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Article

Whole Transcriptome-Based Study to Speculate upon the Silkworm Yellow Blood Inhibitor (I) Gene and Analyze the miRNA-Mediated Gene Regulatory Network

by
Qi Ge
1,2,†,
Yixuan Fan
2,†,
Jia Xu
2,
Liang Chen
2,
Shangshang Ma
2,3,
Rehab Hosny Taha
4,
Qin Yao
2,
Yi Yuan
2,3,* and
Keping Chen
2,*
1
School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China
2
School of Life Sciences, Jiangsu University, Zhenjiang 212013, China
3
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
4
Plant Protection Research Institute, Agricultural Research Center, Dokki-Giza 8655, Egypt
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Processes 2022, 10(8), 1556; https://doi.org/10.3390/pr10081556
Submission received: 9 July 2022 / Revised: 4 August 2022 / Accepted: 4 August 2022 / Published: 8 August 2022
(This article belongs to the Section Biological Processes and Systems)
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Abstract

:
White cocoon is developed and used as a natural fiber, and different silkworm strains have different cocoon colors. Natural-colored cocoons are preferred by people, however, the cocoon color mainly settles on sericin and it basically falls off after reeling. Currently, there are no varieties applied to production due to the formation mechanism of cocoon color is not clear. The formation of cocoon color involves multiple gene regulations. Previous studies have shown that the main genes regulating cocoon traits are the yellow blood (Y) gene, yellow blood inhibitor (I) gene, and yellow cocoon (C) gene. Among them, the products of the Y gene and C gene have been studied, but the I gene is still unclear. In this study, the midgut tissues of the yellow (NB) and the white (306) cocoon silkworm were analyzed by whole transcriptome sequencing. The results showed that there are 1639 DE-circRNAs, 70 DE-miRNAs, and 3225 DE-mRNAs, including 1785 up-regulated genes and 1440 down-regulated genes. GO and KEGG annotation results indicated that DE-mRNAs are mainly involved in intracellular transport, signal transduction, lipid transport, and metabolic processes. Two key genes, KWMTBOMO10339 and KWMTBOMO16553, were screened out according to the annotation results, which were involved in amino acid transport and ion exchange function, respectively. The interaction analysis between ncRNA and target genes showed that there were five miRNAs regulating these two genes. The qPCR analysis showed that the I gene was down-regulated, and the miRNA expression profiles were most up-regulated. Therefore, during the yellow and white cocoon formation, KWMTBOMO10339 and KWMTBOMO16553 may be regulated by miRNA, resulting in the non-expression of KWMTBOMO10339 and KWMTBOMO16553 in yellow cocoon silkworm, and the pigment molecules can enter hemolymph from the midgut to form yellow blood, then transport to the middle silk gland to finally form yellow cocoons.

1. Introduction

The silkworm, Bombyx mori L., is an important economic insect and the foundation of the national silk industry, which has made great contributions to the development of China’s national economy [1]. At present, more than 1000 strains of silkworms are preserved all over the world. Among them, the white silkworm strain is the most extensive variety of sericulture [2]. The natural cocoon colors are white, yellow, pink, green, and so on. Carotenoids and flavonoids are the main pigments of these colored cocoons [3,4], the silkworm cannot synthesize these pigments by itself [5]. The carotenoids in mulberry leaves are absorbed from the midgut into the hemolymph and transported to the middle silk glands to produce silk protein and deposit in the sericin layer [6,7,8]. The permeation and absorption of these pigments are dominated by multiple alleles such as the blood color gene, inhibitory gene, and cocoon color basic gene.
Studies have confirmed that the cocoon traits of silkworms are mainly controlled by three genes, namely the yellow blood gene (Y), yellow blood inhibitor gene (I), and yellow cocoon gene (C). The Y gene mainly controls the absorption and transport of carotenoids in the midgut of the silkworm and directly determines the coloring of silkworm hemolymph [9]. The C gene determines whether the silkworm cocoon is yellow and the coloring of the inner and outer cocoons by controlling the absorption of pigment from the hemolymph to the silk gland [10]. The Y gene encodes a carotenoid binding protein (CBP), which contains a lipid-binding domain called steroidogenesis acute regulatory protein-related lipid transfer domain, which controls the uptake of carotenoids into intestinal mucosa and silk gland [11]. Sakudoh et al. showed that the Y gene was expressed in the midgut and hemolymph of yellow cocoon silkworm and speculated that it might be involved in the transport of carotenoids in the silk gland [12].
The I gene is an important control gene in the formation of the natural yellow cocoon, which prevents the transport of pigment from midgut epithelial cells to hemolymph, thereby inhibiting the expression of the Y gene. Currently, it is not clear about the I gene. The existence of the C gene alone cannot produce yellow cocoons. In other words, only when the Y and C genes are expressed at the same time and the I gene is not expressed then the cocoon will appear yellow. Otherwise, when the I gene exists, carotenoid pigment cannot penetrate the gland cavity through the silk gland tissue and become a white cocoon [13]. The I gene can oxidize and decompose carotenoids, thereby making body fluids colorless. Importantly, in the I gene mutant, the transfer of other lipids from the midgut to lipid proteins is normal, and only the transfer of carotenoids is affected [13].
The dominant I allele mutant silkworm shows outflow defects of lutein and β-carotene from the midgut cells to the hemolymph, resulting in fewer carotenoids in the hemolymph and leading to the larvae forming white cocoons [6,14]. It is speculated that the I gene may promote the efflux of lutein and β-carotene from midgut cells to hemolymph, and is associated with lipids, and may also be excreted from the silk gland cells to the fluid filaments of the silk gland lumen. Therefore, the larvae with I gene mutation have colorless hemolymph. At present, there are not many research reports on the I gene, and its molecular properties and functions are still unknown.
In this study, the I gene was identified from the perspective of omics, and it is speculated that the I gene might play a role in regulating pigment entering hemolymph in the midgut of the silkworm. Moreover, the I gene was not expressed in the yellow cocoon silkworm but highly expressed in the white cocoon silkworm. Based on this speculation, the midgut tissues of the yellow cocoon and white cocoon silkworm were taken for whole transcriptome analysis and verification.

2. Results

2.1. The Whole Transcriptome Sequencing Analysis of Yellow and White Cocoon Strain Silkworm B. mori

The high-throughput specific RNA-seq was performed on the midgut tissues of white blood strain silkworm 306 and yellow blood strain silkworm NB. Each group had three biological replicates. A total of 728,975,596 raw reads were obtained from the six libraries, with an average of 14.15 million reads per sample. The average number of clean reads that can be mapped on the genome is 9.96 million, and the average coverage rate is 79.52% (Table S1). Then the clean reads mapped to the silkworm genome were used for transcript assembly. A total of 16,067 transcripts were generated after filtration (Table S2). Among them, there are 3225 significant DE-mRNAs, 70 DE-miRNAs, and 1639 DE-circRNAs (Table S3). The differentially regulated mRNA, miRNA, and circRNA were generated and clustered using hierarchical clustering analysis (Figure 1).

2.2. Screening and Analysis of Differential Expression of Genes (DEGs)

According to transcriptome sequencing results, Fold Change ≥ 2 and p-value ≤ 0.05 were set as screening conditions. As shown in Figure 2A, there were 3225 DEGs in yellow cocoon silkworm NB compared with white cocoon silkworm 306, of which 1785 up-regulated genes and 1440 down-regulated genes. The results show that some DEGs may be involved in the regulation of the formation of the yellow and white cocoons, and the specific regulatory genes still need to be further analyzed. In addition, Figure 2B shows that there are 70 DE-miRNAs in the yellow cocoon silkworm NB compared with white cocoon silkworm 306, of which 32 up-regulated DE-miRNAs and 38 down-regulated DE-miRNAs. In Figure 2C, there are 1639 DE-circRNAs, of which 1785 are up-regulated and 1440 down-regulated.

2.3. Function Annotation and Enrichment Analysis of DEGs

Based on sequence homology, 2903 DEGs were finally mapped to four different classifications of the Clusters of Orthologous Groups (COG) and 22 COG types (Table S4). Except for the COG with “unknown function” S category (1585 single gene), other genes are mainly involved in cellular processes and signaling, information storage and processing, and metabolisms, such as intracellular trafficking, secretion, and vesicular transport, replication, recombination and repair, and lipid transport and metabolism. Among them, the largest COG group is “replication, recombination and repair” (209 single genes), followed by “post-translational modification, protein conversion” (203 single genes), and “intracellular transport, secretion, and vesicle transport” (172 single genes) and so on (Figure 3A, Table S4).
A total of 2615 genes were annotated by Gene Ontology (GO) analysis (Figure 3B, Table S5), of which 2088 genes were enriched into the category of biological process, including metabolic process and cellular process, biological regulation, localization, and so on; and 2170 genes were classified into the cellular component category, including membrane part, cell part, organelle, and also 2579 genes were classified into molecular function category, including binding, transporter activity, molecular function regulator, molecular translator activity and so on. Kyoto Encyclopedia of Genes and Genomes (KEGG) mainly involves metabolism (655 sequences), genetic information processing (267 genes), environmental information processing (278 genes), cellular processes (280 genes), organic systems (669 genes), and human diseases (828 genes) (Figure 3C, Table S6).

2.4. Screening of Yellow Blood Inhibitor Gene (I gene)

The expression of DEGs in the yellow cocoon silkworm NB was set as 0, while in the white cocoon silkworm, 306 were more than 0. Then the Venn analysis was performed between these two groups; the results show that there are 1392 differentially non-expressed genes in the NB group and 11,365 DEGs in the 306 group, of which 92 DEGs were not expressed in both groups (Figure 4). According to Tsuchida et al. speculation [6], the I gene may be mainly involved in the function of ion pump exchange and transport. According to these 92 DEGs annotation information, KWMTBOMO10339 is mainly located in the inner membrane of cells and performed the function of metal carboxypeptidase activity and metal ion binding, and was similar to the function of sodium-dependent nutritional amino acid transporter in Bombyx mandarina. KWMTBOMO16553 is mainly involved in the signal transduction pathway. Therefore, we finally concluded that KWMTBOMO10339 and KWMTBOMO16553 might be the yellow blood inhibitory gene (I gene) involved in regulating the transport of pigment plasma during the formation of silkworm yellow and white cocoons.

2.5. Sequence Alignment and Phylogenetic Tree Analysis of Yellow Blood Inhibitory Genes

The results show that the amino acid sequence similarity of KWMTBOMO10339 and putative amino acid transporter (NP_001124343.1) in B. mori, sodium-dependent nutrient amino acid transporter (XP_004927040.1), sodium-and chloride-dependent glycine transporter 2 (XP_004929221.1) and sodium- and chloride-dependent GABA transporter 1 (XP_004923491.1) was 100%, 99%, 93%, 88%, respectively (Figure 5A). In addition, the sequence similarity of KWMTBOMO16553, Na(+)/H(+) exchange regulatory cofactor NHE-RF1-like (XP_037877285.1), sorting nexin-27 like (XP_028040228.1), sorting nexin-27-like (XP_028040228.1), tax1-binding protein 3 homolog (XP_004930049.1) was 76%, 78%, 66%, 37%, respectively (Figure 5B). To further analyze the characteristics of KWMTBOMO10339 and KWMTBOMO16553, the phylogenetic tree of silkworm yellow blood inhibitor gene and its homologous sequence was constructed by MEGA software. The result shows that the genetic relationship of KWMTBOMO10339 was close to the sodium-dependent neutral amino acid transporter 1 in B. mori (Figure 6A), and KMWTBOMO16553 was close to Na(+)/H(+) exchange regulatory cofactor NHE-RF1-like protein (Figure 6B).

2.6. Construction of the circRNA-miRNA-mRNA Regulatory Network

Noncoding RNA has a wide range of regulatory effects. It can not only directly regulate DNA structure, RNA transcription, and translation but also bind miRNA as a sponge in competition to inhibit the regulatory effect of miRNA on target genes [15]. Based on the competitive endogenous RNA (ceRNA) theory, a circRNA-miRNA-mRNA regulatory network was constructed at the genome-wide transcription level. Firstly, the DE-mRNAs that regulate the formation of yellow and white cocoons were analyzed through Venn screening, and the target mRNAs corresponding to DE-miRNA was analyzed by using miRanda software (Table S7). Then, the target circRNAs of DE-miRNA were analyzed, and the DE-miRNA-DE-circRNA data (Table S8) was constructed based on the DE-circRNA data (Table S3). Finally, a comprehensive analysis was carried out by using Cytoscape software to build a regulatory network based on DE-circRNA-DE-miRNA-DE-mRNA (Table S8). As shown in Figure 7, two target genes (KWMTBOMO10339 and KWMTBOMO16553) act on five miRNAs, namely bmo-miR-2839-5P, bmo-miR-2808b, bmo-miR-2808a-5P, bmo-miR-745-5P, bmo-miR-274-5P, of which DE-mRNA expression was down-regulated in yellow cocoon line NB compared to white cocoon line 306, while DE-miRNA expression was up-regulated. In addition, these five DE-miRNAs regulate 15 DE-circRNAs, respectively.

2.7. The Expression Profile Verification of I Gene and the Regulated miRNA between the Yellow and White Cocoon Silkworm

To evaluate the differences in the I gene (KWMTBOMO10339 and KWMTBOMO16553) and the five regulated miRNA (bmo-miR-2839-5P, bmo-miR-2808b, bmo-miR-2808a-5P, bmo-miR-745-5P, and bmo-miR-274-5P) relative expression profiles between the yellow and white cocoon silkworm, the RT-qPCR was performed for validation. Compared with the white cocoon silkworm group (306), the I gene, KWMTBOMO10339 and KWMTBOMO16553, were all downregulated in the yellow silkworm group (NB) (Figure 8A). The result of relative expression level was consistent with the RNA-seq data. Besides, except for the down-regulated expression profiles of bmo-miR-2808a-5P, the expression level of bmo-miR-2839-5P, bmo-miR-2808b, bmo-miR-745-5P, and bmo-miR-274-5P in NB group were all up-regulated compared with the 306 group (Figure 8B).

3. Discussion

In this study, the whole transcriptome sequencing was performed on the midgut tissues of the yellow cocoon silkworm NB and the white cocoon silkworm 306. According to the different expression of the I gene in NB and 306 strains, two genes KWMTBOMO10339 (XP_028027180.1) and KWMTBOMO16553 (XP_004923504.1), were selected that may be involved in the flow of pigment from midgut cells to the hemolymph (Figure 4). GO function annotation results show that the I gene is mainly involved in the integral component of membrane (GO:0016021) and has the function of metallocarboxypeptidase activity (GO:0004181), neurotransmitter (GO:0005328), and zinc ion binding (GO:0008270). Non-redundant protein (NR) annotation results show that it is similar to sodium-dependent nutrient amino acid transporter. In addition, the total gene length of KWMTBOMO16553 was 583bp, and the result of GO functional annotation showed that this gene mainly performed the function of protein binding (GO:0005515). KEGG enrichment results showed that KWMTBOMO16553 was mainly involved in aldosterone-regulated sodium reabsorption (map04960). According to Tsuchida and Sakudoh’s analysis [6], based on the expression of these two genes in yellow cocoon NB and White cocoon 306 and the annotation information of their molecular functions, we speculated that these two genes were most likely to be yellow blood inhibitory genes and involved in the transport of pigment from midgut cells to the hemolymph.
Sequence alignment result shows that the amino acid sequence of KWMTBOMO10339 has the highest similarity with the putative amino acid transporter (NP_001124343.1) and sodium-dependent nutrient amino acid transporter (XP_004927040.1) in the silkworm. And the phylogenetic tree results also indicate that this gene is homology to the sodium-dependent neutral amino acid transporter (Figure 5A and Figure 6A). In addition, the amino acid sequence of KWMTBOMO16553 has the highest similarity with the sequence of Na(+)/H(+) exchange regulatory cofactor NHE-RF1-like (XP_037877285.1) in the silkworm, and the phylogenetic tree construction result shows that the KWMTBOMO16553 is homology to Na(+)/H(+) exchange regulatory cofactor NHE-RF1-like protein (Figure 5B and Figure 6B). Martin et al. used a new GFP link reporting system combined with in vitro X-ray crystallography and molecular dynamics studies to describe the molecular determinants of the interaction between Na/H exchange regulatory co-factor NHE-RF1 and the cystic fibrosis transmembrane conductance regulator (CFTR) [16]. Among them, the PDZ domain is one of the most common protein interaction modules, and more than 500 proteins containing the PDZ domain have been identified in the human genome [17]. The PDZ domain performs its scaffolding function by recognizing short linear PDZ-binding primitives, which are usually present at the C-terminus of receptors and ion channels [18]. The KWMTBOMO16553 identified in the silkworm this time also has the PDZ protein domain. Therefore, we speculate that the KWMTBOMO16553 gene may interact with other proteins through this domain and play an ion exchange function.
What’s more, existing reports indicate that only a small part of genes encode proteins in eukaryotic genomes, and more than 97% of transcription products are non-coding RNAs [19], of which miRNA is a 19/25 nt single-stranded small non-coding RNA, widely found in animals, plants and nematodes [20]. miRNA regulates gene expression after transcription through binding to the 3′UTR of mRNA target, leading to its degradation or translation inhibition [21]. Moreover, the transcription of target gene mRNA can be regulated through sequence complementary pairing [22], which regulates many biological pathways and participate in many important life processes, such as cell proliferation, cell differentiation, cell death, metabolism, immunity, development, and so on [23,24]. Wang et al. revealed that a Let-7 microRNA in the silkworm genome could control the cell growth and development of silk glands by coordinating nutrient metabolism and energy signaling pathways [25]. Liu et al. showed that miR-2738 is a secondary regulator of sex-determining genes in silkworms [26]. Liu et al. indicated that miR-14 is a general regulator that maintains the homeostasis of silkworm ecdysone and promotes its normal development and metamorphosis [27]. He et al. also showed that microRNA-14 could be used as an effective inhibitor to shut down ecdysone production after insect molting [28]. So far, a large number of miRNAs have been found in the genomes of higher organisms, but the biological functions of most miRNAs are still unclear, and there are few studies on the functions of miRNAs in the genomes of silkworms. In this study, miRanda software was used to predict and analyze the target genes of miRNA, and a total of five miRNAs that interact with KWMTBOMO10339 and KWMTBOMO16553, which are bmo-miR-2839-5P, bmo-miR-745-5P, bmo-miR-2808a-5P, bmo-miR-2808b, bmo-miR-274-5P, respectively, and also interact with 15 circRNAs (Figure 7). It is speculated that it may be involved in the regulation of yellow blood inhibitor genes during the formation of yellow and white cocoons in silkworms.
The formation of silkworm cocoon color is regulated by multiple gene groups. The wild-type silkworm produces yellow cocoons, yellow hemolymph, and its genotype is [Y +I C]. The other three main mutant genotypes are [Y +I + C], with white cocoons and yellow hemolymph; [Y I C], which has white cocoons and colorless hemolymph; and [+Y + I C], which has one White cocoon, colorless hemolymph, and colorless midgut [6]. Only larvae with phenotype [Y + I C] will produce yellow cocoons, and all other gene combinations will form white cocoons [14]. In other words, only when Y and C genes exist at the same time will the cocoon will appear yellow or only when one of the genes is expressed will the cocoon appear white [12,13,29]. The genetic analysis of silkworms shows that the formation of the yellow cocoon is mainly controlled by the yellow blood gene (Y), yellow inhibitor gene (I), and yellow cocoon gene (C). Among them, the Y gene controls the absorption of carotenoids from the lumen to the midgut epithelium; the I gene inhibits the transport of carotenoids from the midgut epithelium to hemolymph; the C gene regulates the entry of carotenoids from hemolymph into the silk gland. Y gene products can directly bind carotenoids to promote their transport in silkworms [7,12,30]. In addition, during the formation of the yellow cocoon, another important gene, the C gene, also called the Cameo2 gene, belongs to the transmembrane lipoprotein receptor of the CD36 protein family [29]. Further, the pigment can enter the lumen of the middle silk gland from the hemolymph through the action of the C gene and be absorbed by sericin to become a yellow cocoon [6,29,31,32]. To form colorful cocoons in silkworms, carotenoids from mulberry leaves must pass through the midgut into the silk glands. This whole process is coordinated systematically by many factors, among which Cameo2 and CBP are involved in the transportation of yellow cocoons of silkworm and larval carotenoids, both of which are the most important transport carriers for the absorption and transport of xanthophyll cells. The accumulation of cell lutein requires the simultaneous expression of Cameo2 and CBP. To deliver carotenoids to cocoons, the carotenoids contained in mulberry leaf digests in the intestinal lumen must first be ingested into intestinal cells. Whether carotenoids can be ingested into intestinal cells is determined by the gene at the Y gene locus. When the Y locus is dominant Y, carotenoids can be taken into intestinal cells; when the Y locus is recessive +Y, carotenoids cannot be taken into intestinal cells and thus cannot reach the cocoon [30].
Researchers have found that STARD3, which is homologous to CBP, specifically binds to lutein in the macula of the human retina [33]. These proteins with START domain are mainly located in the cytoplasm, nucleus, and Golgi apparatus rather than the plasma membrane [34]. Therefore, CBP acts as a cytoplasmic transporter, binding and transporting lutein from the plasma membrane to the cytoplasm. In this study, Cameo2, as a membrane protein, is responsible for recognizing lutein; CBP, as a cytoplasmic protein, captures lutein from the plasma membrane and makes it diffuse into the cytoplasm. In the yellow cocoon silkworm NB, the I gene may be inhibited by miRNA, making the I gene unable to be expressed, while the CBP is normally expressed in the midgut and mid silk gland [9], and the pigment molecules can be transported by CBP from the midgut to the hemolymph and silk glands. Finally, the yellow cocoon silkworm NB shows yellow in hemolymph, silk gland, and cocoon (Figure 9A). In the white cocoon silkworm 306, the I gene could be normally expressed, which inhibited the carotenoid molecules entering the hemolymph from the midgut and eventually formed white blood and white cocoon character in 306 strain (Figure 9B).

4. Materials and Methods

4.1. Silkworm Strain and Sample Preparation

The yellow cocoon silkworm (NB) (N = 30) and the white cocoon silkworm (306) (N = 30) are both reared in the silkworm room of the School of Life Sciences, Jiangsu University. The silkworm larvae were fed with fresh mulberry leaves at 25 ± 1 °C and 75% ± 3% relative humidity. At the fifth instar, the silkworm was dissected and the midgut and middle silk gland tissues were taken for experiment [10]. For each silkworm tissue, five isolated midgut tissues were combined into one sample to reduce individual genetic differences. The collected sample tissues were quickly placed in liquid nitrogen and stored at −80 °C. The whole transcriptome sequencing analysis was carried out by Shanghai Majorbio Co., Ltd. (Shanghai, China), and the data analysis was performed on the Majorbio Cloud platform free online platform (www.majorbio.com, accessed on 1 July 2021). And all the RNA-seq data were deposited in the National Center for Biotechnology Information (NCBI) database under the accession number PRJNA835915.

4.2. RNA Extraction, Library Construction, and Sequencing

The total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol and the integrity was checked firstly on 1% agarose gels. The quality and concentration of RNA (A260/280 = 1.8 – 2.0; A260/230 > 2.0) were measured using Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) and Qubit RNA Analysis Kit (Life Technologies, Carlsbad, CA, USA). The mRNA was enriched by using Oligo (dT) magnetic beads and a fragmentation buffer was added to break mRNA into short fragments; then, mRNA was used as a template to synthesize one-strand cDNA with random hexamers. After purifying the ds-cDNA, the ends were repaired, the A tail and connected adapter were added, the AMPure XP beads were used for fragment size selection, and finally, PCR was performed to obtain the cDNA library. SeqPrep (https://github.com/jstjohn/SeqPrep, accessed on 1 July 2021) and Sickle (https://github.com/najoshi/sickle, accessed on 1 July 2021) were used to perform quality control on the original sequencing data for obtaining high-quality control data (clean data) and ensure the accuracy of subsequent analysis results. Trimming the bases with low quality (mass value less than 20) at the end of the sequence (3′ end). If there are bases with a mass value less than 10 in the rest of the sequence, the whole sequence will be removed; otherwise, it will be retained. All the clean reads were compared with the silkworm reference genome (https://sgp.dna.affrc.go.jp/KAIKObase/, accessed on 1 July 2021) to obtain mapped data (reads) for subsequent transcript assembly and expression calculation [35]. Meanwhile, evaluating the comparison results of the transcriptome. Based on the selected reference genome sequence, the StringTie (http://ccb.jhu.edu/software/stringtie/, accessed on 1 July 2021) or Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/, accessed on 1 July 2021) software was used to splice Mapped Reads, and compare with the original genome annotation information, search the original unannotated transcription regions, then discovering new transcripts and new genes of the species, thus complementing and perfecting the original genome annotation information. The annotation file (gtf/gff) is included on the online platform of Majorbio Cloud Platform.

4.3. Identification of DEGs, DE-miRNAs, and DE-circRNAs

After the read counts of genes were obtained, DESeq2 [36] was used to analyze the differential expression of genes in various samples. According to the significance level p-adjust > 0.05 and the difference ratio between up-regulated and down-regulated genes was greater than 2, the DEGs were identified. Benjamini and Hochberg’s (BH) method was used for multiple test corrections. StringTie (http://ccb.jhu.edu/software/stringtie/, accessed on 1 July 2021) was used to calculate the fragments per kilobase of transcript per million fragments mapped (FPKM) of both miRNAs and circRNAs in each sample. miRNA expression levels were estimated by transcript per million (TPM). DE-miRNAs was performed using the DESeq R package [37], and the p-value < 0.05 and |log2 (fold change)| > 1 were considered as significantly differential expression. The differential expression analysis of DE-circRNA was also performed by the edgeR [38] software package.

4.4. GO and KEGG Enrichment Analysis

After obtaining the DEGs, GOATOOLS [39] was used to perform GO enrichment analysis on the DEGs and describe its function in combination with the GO annotation result. The number of differential genes included in each GO term was counted, and the significance P-value of differential gene enrichment in each GO term was also calculated according to the hypergeometric distribution test method. p ≤ 0.05 indicated that differential genes were enriched in this GO term. KEGG enrichment analysis was performed for differential genes, and the significant p-value of differential gene enrichment was calculated. p ≤ 0.05 also indicated that differential genes were enriched in the KEGG terms. Through the pathway analysis of differential genes, the enrichment pathways of target genes can be found and explored in the metabolic pathways that DEGs may participate in.

4.5. Screening and Identification of Yellow Blood Inhibitor Gene (I Gene)

The transcriptome sequencing results were normalized with FPKM, and the obtained P-values were compared and adjusted multiple times using the Benjamini and Hochberg methods [40]. According to the previous speculation, we believe that when the I gene is not expressed, silkworm shows the yellow cocoon trait and when the I gene is expressed, silkworm will show the white cocoon trait, and the function of the I gene is mainly related to channel switch and ion transport [10,14]. Therefore, based on the transcriptome sequencing results, the DEGs expression was set to be 0 in yellow cocoon silkworm NB and > 0 in white cocoon silkworm 306. Besides, the NR annotation was mainly involved in ion exchange and transport function. The NCBI BLAST (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 January 2022) and DNAMAN software were used for s multiple sequence alignment of the screened yellow blood inhibitory genes. The Neighbor-Joining tree was generated by MEGA7. One thousand bootstraps were performed to check the reproducibility of the result.

4.6. LncRNA and the Target Gene Interaction Network Construction

LncRNA is a type of non-coding RNA with a length of more than 200 nt, which widely exists in various organisms and can play a critical role in regulating life activities at various levels, including epigenetic, transcription, and post-transcription. The reported lncRNA sequences were downloaded from the lncRNA-related database, including the NCBI databases (https://www.ncbi.nlm.nih.gov/bioproject/634502, accessed on 1 January 2022) (accession number is PRJNA634502). Then the known lncRNAs were identified according to these databases. In addition, the reads of the reference genome were compared with miRBase and Rfam databases to obtain the annotation information of known miRNA and ncRNA, respectively. The Mirdeep2 software (https://www.mdc-berlin.de/content/mirdeep2-documentation, accessed on 1 July 2021) was used to predict new miRNAs for Reads without annotation information. Circular RNA (circRNA) is a special kind of non-coding RNA molecule, which has a closed circular structure, is not affected by RNA exonuclease, has more stable expression, and is not easy to degrade. CircRNAs are mainly formed by the reverse connection between the splicing donor site of the downstream exon and the splicing acceptor site of the upstream splicing exon. Based on Back splice junction (BSJ) reads, CIRI2 [41] and find_circ [42] were used for circRNA prediction and compared with the circBase database. To explain the interaction between ncRNA and target genes, the data of the interaction between ncRNA and target genes were imported into Cytoscape 3.2.1 software [43] through information integration and data mining to construct an interaction network.

4.7. miRNA Reverse Transcription and qRT-PCR Analysis

Total RNA was reverse transcribed into a cDNA template according to the instruction of HiScript®Ⅲ RT SuperMix for qPCR (+gDNA wiper) kit (R323-01) and miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (MR101-01/02) (Vazyme, Nanjing, China). A total of two I genes (KWMTBOMO10339 and KWMTBOMO16553) and five miRNAs (bmo-miR-2808b, bmo-miR-274-5p, bmo-miR-2839-5p, bmo-miR-2808a-5p, and bmo-miR-745-5p) were chosen to verify the expression. The β-actin and U6 were selected as the inner reference. All the primer information is shown in Table S9. The qRT-PCR reaction was performed by using the Applied Biosystems® QuantStudio 3 (ABI, Carlsbad, USA) StepOnePlus™ System with AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The total volume of qRT-PCR reactions was 20 μL, which contains 2 μL cDNA, 0.4 μL specific primers (10 μM), 10 μL 2 × AceQ Universal SYBR qPCR Master Mix, and 7.2 μL ddH2O. The reaction conditions were as follows: Stage 1: 95 °C for 5 min; Stage 2: 95 °C for 10 s, 60 °C for 30 s, 40 cycles; Stage 3: 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s. The 2−ΔΔCt method was used to analyze the gene expression level.

4.8. Statistical Analysis

All of the samples were analyzed in triplicate. ANOVA and Tukey’s tests were used for data analysis, and statistical significance was set at p < 0.05. The histograms of the results were analyzed and created by the GraphPad Prism 7 software (San Diego, CA, USA).

5. Conclusions

In summary, the formation of lutein-related yellow cocoons requires the simultaneous expression of Cameo2 and CBP in the midgut and silk glands of the silkworm. Cameo2 and CBP are located in the cell membrane and cytoplasm, respectively, and interact to mediate the transmembrane transport of lutein. In addition, in the process of transmembrane transport of lutein, the I gene may not be expressed due to the regulation of miRNA. Eventually, under the coordinated action of the three genes, lutein molecules enter the hemolymph and the middle silk gland from the midgut to form yellow blood and yellow cocoons.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr10081556/s1, Table S1: The total reads and coverage rate of each groups; Table S2: The total transcripts information; Table S3: The differentially expressed mRNAs, miRNAs, circRNAs; Table S4: The COG classification of DEGs; Table S5: The GO annotation of DEGs; Table S6: The KEGG annotation of DEGs; Table S7: The prediction of miRNA target genes; Table S8: The interaction network of DE-circRNAs-DE-miRNAs-DE-mRNAs; Table S9: The detailed primers information of the I gene and the miRNAs.

Author Contributions

Writing—Original draft preparation, investigation, data curation., Q.G.; resources, investigation, Y.F.; data curation, J.X.; validation, L.C.; formal analysis, investigation, S.M.; formal analysis, R.H.T.; supervision, Q.Y.; methodology, funding acquisition, Y.Y.; supervision, project administration, funding acquisition, K.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant numbers 31872425, 31861143051, 31900359, and 31802140; The APC was funded by 31861143051.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

In this work, we acknowledge the online platform of Majorbio Cloud Platform (www.majorbio.com, accessed on 1 July 2021).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Hierarchical cluster analysis of whole transcriptome sequencing mRNA, miRNA, and circRNA in the yellow cocoon strain silkworm (NB) and the white cocoon strain silkworm (306). (A) DE-mRNA hierarchical cluster analysis, in which the relative expression level of mRNAs was expressed as a color range from a low level (−2, blue) to a high level (2, red); (B) DE-miRNA hierarchical cluster analysis, in which the relative expression level of miRNAs was expressed as a color range from a low level (−1, blue) to a high level (1, red); (C) DE-circRNA hierarchical cluster analysis, in which the relative expression level of circRNAs was expressed as a color range from a low level (−1, blue) to a high level (0.5, red).
Figure 1. Hierarchical cluster analysis of whole transcriptome sequencing mRNA, miRNA, and circRNA in the yellow cocoon strain silkworm (NB) and the white cocoon strain silkworm (306). (A) DE-mRNA hierarchical cluster analysis, in which the relative expression level of mRNAs was expressed as a color range from a low level (−2, blue) to a high level (2, red); (B) DE-miRNA hierarchical cluster analysis, in which the relative expression level of miRNAs was expressed as a color range from a low level (−1, blue) to a high level (1, red); (C) DE-circRNA hierarchical cluster analysis, in which the relative expression level of circRNAs was expressed as a color range from a low level (−1, blue) to a high level (0.5, red).
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Figure 2. The differential expression analysis of DEGs, DE-miRNAs, and DE-circRNAs in the yellow cocoon silkworm NB compared with white cocoon silkworm 306. (A): The volcano map analysis of DEGs; (B): The volcano map analysis of DE-miRNAs; (C): The volcano map analysis of DE-circRNAs; red dots represent up-regulated differentially expressed profiles, and the green dots represent down-regulated differentially expressed profiles.
Figure 2. The differential expression analysis of DEGs, DE-miRNAs, and DE-circRNAs in the yellow cocoon silkworm NB compared with white cocoon silkworm 306. (A): The volcano map analysis of DEGs; (B): The volcano map analysis of DE-miRNAs; (C): The volcano map analysis of DE-circRNAs; red dots represent up-regulated differentially expressed profiles, and the green dots represent down-regulated differentially expressed profiles.
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Figure 3. The COG, GO, and KEGG annotation of differentially expressed transcripts in the yellow cocoon silkworm NB compared with the white cocoon silkworm 306. (A) Clusters of Orthologous Groups of Protein (COG) classification; (B) Gene Ontology (GO) assignments; (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways annotation.
Figure 3. The COG, GO, and KEGG annotation of differentially expressed transcripts in the yellow cocoon silkworm NB compared with the white cocoon silkworm 306. (A) Clusters of Orthologous Groups of Protein (COG) classification; (B) Gene Ontology (GO) assignments; (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways annotation.
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Figure 4. Venn analysis of DEGs between yellow cocoon silkworm strain NB and white cocoon silkworm strain 306. Among them, the expression of DEGs in the NB group was 0, while the expression of DEGs in the 306 groups was more than 0.
Figure 4. Venn analysis of DEGs between yellow cocoon silkworm strain NB and white cocoon silkworm strain 306. Among them, the expression of DEGs in the NB group was 0, while the expression of DEGs in the 306 groups was more than 0.
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Figure 5. Multiple amino acid sequence alignment. (A) The alignment of KWMTBOMO10339 homologous protein; (B) The alignment of KWMTBOMO16553 homologous protein, the black box indicates the PDZ domain. Multiple sequence alignment columns with no gaps are colored in blue or red. The red color indicates highly conserved columns and blue indicates less conserved ones.
Figure 5. Multiple amino acid sequence alignment. (A) The alignment of KWMTBOMO10339 homologous protein; (B) The alignment of KWMTBOMO16553 homologous protein, the black box indicates the PDZ domain. Multiple sequence alignment columns with no gaps are colored in blue or red. The red color indicates highly conserved columns and blue indicates less conserved ones.
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Figure 6. The phylogenetic tree analysis of yellow blood suppressor genes. (A) Phylogenetic tree of KWMTBOMO10339 and other homologous proteins from B. mori; (B) Phylogenetic tree of KWMTBOMO16553 and other homologous proteins from B. mori.
Figure 6. The phylogenetic tree analysis of yellow blood suppressor genes. (A) Phylogenetic tree of KWMTBOMO10339 and other homologous proteins from B. mori; (B) Phylogenetic tree of KWMTBOMO16553 and other homologous proteins from B. mori.
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Figure 7. The display of circRNAs-miRNAs-mRNA regulatory network for cocoon color formation of the yellow and white cocoon silkworm, B. mori. The purple diamond represents circRNA, the red triangle represents miRNA, and the green circle represents mRNA.
Figure 7. The display of circRNAs-miRNAs-mRNA regulatory network for cocoon color formation of the yellow and white cocoon silkworm, B. mori. The purple diamond represents circRNA, the red triangle represents miRNA, and the green circle represents mRNA.
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Figure 8. The relative expression profile of the I gene and the regulated miRNA between the yellow and white cocoon silkworm. (A): The relative expression level of I gene (KWMTBOMO10339 and KWMTBOMO16553), NB group compared with 306 group; (B): The relative expression level of the miRNA (bmo-miR-2839-5P, bmo-miR-2808b, bmo-miR-2808a-5P, bmo-miR-745-5P, and bmo-miR-274-5P), NB group compared with 306 group. All the data was repeated thrice. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Figure 8. The relative expression profile of the I gene and the regulated miRNA between the yellow and white cocoon silkworm. (A): The relative expression level of I gene (KWMTBOMO10339 and KWMTBOMO16553), NB group compared with 306 group; (B): The relative expression level of the miRNA (bmo-miR-2839-5P, bmo-miR-2808b, bmo-miR-2808a-5P, bmo-miR-745-5P, and bmo-miR-274-5P), NB group compared with 306 group. All the data was repeated thrice. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
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Figure 9. The miRNAs regulate the yellow blood inhibitor gene (I gene) and participate in the absorption of pigment molecules in the yellow and white cocoon silkworm NB and 306. Among them, ACP represents absorbent of carotenoid pigment, CBP represents a carotenoid-binding protein, I gene represents the yellow blood inhibitor genes KWMTBOMO10339 and KWMTBOMO16553 that were identified in this work, and miRNA represents the five miRNAs that were analyzed in this work. (A): The mechanism of miRNA and I gene regulation to form a yellow cocoon in NB silkworm; (B): The mechanism of miRNA and I gene regulation to form a white cocoon in 306 silkworm.
Figure 9. The miRNAs regulate the yellow blood inhibitor gene (I gene) and participate in the absorption of pigment molecules in the yellow and white cocoon silkworm NB and 306. Among them, ACP represents absorbent of carotenoid pigment, CBP represents a carotenoid-binding protein, I gene represents the yellow blood inhibitor genes KWMTBOMO10339 and KWMTBOMO16553 that were identified in this work, and miRNA represents the five miRNAs that were analyzed in this work. (A): The mechanism of miRNA and I gene regulation to form a yellow cocoon in NB silkworm; (B): The mechanism of miRNA and I gene regulation to form a white cocoon in 306 silkworm.
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Ge, Q.; Fan, Y.; Xu, J.; Chen, L.; Ma, S.; Taha, R.H.; Yao, Q.; Yuan, Y.; Chen, K. Whole Transcriptome-Based Study to Speculate upon the Silkworm Yellow Blood Inhibitor (I) Gene and Analyze the miRNA-Mediated Gene Regulatory Network. Processes 2022, 10, 1556. https://doi.org/10.3390/pr10081556

AMA Style

Ge Q, Fan Y, Xu J, Chen L, Ma S, Taha RH, Yao Q, Yuan Y, Chen K. Whole Transcriptome-Based Study to Speculate upon the Silkworm Yellow Blood Inhibitor (I) Gene and Analyze the miRNA-Mediated Gene Regulatory Network. Processes. 2022; 10(8):1556. https://doi.org/10.3390/pr10081556

Chicago/Turabian Style

Ge, Qi, Yixuan Fan, Jia Xu, Liang Chen, Shangshang Ma, Rehab Hosny Taha, Qin Yao, Yi Yuan, and Keping Chen. 2022. "Whole Transcriptome-Based Study to Speculate upon the Silkworm Yellow Blood Inhibitor (I) Gene and Analyze the miRNA-Mediated Gene Regulatory Network" Processes 10, no. 8: 1556. https://doi.org/10.3390/pr10081556

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