Seq
Chenjie Yang a, b, Lequn Kang c, Qiaoling Zhao a, b, *
a
School of Biotechnology, Jiangsu University of Science and Technology, Nanxv Road, Zhenjiang, Jiangsu 212018, China
b
The Sericulture Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212018, China
c
School of Grain Science and Technology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China

A R T I C L E I N F O A B S T R A C T

Keywords: The silkworm (Bombyx mori) is a domesticated holometabolous insect, and more than 400 Mendelian mutations
Bombyx mori have been identified. Investigating the mechanism behind these silkworm mutants is essential for understanding
RNA-Seq the development of silkworms and other lepidopterans, and lethal genes could be used for pest control. The lethal
l-4i mutant
silkworm mutant in the fourth instar (l-4i) has been recently found; however, the underlying mechanism is not
Transcriptome
yet clear. Herein, we studied the l-4i mutant and its wild-type strain P33 using RNA sequencing (RNA-seq). Our
results revealed that 2013 genes were significantly downregulated, and 20 biological processes, including spli­
ceosomal snRNP assembly, protein folding and protein catabolic process, were significantly enriched in these
downregulated genes. Moreover, 2405 genes were significantly upregulated in the l-4i mutant, and 20 biological
processes, including purine nucleobase metabolic process, nucleoside metabolic process and de novo IMP
biosynthetic process, were significantly enriched in these upregulated genes. The study suggests that the
imbalance of multiple biological processes and pathways and abnormal protein generation from RNA alternative
splicing may cause the death of the l-4i mutant.

1. Introduction An advantage of working with domesticated silkworms over other

lepidopteran species is the availability of mutations and inbred lines.
The domesticated silkworm (Bombyx mori) is a domesticated holo­ More than 400 Mendelian mutations have been identified, and most
metabolous insect that has been used for silk production for approxi­ mutants have been found in China and Japan (Goldsmith et al., 2005).
mately 5000 years and is still of great economic importance in Understanding the mechanism behind these silkworm mutants is
developing countries, especially in China (International Silkworm important for investigating the development of silkworms, and lethal
Genome, 2008). Moreover, as being first sequenced lepidopteran (Mita genes could be used for pest control. During the production and pres­
et al., 2004; Xia et al., 2004), it is also a model insect used in various ervation of the bivoltine silkworm variety P33, a lethal mutant in fourth
fields of biology, including physiology, biochemistry, developmental instar larvae (l-4i) was discovered. They feed slowly and stop develop­
biology, neurobiology, screening of antimicrobial drugs, and environ­ ment after the fourth instar and finally die on day 3 or day 4 of the fourth
mental monitoring (Meng et al., 2017; Kawamoto et al., 2019). instar (Kang et al., 2015b). It has been found that the gene expression of
Furthermore, silkworm has also been used as a bioreactor expressing β-glucosidase was significantly lower in larvae of l-4i than P33 (Kang
recombinant protein production (Tomita et al., 2003; Hino et al., 2006; et al., 2015a). In this study, comparative transcriptomic analysis of the l-
Iizuka et al., 2009) and producing novel silk biomaterials (Teulé et al., 4i mutant and P33 was performed using RNA-Seq. Some important
2012; Xu et al., 2018). Significant progress has been achieved in silk­ biological processes and pathways were significantly changed in the l-4i
worm research since the genome was sequenced (Xia et al., 2014), such mutant, and a large number of differential RNA alternative splicing
as constructing a fine genome sequence (Kawamoto et al., 2019) and a events were found between l-4i and P33.
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).

* Corresponding author at: School of Biotechnology, Jiangsu University of Science and Technology, Nanxv Road, Zhenjiang, Jiangsu Province 212018, China.
E-mail address: [email protected] (Q. Zhao).

https://doi.org/10.1016/j.cbd.2021.100800
Received 31 August 2020; Received in revised form 29 January 2021; Accepted 30 January 2021
Available online 6 February 2021
1744-117X/© 2021 Elsevier Inc. All rights reserved.
C. Yang et al. Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 38 (2021) 100800

2. Materials and methods 3. Results

2.1. Silkworm preparation 3.1. RNA-Seq analysis

The wild silkworm strain P33 and l-4i mutant heterozygotes were RNA-Seq generated 25.4–30.3 million reads for l-4i samples and
maintained in our lab (Sericulture Research Institute, Chinese Academy 18.5–27.7 million reads for P33 samples (Table S2). These raw
of Agricultural Sciences, Zhenjiang, Jiangsu Province, China). Their sequencing data have been submitted to SRA in NCBI (BioProject ID:
larvae were fed fresh mulberry leaves under standard conditions with PRJNA681471).
alternating 12 h of light and 12 h of darkness at 25 ± 2 ◦ C and relative Compared to the gene expression in P33, 2013 genes were signifi­
humidity 65 ± 5%. Three silkworm larvae were collected for each cantly downregulated (fold change <0.5 and p value <0.01), and 2405
sample on day 2 of the 4th instar. After removing the mulberry leaves in genes were significantly upregulated (fold change >2 and p value
the midgut, the whole bodies of the silkworm were stored at − 80 ◦ C for <0.01) in the l-4i mutant (Figs. 1 and 2, Figs. S1, S2, S3, S4, and S5,
future use. Tables S2, S3, and S4). Given 16,880 genes in the genome of silkworms,
approximately 25% of genes were significantly affected in the l-4i
2.2. Total RNA extraction, cDNA library construction and RNA mutant, and the overall gene expression changes are shown in Figs. 1
sequencing (RNA-Seq) and 2. For these differentially expressed genes in the l-4i mutant, the fold
changes of the majority of the upregulated genes were less than 24. It has
Total RNA was extracted from the stored whole bodies using RNAiso been found that gene expression increased over 50-fold change in some
Plus (TaKaRa, Dalian, China) and treated with DNase to remove residual genes, such as BGIBMGA008128(3-ketodihydrosphingosine reductase
genomic DNA. Its quality and integrity were evaluated with an Agilent tsc-10), BGIBMGA003095(BMORCPR58), BGIBMGA011759
Bioanalyzer. The cDNA libraries were constructed using the mRNA Seq (BMORCPH9), BGIBMGA010040(Putative uncharacterized protein),
Sample Prep Kit (Illumina). The mRNAs were enriched using oligo (dT) BGIBMGA011727(BMORCPH14), BGIBMGA004780(BMORCPH33),
magnetic beads and then sheared into short fragments, which were then BGIBMGA000045(ENSANGP), BGIBMGA013828(hypothetical protein),
synthesized into double-stranded cDNA using random primers, and BGIBMGA000013(CG15592-PA) and BGIBMGA000010(OSI9).
terminal modification was performed for the purified double-stranded In addition to gene expression changes, differential RNA alternative
cDNA; a base (A) tail was added, and the fragments were ligated. The splicing events were found in the transcriptomes of l-4i; 69 of 357
quality of the cDNA libraries was evaluated using an Agilent 2100 mutually exclusive slicing events and 274 of 2748 exon skipping events
Bioanalyzer. RNA-Seq (paired end 150 bp, PE150) was performed with were significantly different between the transcriptomes of l-4i and P33
the Illumina HiSeq XTen system (Roberts et al., 2011; Li and Jiang, (Fig. 3).
2012; Cristian et al., 2014).
3.2. GO enrichment analysis of differentially expressed genes
2.3. RNA-Seq analysis
For those 2013 significantly downregulated genes, GO enrichment
The quality of the raw reads was evaluated using FastQC, adaptors analysis indicated that 20 biological processes (protein folding, chitin
were removed using FASTQ trimmer, and reads (shorter than 50 bp or metabolic process, protein catabolic process, DNA replication, spliceo­
Phred <20) were filtered out. The trimmed reads were aligned with the somal snRNP assembly, DNA replication initiation, isoprenoid biosyn­
silkworm genome in the Bombyx_mori database in Ensembl (http://m thetic process, nuclear-transcripted mRNA catabolic process, small
etazoa.ensembl.org/). The reads per kb per million reads (RPKM) GTPase-mediated signal transduction, protein methylation, mitotic
were used to indicate the expression level of the genes. The significantly chromosome condensation, mitotic spindle assembly checkpoint,
differentially expressed genes (fold change >2 or <0.5, and p value proteasome-mediated ubiquitin-dependent protein catabolic process,
<0.01) were further analyzed (Anders and Huber, 2010; Chen and microtubule-based process, cell cycle, dsRNA transport, signal peptide
Boutros, 2011; Kolde, 2015). A volcano plot was used to visualize the processing, protein import into the nucleus, and mRNA transport, cilium
overall differential gene expression between the l-4i mutant and P33, or flagellum-dependent cell motility) were significantly enriched,
and it was generated with an online program (http://www.bioinformati especially more than 10 genes in four biological processes, including
cs.com.cn). We used rMATS to analyze RNA alternative splicing events protein folding, the chitin metabolic process, DNA replication and small
in the transcriptomes of the l-4i mutant and P33. GO enrichment and GTPase-mediated signaling transduction (Fig. 4A, Table S5). Of the 2405
KEGG pathway analysis for the differentially expressed genes were significantly upregulated genes, 20 biological processes (translation,
performed (Ashburner et al., 2000; Minoru and Susumu, 2000; Hahne lipid metabolic process, metabolic process, ‘de novo’ IMP biosynthetic
et al., 2010; Yu et al., 2012; Shen et al., 2014; Anders et al., 2015). process, ribosome biogenesis, the neuropeptide signaling pathway, ATP
synthesis-coupled proton transport, transmembrane transport, biosyn­
2.4. Quantitative reverse transcriptase polymerase chain reaction (qRT- thetic process, nucleotide catabolic process, glutathione catabolic pro­
PCR) cess, purine nucleobase biosynthetic process, ‘de novo’ AMP biosynthetic
process, nucleoside metabolic process, tricarboxylic acid cycle, aromatic
Total RNA of l-4i and P33 was reverse transcribed into cDNA using a compound catabolic process, aromatic amino acid family metabolic
Reverse Transcriptase M-MLV (RNase H) kit (TaKaRa, Dalian, China). process, ATP hydrolysis coupled proton transport, the spliceosomal tri-
The cDNA samples were diluted to 50 ng/μL as the template for qRT- snRNP complex assembly, and cellular glucose homeostasis) were
PCR. In brief, the qRT-PCR was carried out in a reaction volume of 20 significantly enriched, and more than 10 genes were enriched in four
μL, consisting of 1 μL of gene-specific primers (10 μmol/L, Table S1), 1 biological processes, including translation, lipid metabolic process,
μL cDNA, and 10 μL SYBR® Premix Ex Taq™ (Tli RNaseH Plus, 2×), metabolic process and transmembrane transport (Fig. 4B, Table S5).
supplemented to 20 μL with ddH2O. The qRT-PCR was run on a Light­
Cycler®96 real-time PCR system (Roche, Switzerland) under the 3.3. KEGG pathway analysis of the differentially expressed genes
following conditions: predenaturation at 95 ◦ C for 10 min, followed by
40 cycles with three steps: 95 ◦ C 10 s, 58 ◦ C 10 s, and 72 ◦ C 10 s. The KEGG pathway analysis indicated that 20 pathways (ECM-receptor
relative gene expression in the larvae of l-4i and P33 was calculated interaction, Lysosome, Protein processing in endoplasmic reticulum,
using the 2-ΔΔCt method (Livak and Schmittgen, 2001), and BmGAPDH Mismatch repair, Nucleotide excision repair, Base excision repair, Pro­
(BGIBMGA007490) was used as a reference gene. tein export, Proteasome, Spliceosome, DNA replication, RNA transport,

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C. Yang et al. Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 38 (2021) 100800

Fig. 1. The volcano plot shows the fold change and the significance of the sequenced genes. The red dots represent the significantly upregulated genes in l-4i, the
green dots represent the significantly downregulated genes in l-4i, and the gray dots represent the genes that are not significant between l-4i and P33. The Padj
represents the adjusted p value. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3000
2748

2500
The number of genes

2000

1500 AS
DE
1000

500 357 274

69
0
MXE SE

Fig. 3. Differential RNA alternative splicing events between l-4i and P33. MXE
= Mutually Exclusive Exon event, SE = Skipping Exon event, AS represents all
RNA alternative splicing events, DE represents differential AS events.

biosynthesis, Nicotinate and nicotinamide metabolism, One carbon pool

by folate, Glyoxylate and dicarboxylate metabolism, Pyruvate meta­
bolism, Glycerolipid metabolism, Starch and sucrose metabolism,
Phenylalanine, tyrosine and tryptophan biosynthesis, Phenylalanine
metabolism, Tyrosine metabolism, Arginine and proline metabolism,
Fig. 2. Clustered heat map of top-ranked differentially expressed transcripts. Glycine, serine and threonine metabolism, Alanine, aspartate and
The columns represent the sequenced samples, WT = P33, HOM = l-4i, and the glutamate metabolism, Galactose metabolism, Fructose and mannose
rows represent transcripts. The color scale bar represents the gene expression metabolism, and Citrate cycle (TCA cycle)) were significantly enriched
level (RPKM). in upregulated genes, and more than 10 genes were enriched in path­
ways, including carbon metabolism, aspartate and glutamate meta­
Aminoacy-tRNA biosynthesis, Terpenoid backbone biosynthesis, Gly­ bolism, galactose metabolism, fructose and mannose metabolism, and
cosphingolipid biosynthesis-ganglio series, Glycosphingolipid the citrate cycle (TCA cycle). (Fig. 5B, Table S6).
biosynthesis-globo and isoglobo series, Glycosaminoglycan degradation,
Other glycan degradation, Selenocompound metabolism, beta-Alanine
metabolism, and Fatty acid degradation) were significantly enriched 3.4. Validating RNA-Seq data by qRT-PCR
in downregulated genes, and more than 10 genes were enriched in
pathways, including proteasome, protein export, protein processing in To confirm the RNA-Seq data, qRT-PCR for 39 significantly differ­
endoplasmic reticulum and nucleotide excision repair pathways entially expressed genes was performed, and the qRT-PCR results were
(Fig. 5A, Table S6). Twenty pathways (Ribosome, Biosynthesis of amino consistent with the RNA-Seq results (Fig. 6, Fig. S6, Tables 1, S7, and
acids, 2-oxocarboxylic acid metabolism, Carbon metabolism, Folate S8).

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C. Yang et al. Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 38 (2021) 100800

Fig. 4. GO enrichment analysis (biological process) of the differentially expressed genes. (A) The enriched biological processes enriched in the downregulated genes.
(B) The enriched biological processes enriched in the upregulated genes. The X-axis represents the number of specific GO-BP items enriched; the Y-axis indicates the
enriched biological processes. The column color represents the P value of the enriched biological processes.

4. Discussion 2019).
Although the current research on eukaryotes has only explained the
In 2015, our laboratory reported the l-4i mutant (Kang et al., 2015b), assembly mechanism of yeast spliceosome snRNP, the theoretical basis
but the molecular mechanism remains unclear. In this study, the whole for variable splicing of higher eukaryotes can be deduced from the
bodies of the l-4i mutant and its original strain P33 were used to perform existing structural evidence the most directly and effectively (Bai et al.,
RNA-Seq, and the results revealed that 2013 genes were significantly 2018). In our study of the silkworm mutant l-4i, in the biological process
downregulated. Twenty biological processes, including spliceosomal of spliceosome snRNP assembly (GO.0000387), small nuclear ribonu­
snRNP assembly, protein folding and protein catabolic processes, were cleoproteins Sm D1 (BGIBMGA003840) and Sm D3 (BGIBMGA013100)
significantly enriched in these downregulated genes, and 2405 genes were significantly downregulated in the l-4i mutant (Table 1), which
were significantly upregulated in the l-4i mutant. Because of the whole might affect RNA alternative splicing. Therefore, silkworm mutations
bodies used in this study, the genes that were differentially expressed in lack the function of the corresponding proteins, eventually leading to
specific tissues may be missed as a result of their opposite expression in the absence of the biological functions of mutants and gradual death
other tissues. (Fig. 3).
As the protein structure determines its function, protein folding af­
fects its structure and function. In the biological process of protein
4.1. Spliceosomal snRNP assembly, protein folding and protein catabolic folding (GO.0006457), calreticulin precursor (BGIBMGA000475), DnaJ
process were significantly enriched in the downregulated genes homolog subfamily A member 4 (BGIBMGA01254) and peptidyl-prolyl
cis-trans isomerase (BGIBMGA002429, PPI) were significantly down­
RNA splicing plays an important role in gene expression regulation in regulated in the l-4i mutant (Table 1). Calreticulin is involved in protein
eukaryotes. As the executor of catalytic premRNA splicing in eukaryotic folding as a molecular chaperone. DnaJ homolog subfamily A member 4
cells, the spliceosome is one of the most basic molecular machines in has been reported to participate in the protein folding process (Li and
eukaryotes. The incorrect RNA splicing and regulation of spliceosomes Bao, 2016). PPI and protein disulfide isomerase (PDI) form a folding
are associated with many diseases (Wan et al., 2016; Wilkinson et al., enzyme involved in protein folding. The downregulation of these genes
2019). Moreover, snRNP (small nuclear ribonucleoprotein) is a kind of might affect protein folding in the l-4i mutant silkworm.
small DNA protein particle and a kind of dynamic RNA-protein complex In the protein catabolic process (GO.0030163), 26S protease
that can accumulate in the nucleus (Stone and Riley, 2014; So et al.,

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C. Yang et al. Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 38 (2021) 100800

Fig. 5. The enriched KEGG pathways in the differentially expressed genes. (A) The enriched pathways enriched in the downregulated genes, (B) the enriched
pathways enriched in the upregulated genes. The X-axis is the enrichment value, and the Y-axis is the specific function entry. The scatter size represents the number of
genes enriched in a specific functional entry. The scatter color represents the negative logarithm of the p-value of functional enrichment, and the more purple the
color is, the more significant the enrichment analysis results are. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)

downregulated in l-4i (Table 1). Their downregulation might affect the

decomposition of proteins in l-4i.
Consistent with these affected biological processes, differential RNA
alternative splicing events were found between l-4i and P33 (Fig. 3);
they might produce different proteins with different protein sequences
and structures, and incorrect expression or folding of the essential pro­
teins might lead to maldevelopment and even death. Through map-
based cloning, our laboratory also found that topoisomerase 3α has
sequence deletions in its transcript (Kang et al., unpublished).

4.2. The proteasome, protein processing in the endoplasmic reticulum,

protein export and nucleotide excision repair pathways were significantly
enriched in the downregulated genes in l-4i

The production and degradation of proteins are basic metabolic

processes in organisms and are involved in almost all important life
Fig. 6. Pearson correlation analysis of qRT-PCR and RNA-Seq data. The black
dots represent the genes, the values on the x-axis represent the gene expression activities in organisms. It is not difficult to imagine that the great dif­
in RNA-seq, and the values on the y-axis represent the gene expression in ferences in body shape and physiological activities between l-4i and P33
qRT-PCR. must be inseparable from the related functions of proteins. Hence, the
relevant metabolic pathways were analyzed. Contrary to the ribosome
regulatory subunit 6A (BGIBMGA004782), subunit 4 function of the molecular machine responsible for protein synthesis, the
(BGIBMGA004908), subunit 7 (BGIBMGA007332), subunit S10B proteasome is the molecular machine responsible for protein degrada­
(BGIBMGA010794) and subunit 8 (BGIBMGA014177) were significantly tion (Lub et al., 2015; Shi et al., 2016; Nguyen et al., 2017). The pro­
teasome is widely distributed in the cytoplasm and nucleus of eukaryotic

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Table 1 Table 1 (continued )

Gene expression comparison between RNA-Seq and qRT-PCR. Gene id. Description Fold (l-4i/P33) Consistency
Gene id. Description Fold (l-4i/P33) Consistency
FPKM qRT- Up Down
FPKM qRT- Up Down PCR
PCR
BGIBMGA012203 Translocon-associated 0.29 0.47 √
BGIBMGA003840 Probable small nuclear 0.35 0.14 √ protein
ribonucleoprotein Sm D1
BGIBMGA013100 Small nuclear 0.22 0.86 √
ribonucleoprotein Sm D3 cells and has a variety of catalytic functions. It can selectively degrade
BGIBMGA000475 Calreticulin precursor 0.18 0.10 √ newborn proteins with short half-lives and misfolded, mutated struc­
BGIBMGA012541 DnaJ homolog subfamily A 0.39 0.40
tural and functional proteins, stress-damaged proteins and other specific
√
member 4
BGIBMGA002429 Peptidyl-prolyl cis-trans 0.98 0.60 √ proteins that need to be removed by the cell itself, which are closely
isomerase related to important life activities, such as DNA damage repair, gene
BGIBMGA004782 26S protease regulatory 0.61 0.46 √ transcription, cell growth, apoptosis and antigen presentation (Jia and
subunit 6A Sun, 2009; Jiang et al., 2017; Bai et al., 2018; Yin et al., 2020).
BGIBMGA004908 26S protease regulatory 0.50 0.66 √
subunit 4
In this study, 12 genes in the proteasome pathway (ko03050) were
BGIBMGA007332 26S protease regulatory 0.56 0.26 √ significantly enriched among the downregulated genes. The following
subunit 7 four genes (BGIBMGA004782, BGIBMGA004908, BGIBMGA007332 and
BGIBMGA010794 26S protease regulatory 0.53 0.31 √ BGIBMGA014177) were significantly downregulated in the biological
subunit S10B
process of protein catabolism (GO.0030163) (Fig. 5A, Table 1). It has
BGIBMGA014177 26S protease regulatory 0.49 0.19 √
subunit 8 been mentioned earlier in this paper that these four genes are all func­
BGIBMGA001333 Uricase 3.12 3.15 √ tional genes related to proteasome regulatory subunits, which are
BGIBMGA007647 Amidophosphoribosyl 6.39 4.01 √ responsible for the catabolism of proteins. The significantly down­
transferase precursor regulated expression of related genes in the proteasome pathway in the
BGIBMGA003319 Aspartate 1.91 1.40 √
aminotransferase
l-4i mutant might affect the physiological activity of silkworms. In
BGIBMGA004655 CG3523 15.55 4.98 √ addition, 15 genes in the two pathways of protein export (ko03060) and
BGIBMGA014118 Trifunctional purine 4.77 2.99 √ protein processing in the endoplasmic reticulum (ko04141) were
biosynthetic protein significantly downregulated in l-4i. Contrary to the process of protea­
adenosine-3
some decomposition, proteins are synthesized on free ribosomes and
BGIBMGA013774 CG6330 2.55 1.45 √
BGIBMGA011774 CG16758-PB 6.85 4.44 √ further exported to the endoplasmic reticulum for modification and
BGIBMGA012947 ENSANGP00000007063 2.10 2.31 √ folding (Ciobanu et al., 2018; Liu et al., 2018; Sowers et al., 2018). The
BGIBMGA000971 Proteasome maturation 0.45 0.14 √ downregulation of the above two pathways may cause difficulties in
protein protein modification and folding. Simultaneously, it was also found that
BGIBMGA002953 26S proteasome non- 0.55 0.35 √
ATPase regulatory subunit
BGIBMGA000475 and BGIBMGA012541 were downregulated in the
3 biological process of protein folding (GO.0006457) analyzed above
BGIBMGA003009 Proteasome subunit beta 0.48 0.44 √ (Fig. 5 A, Table 1). This finding is consistent with the results of our
type-7 precursor analysis.
BGIBMGA003028 Proteasome subunit alpha 0.45 0.33 √
In addition to protein metabolism, DNA repair can enhance the
type-7
BGIBMGA003212 similar to 26S proteasome 0.49 0.29 √ ability of cells to resist external stimuli and maintain the normal life
non-ATPase regulatory activities of cells and the stability of genetic material (Hamdi et al.,
subunit 6 2019; Santos et al., 2019; Zebian et al., 2019). It was also detected that
BGIBMGA003335 Proteasome subunit beta 0.52 0.67 √ the related genes in the nucleotide excision repair pathway (ko03420)
type-6 precursor
BGIBMGA004657 Proteasome subunit alpha 0.53 0.79 √
were significantly downregulated in l-4i. Genetic mutations in such
type-6 mutants are inevitable.
BGIBMGA005994 26S proteasome non- 0.47 0.47 √
ATPase regulatory subunit 4.3. Nucleoside metabolic processes and de novo IMP biosynthetic
7
processes were enhanced in l-4i
BGIBMGA003048 Similar to signal peptidase 0.38 0.36 √
complex subunit 2
homolog Purines in the body play a significant role in the energy supply,
BGIBMGA004993 Signal recognition particle 0.66 0.29 √ metabolic regulation and composition of coenzymes and are maintained
54 kDa protein by the synergistic action of the salvage biosynthetic signal pathway and
BGIBMGA006209 Zgc 0.26 0.42 √
BGIBMGA007043 ENSANGP 0.57 0.49 √
de novo synthesis biological signal pathway in mammalian cells (Yasu­
BGIBMGA012687 Protein transport protein 0.48 0.59 √ jima et al., 2018; Dos Santos et al., 2019). Salvage biosynthetic signaling
Sec61 subunit alpha pathways maintain purine nucleotide levels under normal physiological
isoform 2 conditions, while de novo synthesis biosynthetic pathways are upregu­
BGIBMGA001232 RING-box protein 1 0.57 0.27
lated and changed in cancer cells during growth (Jinhe et al., 2015;
√
BGIBMGA002569 Protein disulfide-isomerase 0.47 0.48 √
A6 precursor Wang et al., 2015). The expression of the purine metabolism process
BGIBMGA003587 Multifunctional 0.47 0.45 √ (GO.0006144, GO.0009113) was demonstrated to be more active in l-4i
endoplasmic reticulum (Fig. 5B, Table 1).
luminal polypeptide Oversynthesis of purines may cause the accumulation of harmful
BGIBMGA003985 Transitional endoplasmic 0.32 0.22 √
reticulum ATPase
substances. Moreover, it was also revealed that the expression of
BGIBMGA005769 CG5474-PA 0.36 0.45 √ biosynthesis processes (GO.0006189, GO.0009058) such as de novo IMP
BGIBMGA006819 Putative RAD23-like B 0.67 0.40 √ biosynthesis was more active in l-4i (Fig. 5B, Table 1). Because of the
BGIBMGA011844 Protein disulfide-isomerase 0.57 0.51 √ feedback inhibition of IMP and GMP on purine synthesis, we speculate
precursor
that the active expression of genes related to the above biological pro­
cesses in l-4i is a self-repairing regulation of bad constitution by mutants;

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however, the effect of this regulation is extremely weak. Similarly, the Hahne, F., Huber, W., Gentleman, R, Falcon, S., 2010. Hypergeometric Testing Used for
Gene Set Enrichment Analysis. Springer New York.
active expression of nucleoside catabolism (GO.0009166) was also
Hamdi, Y., Jerbi, M., Romdhane, L., Ben Rekaya, M., El Benna, H., Chouchane, L.,
detected in l-4i, which agrees well with the previous conjecture. Boubaker, M.S., Abdelhak, S., Yacoub-Youssef, H., 2019. Genetic diversity and
Disorders of purine metabolism lead to immunodeficiency, and the functional effect of common polymorphisms in genes involved in the first
most commonly encountered disorder of purine metabolism is gout. This heterodimeric complex of the nucleotide excision repair pathway. DNA Repair
(Amst) 86, 102770.
disorder affects approximately 1–2% of the population and is charac­ Hino, R., Tomita, M., Yoshizato, K., 2006. The generation of germline transgenic
terized by hyperuricemia with urate crystal deposition resulting in silkworms for the production of biologically active recombinant fusion proteins of
nephrolithiasis and inflammatory arthritis (Schlesinger, 2010). The fibroin and human basic fibroblast growth factor. Biomaterials 27, 5715–5724.
Iizuka, M., Ogawa, S., Takeuchi, A., Nakakita, S., Kubo, Y., Miyawaki, Y., Hirabayashi, J.,
nucleoside metabolic process and de novo IMP biosynthetic process were Tomita, M., 2009. Production of a recombinant mouse monoclonal antibody in
enhanced in l-4i, suggesting that nucleosides may be overproduced in transgenic silkworm cocoons. FEBS J. 276, 5806–5820.
the l-4i mutant and lead to silkworm death. International Silkworm Genome, C, 2008. The genome of a lepidopteran model insect,
the silkworm Bombyx mori. Insect Biochem. Mol. Biol. 38, 1036–1045.
Jia, L., Sun, Y., 2009. RBX1/ROC1-SCF E3 ubiquitin ligase is required for mouse
5. Conclusion embryogenesis and cancer cell survival. Cell Div 4, 16.
Jiang, B., Shi, Y., Zhang, X., Xin, X., Qi, L., Guo, H., Li, J., Yang, S., 2017. PIF3 is a
negative regulator of the CBF pathway and freezing tolerance in Arabidopsis. Proc.
In this study, a total of 4418 differentially expressed genes were Natl. Acad. Sci. U. S. A. 114, E6695–E6702.
identified in the l-4i mutant; compared to P33, 2405 genes were upre­ Jinhe, Z., Fei, F., iLei, S.L., Ying, Z., 2015. Relationship among abnormal glucose
gulated and 2013 genes were downregulated in the l-4i mutant. Twenty metabolism and serum uric acid level and xanthine oxidase. China Geriatrics Res
(Electronic Edition) 2, 24–27.
biological processes, including spliceosomal snRNP assembly, protein Kang, L., Huang, F., Wu, F., Zhao, Q., 2015a. Transcription analysis of the beta-
folding and protein catabolic processes, were significantly enriched in glucosidase precursor in wild-type and l-4i mutant Bombyx mori (Lepidoptera:
these downregulated genes, and 20 biological processes, including pu­ Bombycidae). Journal of insect science (Online) 15.
Kang, L., Zhao, Q., Shen, X., Tang, S., Qiu, Z., Xia, D., 2015b. The discovery of a novel
rine nucleobase metabolic process, nucleoside metabolic process and de
bombyx mori mutant,the lethal mutation in the fourth instar larvae. Science of
novo IMP biosynthetic process, were significantly enriched in these Sericulture 41, 53–56.
upregulated genes. A large number of differential RNA alternative Kawamoto, M., Jouraku, A., Toyoda, A., Yokoi, K., Minakuchi, Y., Katsuma, S., Fujiyama,
splicing events were found between l-4i and P33. This study suggests A., Kiuchi, T., Yamamoto, K., Shimada, T., 2019. High-quality genome assembly of
the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 107, 53–62.
that multiple biological processes and pathways and differential RNA Kolde, R., 2015. Pheatmap: Pretty Heatmaps.
alternative splicing cause the lethal phenotype of the l-4i mutant. Li, Y., Bao, Y., 2016. Research progress of the heat shock protein 40 in lung cance. j.smhx
Supplementary data to this article can be found online at https://doi. 36, 487–490.
Li, W., Jiang, T., 2012. Transcriptome Assembly and Isoform Expression Level Estimation
org/10.1016/j.cbd.2021.100800. from Biased RNA-Seq Reads.
Liu, Y.S., Guo, X.Y., Hirata, T., Rong, Y., Motooka, D., Kitajima, T., Murakami, Y., Gao, X.
D., Nakamura, S., Kinoshita, T., Fujita, M., 2018. N-glycan-dependent protein folding
Declaration of competing interest and endoplasmic reticulum retention regulate GPI-anchor processing. J. Cell Biol..
217, 585–599.
The authors declare that they have no known competing financial Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-
time quantitative PCR and the 2 − ΔΔ C T method. Methods 25.
interests or personal relationships that could have appeared to influence Lu, F., Wei, Z., Luo, Y., Guo, H., Zhang, G., Xia, Q., Wang, Y., 2020. SilkDB 3.0:
the work reported in this paper. visualizing and exploring multiple levels of data for silkworm. Nucleic Acids Res..
48, D749-d755.
Lub, S., Maes, K., Menu, E., Bruyne, E.D., Valckenborgh, E.V., 2015. Novel strategies to
Acknowledgment target the ubiquitin proteasome system in multiple myeloma. Oncotarget 7,
6521–6537.
This work was supported by the National Natural Science Foundation Meng, X., Zhu, F., Chen, K., 2017. Silkworm: a promising model organism in life science.
Journal of insect science (Online) 17.
of China (grant number 31972616, Qiaoling Zhao). Minoru, K., Susumu, G., 2000. KEGG: kyoto encyclopedia of genes and genomes. Nucleic
Acids Res. (1).
References Mita, K., Kasahara, M., Sasaki, S., Nagayasu, Y., Yamada, T., Kanamori, H., Namiki, N.,
Kitagawa, M., Yamashita, H., Yasukochi, Y., Kadono-Okuda, K., Yamamoto, K.,
Ajimura, M., Ravikumar, G., Shimomura, M., Nagamura, Y., Shin, I.T., Abe, H.,
Anders, S., Huber, W., 2010. Differential expression analysis for sequence count data.
Shimada, T., Morishita, S., Sasaki, T., 2004. The genome sequence of silkworm,
Genome Biol. 11, R106.
Bombyx mori. DNA Res. 11, 27–35.
Anders, S., Pyl, P.T., Huber, W., 2015. HTSeq—A Python Framework to Work with High-
Nguyen, A.T., Prado, M.A., Schmidt, P.J., Sendamarai, A.K., Wilson-Grady, J.T., Min, M.,
throughput Sequencing Data. 31, 166–169.
Campagna, D.R., Tian, G., Shi, Y., Dederer, V., Kawan, M., Kuehnle, N., Paulo, J.A.,
Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P.,
Yao, Y., Weiss, M.J., Justice, M.J., Gygi, S.P., Fleming, M.D., Finley, D., 2017.
Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L.,
UBE2O remodels the proteome during terminal erythroid differentiation. Science
Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M.,
357.
Sherlock, G., 2000. Gene ontology: tool for the unification of biology. The gene
Roberts, A., Pimentel, H., Trapnell, C., Pachter, L., 2011. Identification of novel
ontology consortium. Nat. Genet. 25, 25–29.
transcripts in annotated genomes using RNA-Seq. Bioinformatics 27, 2325–2329.
Bai, R., Wan, R., Yan, C., Lei, J., Shi, Y., 2018. Structures of the fully assembled\\r
Santos, H.B.P., Morais, E.F., Cavalcante, R.B., Nogueira, R.L.M., Nonaka, C.F.W., Souza,
<i>Saccharomyces cerevisiae</i>\\r spliceosome before activation. Science 360,
L.B., Freitas, R.A., 2019. Immunoexpression of DNA base excision repair and
1423–1429.
nucleotide excision repair proteins in ameloblastomas, syndromic and non-
Chen, H., Boutros, P.C., 2011. VennDiagram: a package for the generation of highly-
syndromic odontogenic keratocysts and dentigerous cysts. Arch. Oral Biol. 110,
customizable Venn and Euler diagrams in R. BMC Bioinformatics 12, 35.
104627.
Ciobanu, L.G., Sachdev, P.S., Trollor, J.N., Reppermund, S., Thalamuthu, A., Mather, K.
Schlesinger, N., 2010. Diagnosing and treating gout: a review to aid primary care
A., Cohen-Woods, S., Stacey, D., Toben, C., Schubert, K.O., Baune, B.T., 2018. Co-
physicians. Postgrad. Med. 122, 157–161.
expression network analysis of peripheral blood transcriptome identifies
Shen, S., Park, J.W., Lu, Z., Lin, L., Henry, M.D., Wu, Y.N., Zhou, Q., Xing, Y., 2014.
dysregulated protein processing in endoplasmic reticulum and immune response in
rMATS: robust and flexible detection of differential alternative splicing from
recurrent MDD in older adults. J. Psychiatr. Res. 107, 19–27.
replicate RNA-Seq data. Proc Natl Acad U S A 111, 5593–5601.
Cristian, G.E., Valentina, V.-M.o., Gustavo, N.e.-A.a., Cynthia, G., 2014. RNA-Seq
Shi, Y., Chen, X., Elsasser, S., Stocks, B.B., Tian, G., Lee, B.H., Shi, Y., Zhang, N., de
analysis using De novo transcriptome assembly as a reference for the Salmon louse
Poot, S.A., Tuebing, F., Sun, S., Vannoy, J., Tarasov, S.G., Engen, J.R., Finley, D.,
Caligus rogercresseyi. PLoS One 9, e92239-.
Walters, K.J., 2016. Rpn1 provides adjacent receptor sites for substrate binding and
Dos Santos, I.F., Sheriff, S., Amlal, S., Ahmed, R.P.H., Thakar, C.V., Amlal, H., 2019.
deubiquitination by the proteasome. Science 351.
Adenine acts in the kidney as a signaling factor and causes salt- and water-losing
So, B.R., Di, C., Cai, Z., Venters, C.C., Guo, J., Oh, J.M., Arai, C., Dreyfuss, G., 2019. A
nephropathy: early mechanism of adenine-induced renal injury. Am. J. Physiol.
complex of U1 snRNP with cleavage and polyadenylation factors controls
Renal Physiol. 316, F743–F757.
telescripting, regulating mRNA transcription in human cells. Mol. Cell 76, 590–599
Duan, J., Li, R., Cheng, D., Fan, W., Zha, X., Cheng, T., Wu, Y., Wang, J., Mita, K.,
e594.
Xiang, Z., Xia, Q., 2010. SilkDB v2.0: a platform for silkworm (Bombyx mori)
Sowers, C.R., Wang, R., Bourne, R.A., McGrath, B.C., Hu, J., Bevilacqua, S.C., Paton, J.C.,
genome biology. Nucleic Acids Res. 38, D453–D456.
Paton, A.W., Collardeau-Frachon, S., Nicolino, M., Cavener, D.R., 2018. The protein
Goldsmith, M.R., Shimada, T., Abe, H., 2005. The genetics and genomics of the silkworm,
Bombyx mori. Annu. Rev. Entomol. 50, 71–100.

7
C. Yang et al. Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 38 (2021) 100800

kinase PERK/EIF2AK3 regulates proinsulin processing not via protein synthesis but S., Wang, J., Wong, G.K., Yang, H., 2004. A draft sequence for the genome of the
by controlling endoplasmic reticulum chaperones. J. Biol. Chem. 293, 5134–5149. domesticated silkworm (Bombyx mori). Science 306, 1937–1940.
Stone, L.B., Riley, K.J., 2014. Small Nuclear Ribonucleoproteins (snRNPs). John Wiley & Xia, Q., Guo, Y., Zhang, Z., Li, D., Xuan, Z., Li, Z., Dai, F., Li, Y., Cheng, D., Li, R.,
Sons, Ltd. Cheng, T., Jiang, T., Becquet, C., Xu, X., Liu, C., Zha, X., Fan, W., Lin, Y., Shen, Y.,
Teulé, F., Miao, Y.G., Sohn, B.H., Kim, Y.S., Hull, J.J., Fraser Jr., M.J., Lewis, R.V., Jiang, L., Jensen, J., Hellmann, I., Tang, S., Zhao, P., Xu, H., Yu, C., Zhang, G., Li, J.,
Jarvis, D.L., 2012. Silkworms transformed with chimeric silkworm/spider silk genes Cao, J., Liu, S., He, N., Zhou, Y., Liu, H., Zhao, J., Ye, C., Du, Z., Pan, G., Zhao, A.,
spin composite silk fibers with improved mechanical properties. Proc. Natl. Acad. Shao, H., Zeng, W., Wu, P., Li, C., Pan, M., Li, J., Yin, X., Li, D., Wang, J., Zheng, H.,
Sci. U. S. A. 109, 923–928. Wang, W., Zhang, X., Li, S., Yang, H., Lu, C., Nielsen, R., Zhou, Z., Wang, J.,
Tomita, M., Munetsuna, H., Sato, T., Adachi, T., Hino, R., Hayashi, M., Shimizu, K., Xiang, Z., Wang, J., 2009. Complete resequencing of 40 genomes reveals
Nakamura, N., Tamura, T., Yoshizato, K., 2003. Transgenic silkworms produce domestication events and genes in silkworm (Bombyx). Science 326, 433–436.
recombinant human type III procollagen in cocoons. Nat. Biotechnol. 21, 52–56. Xia, Q., Li, S., Feng, Q., 2014. Advances in silkworm studies accelerated by the genome
Wan, R., Yan, C., Bai, R., Wang, L., Huang, M., Wong, C.C.L., Shi, Y., 2016. The 3.8 a sequencing of Bombyx mori. Annu. Rev. Entomol. 59, 513–536.
structure of the U4/U6.U5 tri-snRNP: insights into spliceosome assembly and Xiang, H., Zhu, J., Chen, Q., Dai, F., Li, X., Li, M., Zhang, H., Zhang, G., Li, D., Dong, Y.,
catalysis. Science 351, 466–475. Zhao, L., Lin, Y., Cheng, D., Yu, J., Sun, J., Zhou, X., Ma, K., He, Y., Zhao, Y., Guo, S.,
Wang, J., Xia, Q., He, X., Dai, M., Ruan, J., Chen, J., Yu, G., Yuan, H., Hu, Y., Li, R., Feng, Ye, M., Guo, G., Li, Y., Li, R., Zhang, X., Ma, L., Kristiansen, K., Guo, Q., Jiang, J.,
T., Ye, C., Lu, C., Wang, J., Li, S., Wong, G.K., Yang, H., Wang, J., Xiang, Z., Zhou, Z., Beck, S., Xia, Q., Wang, W., Wang, J., 2010. Single base-resolution methylome of the
Yu, J., 2005. SilkDB: a knowledgebase for silkworm biology and genomics. Nucleic silkworm reveals a sparse epigenomic map. Nat. Biotechnol. 28, 516–520.
Acids Res.. 33, D399–402. Xu, J., Dong, Q., Yu, Y., Niu, B., Ji, D., Li, M., Huang, Y., Chen, X., Tan, A., 2018. Mass
Wang, X., Zhang, P., Zhang, X., 2015. Abnormal purine metabolism and hearing loss. spider silk production through targeted gene replacement in Bombyx mori. Proc.
Chinese Journal of Otology 13, 754–756. Natl. Acad. Sci. U. S. A. 115, 8757–8762.
Wilkinson, M.E., Charenton, C., Nagai, K., 2019. RNA splicing by the spliceosome.Annu. Yasujima, T., Murata, C., Mimura, Y., Murata, T., Ohkubo, M., Ohta, K., Inoue, K.,
Rev. Biochem. Yuasa, H., 2018. Urate transport function of rat sodium-dependent nucleobase
Xia, Q., Zhou, Z., Lu, C., Cheng, D., Dai, F., Li, B., Zhao, P., Zha, X., Cheng, T., Chai, C., transporter 1. Physiol Rep 6, e13714.
Pan, G., Xu, J., Liu, C., Lin, Y., Qian, J., Hou, Y., Wu, Z., Li, G., Pan, M., Li, C., Shen, Yin, F., Wang, J., Zhao, K., Xin, C., Shi, Y., Zeng, X., Xu, H., Li, J., Chen, Q., 2020. The
Y., Lan, X., Yuan, L., Li, T., Xu, H., Yang, G., Wan, Y., Zhu, Y., Yu, M., Shen, W., Wu, significance of PA28gamma and U2AF1 in oral mucosal carcinogenesis. Oral Dis. 26,
D., Xiang, Z., Yu, J., Wang, J., Li, R., Shi, J., Li, H., Li, G., Su, J., Wang, X., Li, G., 53–61.
Zhang, Z., Wu, Q., Li, J., Zhang, Q., Wei, N., Xu, J., Sun, H., Dong, L., Liu, D., Zhao, Yu, G., Wang, L.-G., Han, Y., He, Q.-Y., 2012. clusterProfiler: an R package for comparing
S., Zhao, X., Meng, Q., Lan, F., Huang, X., Li, Y., Fang, L., Li, C., Li, D., Sun, Y., biological themes among gene clusters. OMICS-A JOURNAL OF INTEGRATIVE
Zhang, Z., Yang, Z., Huang, Y., Xi, Y., Qi, Q., He, D., Huang, H., Zhang, X., Wang, Z., BIOLOGY 16, 284–287.
Li, W., Cao, Y., Yu, Y., Yu, H., Li, J., Ye, J., Chen, H., Zhou, Y., Liu, B., Wang, J., Ye, Zebian, A., Shaito, A., Mazurier, F., Rezvani, H.R., Zibara, K., 2019. XPC beyond
J., Ji, H., Li, S., Ni, P., Zhang, J., Zhang, Y., Zheng, H., Mao, B., Wang, W., Ye, C., Li, nucleotide excision repair and skin cancers. Mutat. Res. 782, 108286.