Overexpression of Ultrabithorax Changes the Development of Silk Gland and the Expression of Fibroin Genes in Bombyx mori

Ultrabithorax (Ubx) is a member of the Hox gene group involved in cell fate decisions, cell proliferation and organ identity. Its function has been extensively researched in Drosophila melanogaster but little is known about it in Lepidoptera. To uncover the function of Ubx in the development of lepidopterans, we constructed the Ubx overexpression (UbxOE) strain based on the Nistari strain of Bombyx mori. The UbxOE strain showed a small body size, transparent intersegmental membrane and abnormal posterior silk gland (PSG). In the current study, we focused on the effect of Ubx overexpression on the posterior silk gland. As the major protein product of PSG, the mRNA expression of fibroin heavy chain (Fib-H) and fibroin light chain (Fib-L) was upregulated three times in UbxOE, but the protein expression of Fib-H and Fib-L was not significantly different. We speculated that the overexpression of Ubx downregulated the expression of Myc and further caused abnormal synthesis of the spliceosome and ribosome. Abnormalities of the spliceosome and ribosome affected the synthesis of protein in the PSG and changed its morphology.


Introduction
Hox genes, a subset of homeobox genes, play a core role in regulating insect body patterns along the head-tail axis. Hox genes were initially discovered in Drosophila melanogaster (D. melanogaster), but it was later discovered that they can also control body morphology in mammals, including Mus musculus and Homo sapiens [1]. Hox genes are a group of transcription factor genes encoding a DNA binding motif containing 60 amino acids (Homeodomain), which is remarkably conserved throughout evolution [2][3][4]. Ubx, a member of the Hox gene group that interacts with Exd and Hth, has different functions in different regions of the wing imaginal disc in D. melanogaster [5,6]. The Hox genes of Bombyx mori (B. mori) and the Hox genes of D. melanogaster show high structural and sequential homology.
Ultrabithorax (Ubx) is a member of the bithorax complex [7][8][9] (consisting of Ultrabithorax, Abdominal A and Abdominal B in B. mori) [10], which plays a prominent role in the body plan. In D. melanogaster, Ubx plays a significant role in many areas. For instance, Ubx regulates forewing/hindwing differentiation [11] and is involved in autophagy [12] and the splicing biological pathway [13]. However, there have been few studies on Ubx in B. mori, although it is known that knockdown of Ubx impacts the development of the embryo [14].
Myc is targeted by Ubx, according to previous ChIP-seq [15], which was performed in wing discs of B. mori and D. melanogaster. Myc is a central regulator of growth and/or proliferation of many cell types [16], such as imaginal disc cells [17], polyploid cells [18,19], stem cells [20,21] and blood cells [22]. As an oncogene in mammalian tumour cells [23], Myc takes part in cell proliferation and differentiation, mainly by affecting DNA replication and the transition from the G1 to S phase [24][25][26]. Previous studies in B. mori also confirmed that Myc is involved in the regulation of the cell cycle and DNA replication [27]. 2 of 14 In B. mori, knockdown of Ubx at the embryonic stage causes an additional pair of thoracic leg-like protuberances in A1 [14]. However, the actual functions of Ubx remain unclear. In this study, the overexpression of Ubx in B. mori affected the shape of the posterior silk gland (PSG), the transparency of the intersegmental membrane and the body size of the pupa. The production of fibroin heavy chain and light chain increased, but there was no significant effect on the production of silk protein. It is possible that the overexpression of Ubx inhibited Myc and related biological processes including ribosome biogenesis and the spliceosome. Our results suggest that Ubx plays a crucial role during the development of B. mori, especially of the silk gland.

Construction of Ubx Overexpression Strain
The whole coding sequence (CDS) of Ubx was cloned from the PSG of Nistari and was the same as the sequence in the NCBI (ID: NM_001114160.1). The structure of the plasmid for overexpressing Ubx is shown in Figure 1A. The plasmid was injected into eggs of Nistari. Transgenic individuals were screened using the red fluorescent labelling protein ( Figure 1B).
in wing discs of B. mori and D. melanogaster. Myc is a central regulator of growth and/or proliferation of many cell types [16], such as imaginal disc cells [17], polyploid cells [18,19], stem cells [20,21] and blood cells [22]. As an oncogene in mammalian tumour cells [23], Myc takes part in cell proliferation and differentiation, mainly by affecting DNA replication and the transition from the G1 to S phase [24][25][26]. Previous studies in B. mori also confirmed that Myc is involved in the regulation of the cell cycle and DNA replication [27].
In B. mori, knockdown of Ubx at the embryonic stage causes an additional pair of thoracic leg-like protuberances in A1 [14]. However, the actual functions of Ubx remain unclear. In this study, the overexpression of Ubx in B. mori affected the shape of the posterior silk gland (PSG), the transparency of the intersegmental membrane and the body size of the pupa. The production of fibroin heavy chain and light chain increased, but there was no significant effect on the production of silk protein. It is possible that the overexpression of Ubx inhibited Myc and related biological processes including ribosome biogenesis and the spliceosome. Our results suggest that Ubx plays a crucial role during the development of B. mori, especially of the silk gland.

Construction of Ubx Overexpression Strain
The whole coding sequence (CDS) of Ubx was cloned from the PSG of Nistari and was the same as the sequence in the NCBI (ID: NM_001114160.1). The structure of the plasmid for overexpressing Ubx is shown in Figure 1A. The plasmid was injected into eggs of Nistari. Transgenic individuals were screened using the red fluorescent labelling protein ( Figure 1B). To detect whether the imported sequence interrupted other genes, we detected the insertion site of the Ubx overexpression plasmid in the silkworm genome using inverse PCR. The PCR results showed one insertion site at 10,920,272 bp of chromosome Chr13 ( Figure 1C, details in Figure S1). Within 5 kb upstream and downstream of the insertion site, there were only two tRNAs but no coding gene. This suggested that the insertion of the Ubx overexpression plasmid had no influence on known coding genes in our Ubx OE strain.
To confirm whether Ubx was successfully overexpressed in transgenic individuals, we compared the expression levels in Ubx OE and Nistari strains in the embryo and fifth-instar stages. Given the important role of Ubx in the development of the embryo, we collected 4-day-old eggs from Ubx OE and Nistari. Each group contained three samples and each sample contained 30 eggs. The qRT-PCR results showed that although the expression of Ubx in Ubx OE was slightly higher than that in Nistari, the difference was no more than 1.5 times. There was no statistically significant difference between Ubx OE and Nistari ( Figure 2A). It has been demonstrated that the A3 promoter can activate the expression of exogenous genes at the embryo stage [28,29]. Therefore, we inferred that the inactivated overexpression of Ubx might be related to the epigenetic modification and chromatin activity [11]. Next, we chose to detect the expression levels of Ubx at the larval stage. It was extremely highly expressed in the head, midgut, middle silk gland, posterior silk gland and epidermis of Ubx OE fifth-instar larvae ( Figure 2B-F). According to the expression patterns of Hox genes, Ubx should not be expressed in the anterior of the body (e.g., head); however, expression was enhanced in the head of Ubx OE larvae. In the middle silk gland of Ubx OE , its expression level was 140 times higher than that of the wild type. In the posterior silk gland where Ubx was originally expressed, the overexpression of Ubx was nearly 40-fold. Ubx expression also increased in some other tissues at the fifth-instar stage, but with a low and non-significant fold change.

Phenotype of UbxOE Strain
Ubx OE and Nistari showed different phenotypes at the larval stage ( Figure 3). Firstly, the Ubx OE larvae were thinner than the Nistari larvae and the intersegmental membrane was transparent ( Figure 3A). The posterior silk gland of Ubx OE was shorter and had less curvature than that of Nistari ( Figure 3B). In addition, the pupa of Ubx OE was smaller and thinner than that of Nistari, and this difference was more significant in the female pupae ( Figure 3C). The pupal weight of Ubx OE was significantly lighter than that of Nistari, in both males and females ( Figure 3D). These changes in phenotype suggest that the overexpression of Ubx influences the development of silkworms.

Overexpression of Ubx Upregulated the Expression of Fibroin Genes but Inhibited the Synthesis of Fibroin Protein
We observed an abnormality in the posterior silk gland of Ubx OE . This might be associated with the content of fibroin proteins. Fibroin protein is composed of fibroin heavy chain (Fib-H), fibroin light chain (Fib-L) and P25 protein. We detected the mRNA expression level of Fib-H, Fib-L and P25 in the PSG of three-day-old fifth-instar Ubx OE larvae, and found that Ubx upregulated the mRNA expression level of these fibroin genes ( Figure 4A). RNA-seq data also supported this result (Section 2.4 and Table S1). However, Western blot analysis showed that there was no significant difference in protein level ( Figure 4B). This indicates that the overexpression of Ubx upregulated the expression of fibroin genes but not the synthesis of fibroin protein in Ubx OE . To further verify whether the overexpression of Ubx can activate the expression of Fib-H and Fib-L, Ubx was overexpressed in BmN cells and a dual-luciferase reporter (DLR) assay system was constructed, including the promoter of Fib-H and Fib-L ( Figure 4D). BmN cells derived from the ovaries of B. mori do not express fibroin genes. After overexpressing Ubx ( Figure 4C), the expression of Luciferase was initiated by the Fib-L promoter and the fluorescence signal was significantly enhanced, but that of the Fib-H promoter only increased slightly ( Figure 4D,E). This implies that Fib-L might be activated by Ubx. Ubx-OE, the transgenic strain with Ultrabithorax overexpressed, namely Ubx OE . Nistari, the control of Ubx OE . Error bars indicate the standard error of the mean (n = 3). Significant differences were assessed using Student's t-test (ns p > 0.05, ** p < 0.01, and *** p < 0.001).

Phenotype of UbxOE Strain
Ubx OE and Nistari showed different phenotypes at the larval stage ( Figure 3). Firstly, the Ubx OE larvae were thinner than the Nistari larvae and the intersegmental membrane was transparent ( Figure 3A). The posterior silk gland of Ubx OE was shorter and had less curvature than that of Nistari ( Figure 3B). In addition, the pupa of Ubx OE was smaller and thinner than that of Nistari, and this difference was more significant in the female pupae ( Figure 3C). The pupal weight of Ubx OE was significantly lighter than that of Nistari, in both males and females ( Figure 3D). These changes in phenotype suggest that the overexpression of Ubx influences the development of silkworms. (F) The difference in Ubx expression level in epidermis on 5L5D. Ubx-OE, the transgenic strain with Ultrabithorax overexpressed, namely Ubx OE . Nistari, the control of Ubx OE . Error bars indicate the standard error of the mean (n = 3). Significant differences were assessed using Student's t-test (ns p > 0.05, ** p < 0.01, and *** p < 0.001).

Transcriptome of PSG Revealed Differences in mRNA Level
To explore how Ubx overexpression affects the development of the posterior silk gland, we compared the differences in transcriptome between Ubx OE and Nistari. In total, 2904 differentially expressed genes were identified. Compared to Nistari, 1213 genes were upregulated and 1691 genes were downregulated in the posterior silk gland of three-day-old fifth-instar larvae ( Figure 5A, Table S1). After functional annotation and pathway enrichment analyses, 10 KEGG pathways and 56 enriched GO terms were screened ( Figure 5B,C, Table S2).
Most notably, we found that the results of GO enrichment analysis and KEGG enrichment analysis showed that most of the differentially expressed genes (DEGs) in Ubx overexpressed tissues were related to protein synthesis ( Figure 5B,C), suggesting that the abnormality of silk glands may be related to protein synthesis.

Identification of DEGs Potentially Regulated by Ubx and Myc
As a transcription factor, Ubx plays a crucial role in the regulation of transcription [30,31]. To identify the genes potentially regulated by Ubx directly, we performed an analysis combining RNA-seq data and ChIP-seq data ( Figure 6). Using the ChIP-seq data, 924 genes were identified as Ubx targets (Figures 6A and S2). Compared to RNA-seq, 203 DEGs out of 924 Ubx targets were identified (Table S3). In total, 108 genes were upregulated and 95 genes were downregulated in the PSG of the Ubx OE strain.  Statistical samples > 30. Significant differences were assessed using Student's t-test (*** p < 0.001).

Overexpression of Ubx Upregulated the Expression of Fibroin Genes but Inhibited the Synthesis of Fibroin Protein
We observed an abnormality in the posterior silk gland of Ubx OE . This might be associated with the content of fibroin proteins. Fibroin protein is composed of fibroin heavy chain (Fib-H), fibroin light chain (Fib-L) and P25 protein. We detected the mRNA expression level of Fib-H, Fib-L and P25 in the PSG of three-day-old fifth-instar Ubx OE larvae, and found that Ubx upregulated the mRNA expression level of these fibroin genes ( Figure 4A). RNA-seq data also supported this result (Section 2.4 and Table S1). However, Western blot  was initiated by the Fib-L promoter and the fluorescence signal was significantly enhanced, but that of the Fib-H promoter only increased slightly ( Figure 4D, E). This implies that Fib-L might be activated by Ubx.

Transcriptome of PSG Revealed Differences in mRNA Level
To explore how Ubx overexpression affects the development of the posterior silk gland, we compared the differences in transcriptome between Ubx OE and Nistari. In total, 2904 differentially expressed genes were identified. Compared to Nistari, 1213 genes were upregulated and 1691 genes were downregulated in the posterior silk gland of three-dayold fifth-instar larvae ( Figure 5A, Table S1). After functional annotation and pathway enrichment analyses, 10 KEGG pathways and 56 enriched GO terms were screened ( Figure  5B,C, Table S2).
Most notably, we found that the results of GO enrichment analysis and KEGG enrichment analysis showed that most of the differentially expressed genes (DEGs) in Ubx overexpressed tissues were related to protein synthesis ( Figure 5B,C), suggesting that the abnormality of silk glands may be related to protein synthesis.

Identification of DEGs Potentially Regulated by Ubx and Myc
As a transcription factor, Ubx plays a crucial role in the regulation of transcription [30,31]. To identify the genes potentially regulated by Ubx directly, we performed an anal-  in Ubx OE and Nistari. Ubx-OE, the transgenic strain with Ultrabithorax overexpressed, namely Ubx OE . Nistari, the wild type of Ubx OE . Error bars indicate the standard error of the mean (n = 3). Significant differences were assessed using Student's t-test (*** p < 0.001).
We further analysed the function of DEGs potentially targeted by Myc. The GO and KEGG enrichment results showed high consistency for all DEGs ( Figure S7, details in Table S6). In the top ten GO enrichment terms of DEGs targeted by Myc, all biological process terms, 90% of the cellular component terms and half of the molecular function terms were found in all DEGs. The top four KEGG pathways enriched in Myc target DEGs were also found in all DEGs. This result suggests that a large proportion of changes caused by overexpressing Ubx at the transcriptome level might act via the inhibition of Myc.

Discussion
Hox genes regulate the expression of many genes that produce transcription factors and determine the specificity of body segments in insects and mammals, and even play an important role in humans [32][33][34]. Ultrabithorax is a member of the bithorax complex and features in body segment specificity [11,35]. In this study, we constructed a Ubx overexpression silkworm using the piggyBac transposon system and named the resulting strain Ubx OE . Ubx OE showed a series of phenotypes different from the wild type such as small larvae, small pupae and a shorter PSG with less curvature. Nistari, the wild type of Ubx OE . Error bars indicate the standard error of the mean (n = 3). Significant differences were assessed using Student's t-test (*** p < 0.001).
We analysed the function of DEGs targeted by Ubx based on GO and KEGG ( Figure S3, details in Table S4). The GO enrichment results showed that the function of DEGs targeted by Ubx is mainly related to "purine nucleotide binding", "DNA-binding transcription factor activity", "nucleoside-triphosphatase activity", "pyrophosphatase activity" and "hydrolase activity". These DEGs participate in RNA, ncRNA, rRNA processing, and ribosome biogenesis. The KEGG enrichment analysis suggested that DEGs targeted by Ubx are mainly involved in "protein processing in endoplasmic reticulum", "apoptosis", "phagosome", the "MAPK signalling pathway", the "FoxO signalling pathway", "mitophagy" and the "Hippo signalling pathway".
However, DEGs potentially regulated by Ubx only accounted for 7.92% (230/2904) of all DEGs. Their annotation was also different from the annotation of all DEGs. Myc, a crucial transcription factor, can potentially regulate 1065 DEGs, according to ChIP-seq data for the D. melanogaster homologue, accounting for 36.67% (1065/2904) of all DEGs (Table S5). The RNA-seq and qRT-PCR results showed that it was significantly downregulated in the Ubx OE strain ( Figure 6D). The expression level of Myc in Nistari was approximately eight times higher than that of Ubx OE . To further confirm the regulation effect of Ubx on Myc, we knocked down the expression of Ubx in BmN cells and detected the change in Myc expression using qPCR. The result showed that the downregulation of Ubx enhanced the expression of Myc ( Figure S4). This is consistent with the observation at the individual level. Pathway analysis showed that it can be potentially regulated by the TGF-beta signalling pathway ( Figure S5), Wnt signalling pathway ( Figure S6), Hippo signalling pathway and MAPK pathway. Meanwhile, we also observed that the expression of Smad3, an upstream gene of Myc in the TGF-beta signalling pathway and Wnt signalling pathway, was also strongly inhibited in Ubx OE to approximately 10% of that in Nistari ( Figure 6E).
We further analysed the function of DEGs potentially targeted by Myc. The GO and KEGG enrichment results showed high consistency for all DEGs ( Figure S7, details in Table  S6). In the top ten GO enrichment terms of DEGs targeted by Myc, all biological process terms, 90% of the cellular component terms and half of the molecular function terms were found in all DEGs. The top four KEGG pathways enriched in Myc target DEGs were also found in all DEGs. This result suggests that a large proportion of changes caused by overexpressing Ubx at the transcriptome level might act via the inhibition of Myc.

Discussion
Hox genes regulate the expression of many genes that produce transcription factors and determine the specificity of body segments in insects and mammals, and even play an important role in humans [32][33][34]. Ultrabithorax is a member of the bithorax complex and features in body segment specificity [11,35]. In this study, we constructed a Ubx overexpression silkworm using the piggyBac transposon system and named the resulting strain Ubx OE . Ubx OE showed a series of phenotypes different from the wild type such as small larvae, small pupae and a shorter PSG with less curvature.
A previous study demonstrated that Hox genes participate in the development of the silk gland during embryonic and larval development. The expression of Antp was restricted in the middle silk gland (MSG), whereas Ubx was specifically expressed in the PSG [36]. This indicated that Ubx might be involved in regulating the expression of genes in the PSG. Our results demonstrated that overexpressing Ubx increased the expression of Fib-H and Fib-L. Although we speculated that the expression of Fib-H and Fib-L was enhanced after overexpressing Ubx, the content of fibroin protein in Ubx OE did not differ significantly from the wild type. This may be due to the low expression of Myc (Figure 7). We observed that overexpressing Ubx significantly inhibited the expression of Myc in the PSG. According to the GO and KEGG pathway analyses, the downregulation of Myc would impact the synthesis of the spliceosome and ribosomes that participate directly in the synthesis of fibroin proteins. Meanwhile, aberrantly expressed spliceosome and ribosomes also affect the synthesis of other proteins in the cells of PSG. The reduction in protein content would lead to a shorter PSG. Other evidence also suggests that the overexpression of dMyc can increase nucleolar size and cellular ribosome content in the salivary gland of D. melanogaster [37]. The overexpression of Myc enhanced the DNA replication and synthesis of silk protein in the silk gland of B. mori [27]. This proves our viewpoint from the opposite side. A well-known function of Myc is that it is involved in controlling organism size. For example, overexpressing dMyc increased the adult size of D. melanogaster while the hypomorphic dMyd mutation resulted in small adult flies [17,38]. In our research, the small body size of Ubx OE might also be related to the low expression of Myc.
Although we demonstrated that the expression of Myc was significantly inhibited in Ubx OE , the details are still unknown. Many regulators can activate or inhibit the expression of Myc. A famous pathway is the TGF-beta signalling pathway in which Smad3 activates p107, E2F4/5 and DP1 to inhibit the expression of Myc [39]. However, our result showed that both Smad3 and Myc were downregulated in the Ubx OE strain. The expression of Myc can also be activated by Yki/Sd via the Hippo signalling pathway [40]. However, we found that the expression of Yki and Sd was not significantly different between Ubx OE and Nistari. Previous studies showed that LEF/TCF can activate Myc via the Wnt signalling pathway in mammals but there is still no evidence for it in insects [41,42]. gland of D. melanogaster [37]. The overexpression of Myc enhanced the DNA replication and synthesis of silk protein in the silk gland of B. mori [27]. This proves our viewpoint from the opposite side. A well-known function of Myc is that it is involved in controlling organism size. For example, overexpressing dMyc increased the adult size of D. melanogaster while the hypomorphic dMyd mutation resulted in small adult flies [17,38]. In our research, the small body size of Ubx OE might also be related to the low expression of Myc. Although we demonstrated that the expression of Myc was significantly inhibited in Ubx OE , the details are still unknown. Many regulators can activate or inhibit the expression of Myc. A famous pathway is the TGF-beta signalling pathway in which Smad3 activates p107, E2F4/5 and DP1 to inhibit the expression of Myc [39]. However, our result showed Another possible explanation for the formation of a shorter PSG is a reduction in the number of PSG cells. The cell number in the silk gland is determined at the early and middle embryonic stages in B. mori [43]. A previous study reported that knockdown of Bmsage and BmDfd at the early embryonic stage decreased the number of cells in the PSG [44,45]. However, the effect of overexpressing Ubx on the morphology of the silk glands may not have been apparent at the embryonic stage in our study. In B. mori, the silk gland was formatted in the embryonic stage on the fourth day [46]. We detected the expression level of Ubx to explore whether overexpressing Ubx affects the development of the embryo. Contrary to expectations, the overexpression of Ubx had no significant effect in Ubx OE embryos. This may be associated with H3K27me3 modification at the embryonic stage [47]. Our inserted Ubx was not activated at the embryonic stage. Based on the above analysis, we suggest that abnormalities in the PSG were more likely due to the downregulation of Myc caused by the overexpression of Ubx.

Silkworm Strains
The non-diapause silkworms of the strain Nistari were obtained from the Sericulture Research Institute at the Chinese Academy of Agriculture Science, Zhenjiang, Jiangsu province. The silkworm eggs were cultured at a standard temperature of 25 • C under a photoperiod of 12 h light and 12 h dark. The larvae were reared on fresh mulberry leaves and maintained in a 12 h light and 12 h dark cycle at 25 • C with 80 ± 5% relative humidity.

Plasmids in This Study and Constructions of Transgenic Strain
The whole coding sequence of Ubx was cloned from RNA that was extracted from the posterior silk gland of Nistari. The piggyBac-based transgenic plasmid of pBac-DsRed-A3-Ubx was used to construct the overexpression strain of Ubx. The pBac-DsRed-A3-Ubx plasmid was microinjected into 0th generation (G 0 ) eggs of Nistari, which were then incubated at 25 • C in a humidified chamber until hatching. The G 1 silkworms were obtained from G 0 selfing. After hatching of the G 1 generation, successful transgenic silkworms were screened based on the DsRed protein carried on the plasmid. The plasmids of pIZT-mCherry-Ubx, pIZT-mCherry-V5-his-EGFP, pGL3-Fib-L, PGL3-Fib-H and pGL3-basic were used to verify the promoter activity in the BmN cells that were overexpressing Ubx.

Detection of Plasmid Insertion Site
Genome DNA was extracted from the PSG of the Ubx OE strain in third-day fifth-instar larva, and its quality is tested with a Nanodrop1000 (Thermo Fisher Scientific, Waltham, MA, USA). The genome was extensively digested by DpnII (NEB, Ipswich, MA, USA) and purified immediately using an AxyPrep PCR cleanup Kit (Axygen, Corning, NY, USA). The recovered fragments were cyclized using T4 ligase (Takara, Kusatsu, Japan). The cyclized product was used as a template, which used a pair of primers set inside the plasmid, for amplification. The primers are listed in Table S7. Finally, the amplified fragments were sequenced and aligned to the genome using NCBI BLAST to obtain the genome location.

Cell Culture, Transfection and Dual Luciferase Reporter Assay System
The BmN cells (provided by the Sericulture Research Institute at the Chinese Academy of Agriculture Science) were cultured in TC-100 insect medium (AppliChem, Darmstadt, Germany) that contained 10% foetal bovine serum (Gibco, Life technologies, New York, NY, USA) and 1% penicillin-streptomycin solution (Gibco) at 27 • C.
The plasmid of pIZT-mCherry-V5-his-Ubx was transfected into BmN cells using the Neofect DNA Transfection Reagent (Neofect biotech, Beijing, China) as described in the protocol, and the plasmid of pIZT-mCherry-V5-his-EGFP was used as a negative control. Cells were collected separately 0 h and 48 h after transfection. Collected cells were washed with PBS and soaked in Trizol (Takara, Japan) for storage at −80 • C.
For the dual luciferase reporter assay (DLR) experiment, pIZT-mCherry-V5-his-Ubx and pIZT-mCherry-V5-his-EGFP were separately co-transfected in BmN cells with pGL3-Fib-L, pGL3-Fib-H and pGL3-basic (negative control without promoter regions) plasmids (2 × 3 = 6 groups in total, with 3 replicates for each group) and the PRL-CMV plasmid as an internal reference in each repeat. The intensity of luciferase was detected with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and GLOMAX 20/20 LUMINOMETER (Promega) as described in the protocol.

Transgenic Strain Phenotypic Analysis
Through the microinjection technique, we obtained a Ubx overexpression transgenic strain and named it Ubx OE . For silk glands, silkworms on the 4th day of the fifth larva were dissected to obtain their complete silk glands. The silk glands were placed in phosphatebuffered saline and photographed after dissecting. Statistical differences were evaluated using Student's t-tests for unpaired samples. The level of statistical significance was set as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

RNA Extraction for qRT-PCR
Total RNA of Nistari and Ubx OE was extracted as described in the manual of Trizol (Takara). The RNA was then reverse-transcribed into cDNA using a PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara). The cDNA can only be used after the amplification of internal reference genes is verified to be free of problems. The qRT-PCR experiment was performed using a NovoStart SYBR qPCR SuperMix (Novoprotein, Beijing, China). The data were processed using lightcycler96 software (Roche, Basel, Switzerland) based on the 2 −∆∆ct method. All primers for qRT-PCR are listed in Table S7. The gene expression levels were compared using Student's t-tests.

RNA Library Construction for RNA-Seq
Posterior silk glands in fourth-day fifth-instar larva were applicated in the RNA-seq. After RNA extraction, a Fragment Analyzer 5400 (Agilent Technologies, Santa Clara, CA, USA) was used to ensure the integrity of total RNA. A NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB) was used to generate the sequencing libraries as per the manufacturer's recommendations, PCR products were purified (AMPure XP system) and library quality was assessed using the Agilent Bioanalyzer 2100 system. The cDNA libraries were sequenced on an Illumina Novaseq 6000 platform and paired-end reads with a length of 150 bp were generated. The sequencing was performed using Novogene (Beijing, China).

Analyzing Ubx Target Genes Based on ChIP-Seq and RNA-Seq Data
Cleaned reads were mapped to a reference genome from Kaikobase using hisat2 [48,49], and FeatureCounts was used to perform gene expression quantification [50]. DEseq2 was used to perform differential gene expression analysis [51]. We defined genes with |log2 Fold Change (Log 2 FC)| ≥ 1 and adjusted p value ≤ 0.05 as differentially expressed genes (DEGs), which were used to perform GO and KEGG enrichment analysis with the R package ClusterProfiler [52].
For ChIP-seq, raw data of B. mori Ubx were obtained from the NCBI SRA database under the accession PRJNA292691. The peak calling was performed using MACS2, while HOMER was used for motif analysis [53]. The GO and KEGG enrichment analysis were performed in the same way as RNA-seq. Finally, the peak of the hind wing and fore wing were merged for conjoint analysis with RNA-seq to compare the similarities and differences between RNA-seq and ChIP-seq [54].
To identify the target DEGs of Myc, the target gene of Myc of D. melanogaster was obtained from the ChIP-Atlas and converted with the corresponding silkworm homolog by Orthodb [55,56]. The circus plot was visualized with the R package circlize [57].

Protein Extraction and Western-Blot
Total proteins were extracted from the 3rd day of 5th larva using lysis buffer with prohibitor and PMSF. The mixture of ground PSG and lysis buffer was purified by centrifugation after stewing for 30 min. A Bradford Protein Assay Kit (Sangon, Shanghai, China) was used to detected the concentration of protein solution right after the centrifugation. The supernatant was added with 5× loading buffer with a volume of 1/5 then the sample was boiled for 10 min and stored at −80 • C. Samples were separated using SDS-PAGE at 80 V for 0.5 h and 120 V for 1 h, and then transferred to PVDF membranes. The primary antibodies of Fib-L and Fib-H (obtained from Professor Tan Anjiang, Science of Plant Physiology and Ecology, Chinese Academy of Sciences [58]) were incubated with target proteins, and tubulin antibody (1:4000, Bioss ANTIBODIES, Beijing, China) was used as an internal reference. Additionally, secondary antibody (HRP Goat Anti-Rabbit IgG, 1:5000, ABclonal Technology, Wuhan, China) was incubated with PVDF membranes combined with the first antibody. The membranes were incubated with an eECL Western Blot Kit (Vazyme, Nanjing, China) and photographed in the ChemiScope Western Blot Imaging System (CLINX, Shanghai, China).