Characterization of BmCeP, a Salivary Gland-Predominant Expression Promoter in the Silkworm Bombyx mori

TL;DR

Researchers identified a silkworm gene promoter that is active mainly in salivary glands, which could help study insect digestion and plant interactions.

Contribution

A salivary gland-specific promoter (BmCeP) was cloned and validated in transgenic silkworms for targeted gene expression.

Findings

BmCe is a salivary gland-specific gene in Bombyx mori.

BmCeP, a 2152 bp promoter region, drives DsRed expression predominantly in salivary glands.

BmCeP is a valuable tool for studying silkworm salivary gland function and insect-plant interactions.

Abstract

In this study, we identified a gene (BmCe, BGIBMGA010988) that is specifically expressed in salivary glands of the silkworm Bombyx mori. The promoter region of this gene (BmCeP) was cloned and used to construct a transgenic vector driving DsRed expression, and we duly obtained transgenic-positive individuals. Transcript analysis confirmed BmCeP as a salivary gland-predominant promoter, revealing its suitability for further subsequent functional studies of genes associated with the silkworm salivary gland.

The salivary gland is a key organ in insects that plays essential roles in food digestion, nutrient absorption, and energy metabolism, thereby highlighting the importance of studying salivary gland function for gaining a better understanding of nutritional utilization and insect–plant interactions. To date, however, a lack of salivary gland-specific promoters has limited functional analyses of salivary gland genes in Lepidoptera. In this study, based on microarray and salivary gland transcriptome data, we identified nine candidate genes characterized by high salivary gland expression. Semi-quantitative PCR analysis confirmed cholinesterase (BmCe, BGIBMGA010988) as the optimal candidate for promoter cloning. Temporal expression analysis revealed that the expression of BmCe reaches a peak during days 2–4 of the fifth larval instar. A 2152 bp fragment upstream of the transcription initiation site of BmCe was selected as the putative promoter sequence (designated BmCeP) and cloned to construct a piggyBac transgenic vector driving DsRed expression. Transgenic silkworms were obtained via embryonic microinjection and tissue expression analysis on day three of fifth-instar larvae revealed the predominant localization of DsRed expression in the salivary glands. In this study, we thus identified a gene promoter characterized by salivary gland-predominant expression in Bombyx mori, which we believe could serve as a valuable genetic tool for investigating the molecular mechanisms underlying silkworm nutritional utilization and interactions with its host plant, mulberry.

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1. Introduction

The saliva secreted by salivary glands contains a diverse array of enzymes, including α-glucosidase, amylase, protease, and lipase, that contribute to the initial breakdown and hydrolysis of food components. These enzymes partially degrade macromolecular nutrients in the oral cavity or external environment, thereby enhancing feeding efficiency and facilitating subsequent nutrient absorption in the gut [1]. Within the digestive system, salivary glands and the intestine, particularly the midgut, often function in a coordinated manner, with saliva initiating the preliminary degradation of food, after which intestinal enzymes further hydrolyze the smaller products of degradation, thereby facilitating absorption and subsequent transport. In addition to these digestive functions, saliva also plays multiple non-digestive roles, including lubrication, pH buffering, maintenance of the oral microenvironment, and participation in immune defense, all of which can indirectly influence feeding behavior and nutrient utilization [2]. Beyond their direct role in nutrient breakdown, insect oral secretions contain effector molecules and regulatory proteins that can suppress or modulate host plant defense responses, thereby indirectly enhancing feeding efficiency [3]. Conversely, some salivary components recognized by host plants can trigger defense responses, which in turn can influence insect feeding preference and growth [4].

Aphid saliva, secreted by the salivary glands during feeding, contains numerous soluble enzymes that play essential roles in establishing and maintaining feeding sites, suppressing plant defenses, and inducing physiological changes in host plants, thereby facilitating aphid feeding and nutrient uptake. Certain constituents of aphid saliva can also induce toxicity in host plants, highlighting the dynamic regulation of aphid–plant interactions [5]. The salivary glands of Drosophila, which have been extensively studied, consist of two major cell types, namely, secretory and duct cells, the former of which are columnar epithelial cells that synthesize and secrete large amounts of proteins, whereas the latter are cuboidal epithelial cells that form simple tubes connecting secretory cells to the larval mouth [6]. The contemporary domesticated silkworm (Bombyx mori), belonging to the order Lepidoptera and family Bombycidae, is derived from the wild silkworm (Bombyx mandarina), having undergone a prolonged period of domestication [7]. In China, the production of silk using the larvae of B. mori is of substantial economic and cultural significance, highlighting its status as a key economic insect species [8].

From the perspective of breeding improved silkworm strains, it is essential to gain a comprehensive understanding of the mechanisms underlying nutrient utilization. In this regard, long-term artificial selection focusing on economically important characteristics has markedly enhanced traits such as body weight, cocoon silk yield, and rate of egg hatching; consequently, modern silkworm strains require higher levels of material and energy input to sustain rapid larval growth and development [7]. Within the insect digestive system, the midgut serves as the primary organ for digestion and nutrient absorption and performs essential roles in food breakdown and nutrient uptake. Accordingly, most studies on the utilization of nutrients by B. mori have focused on the midgut [9]. However, a comprehensive understanding of nutrient utilization in silkworms also requires consideration of the salivary glands. Indeed, the findings of recent studies have revealed that the salivary gland of silkworms contains the signaling molecule cis-vaccenic acid, which plays an important role in regulating mulberry intake. Notably, compared with B. mandarina, the Bmdesat5 gene, which plays a key role in the synthesis of cis-vaccenic acid, is suppressed in domesticated silkworms. As a consequence of this suppressed expression, these silkworms consume larger quantities of mulberry leaves, thereby providing sufficient energy and materials for larval growth, development, and the spinning of silk, ultimately promoting rapid body enlargement and elevated levels of silk protein production [10].

By facilitating the spatial control or silencing of gene expression, tissue-specific promoters are widely used for investigating gene function in specific tissues, thereby avoiding systemic effects that may cause developmental abnormalities or lethality. Consequently, tissue-specific promoters play a central role in functional genomics research. Numerous such promoters have been identified and applied in insects. For example, in Drosophila, the muscle-specific promoter Mhc and neuron-specific promoter elav are widely used to drive tissue-specific transgene expression [11,12], and in Aedes aegypti, midgut-specific promoters have facilitated studies of parasite transmission and host response mechanisms [13]. In B. mori, several tissue-specific promoters have been successfully applied, including silk gland-specific promoters such as FibH, P25, and Ser1, which direct transgene expression to distinct silk gland regions and are widely used in studies examining the regulation of silk protein production, transgenic silkworm construction, and heterologous protein production [14,15,16]. Furthermore, the midgut-specific promoter P2P has been used to investigate digestion-related gene functions, pathogen infection, and the regulation of antimicrobial peptides [17], whereas the fat-body-specific promoter LP3 has been used to facilitate targeted gene expression in fat bodies and examine lipid metabolism and immune regulation [18]. Collectively, these examples serve to emphasize the importance of tissue-specific promoters in insect molecular biology and functional genomics.

However, despite the acknowledged importance of salivary glands, there have to date been no reports of highly active tissue-specific promoters in lepidopteran insects, thereby hampering functional studies. At present, such studies in B. mori are primarily dependent on the application of constitutive promoters, such as the cytoplasmic actin 3 promoter, which lack tissue specificity. The use of constitutive promoters can result in ectopic gene expression in non-target tissues, thus leading to nonphysiological effects and complicating biological interpretation. In this study, we identified a salivary gland-specific gene, BmCe, and cloned its candidate promoter sequence (BmCeP) based on RNA-sequencing (RNA-seq) transcriptome data from silkworm salivary gland and microarray expression profiles of different tissues and organs. A transgenic vector was constructed using BmCeP to drive expression of the marker DsRed, and positive transgenic lines were obtained. Our findings confirmed that in silkworms, BmCeP functions as a promoter with predominant salivary gland-specific activity, which could thus provide a foundation for further functional studies on silkworm salivary glands.

2. Materials and Methods

2.1. Experimental Materials

The silkworm strain D9L was maintained under laboratory conditions. Larvae were reared on freshly harvested mulberry leaves at approximately 25 °C and 75% ± 5% relative humidity. On day 3 of the fifth instar, larvae were dissected to collect multiple tissues (head, salivary glands, silk glands, fat bodies, Malpighian tubules, epidermis, midgut, testes, and ovaries). All samples were immediately frozen in liquid nitrogen and stored at −80 °C until used for further analysis.

2.2. Identification of Salivary Gland-Specific Genes

Genes showing exclusive expression in the head based on microarray data [19] and high expression indicated by silkworm salivary gland transcriptomic data [10] were selected as candidate genes, among which we identified the following nine highly expressed salivary gland-specific candidates: BGIBMGA010988, BGIBMGA012697, BGIBMGA00778, BGIBMGA012195, BGIBMGA009101, BGIBMGA011595, BGIBMGA003769, BGIBMGA001026, and BGIBMGA009568. Gene-specific primers were designed using Primer Premier 6.0 based on the B. mori genome database (SilkDB 3.0) and commercially synthesized by BGI (Shenzhen, China). PCR amplification was performed using B. mori genomic DNA as the template. The reaction conditions were as follows: an initial denaturation at 94 °C for 4 min; followed by 30 cycles of denaturation at 94 °C for 40 s, annealing at 60 °C for 40 s, and extension at 72 °C for 80 s; with a final extension at 72 °C for 10 min and subsequent storage at 4 °C. The PCR products were analyzed by 1.2% agarose gel electrophoresis, purified, TA-cloned, and verified by sequencing.

2.3. RT-PCR Analysis

Total RNA was extracted from 12 types of silkworm tissue and reverse transcribed to cDNA using a one-step reverse transcription kit (TransGen Biotech, Beijing, China), with the BmActin3 gene serving as an internal reference to assess transcription efficiency. Semi-quantitative PCR was conducted using cDNA from nine tissue types as templates with gene-specific primers designed for the nine candidate genes. The PCR program consisted of an initial denaturation at 94 °C for 4 min; followed by 35 cycles of denaturation at 94 °C for 40 s, annealing at 55 °C for 30 s, and extension at 72 °C for 40 s; with a final extension at 72 °C for 10 min. The amplified products were stored at 12 °C. To identify genes with salivary gland-specific expression patterns, we compared the levels of relative expression among the different tissues.

2.4. Construction of a Transgenic Expression Vector

Purified PCR products were ligated into a pEASY-T5 zero vector for TA cloning. Based on the structure of the overexpression vector pSL1180[Hr3-A4-DsRed-SV40] [20,21] and the genomic features of BmCe, we selected sites for the two restriction enzymes, BamHI and SalI. The following promoter-specific primers were designed using Primer Premier 6.0: BGIBMGA010988-pro-SalI-F (5′-ACGCGTCGACCAGTACTTGTCTACTGGGAAG-3′) and BGIBMGA010988-pro-BamHI-R (5′-CGGGATCCATCCTAAGTGCATGCG-3′). The candidate promoter region, designated BmCeP, was amplified from the genomic DNA of B. mori under the following PCR conditions: an initial denaturation at 98 °C for 3 min; followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s, and extension at 72 °C for 15 s/kb; with a final extension at 72 °C for 10 min, and holding at 12 °C. The amplified fragments were resolved by gel electrophoresis, visualized using a Bio-Rad gel imaging system (Hercules, CA, USA), and bands of the appropriate size were excised and purified. The BmCeP fragment and pSL1180 vector were digested with BamHI and SalI, ligated using T4 DNA ligase, and used to generate the recombinant plasmid pSL1180[Hr3-BmCeP-DsRed-SV40]. This construct was further digested using AscI and ligated into the dephosphorylated piggyBac[3×P3-EGFP] [22], yielding the final construct piggyBac-3×P3-EGFP[Hr3-BmCeP-DsRed].

2.5. Microinjection and Fluorescence Screening

The recombinant piggyBac-3×P3-EGFP[Hr3-BmCeP-DsRed] plasmid and a helper plasmid encoding piggyBac transposase [23] were mixed in equimolar ratios to a final concentration exceeding 500 μg/μL to ensure efficient transposition. The mixture was microinjected into D9L silkworm eggs using a microinjection system (Eppendorf AG, Hamburg, Germany), with the injection sites being sealed with non-toxic glue. The injected eggs were subsequently incubated at 25 °C and 85% relative humidity until hatching to obtain F_0_ individuals, which were self-crossed, and stable transgene inheritance was assessed in the F_1_ generation. F_1_ eggs at 5–6 days of development were screened using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) (excitation wavelength 460–490 nm), with offspring characterized by eye-specific green fluorescence being identified as transgenic.

2.6. Quantitative Real-Time PCR (qRT-PCR)

From onset of the fifth instar until day 6, salivary glands were collected daily from wild-type D9L silkworms. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse transcribed to cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Gene-specific primers were designed based on target mRNA sequences, and the housekeeping gene sw22934 was used as an internal control. qRT-PCR was performed using SYBR Premix Ex Taq II (Takara, Japan), with amplification of BmCeP facilitated using the primers qRT-BmCeP-F (5′-CAGCGAAGACCTAGGATAG-3′) and qRT-BmCeP-R (5′-GCTTACGGAAACCACAAC-3′), whereas sw22934-F (5′-TTCGTACTGGCTCTTCTCGT-3′) and sw22934-R (5′-CAAAGTTGATAGCAATTCCCT-3′) were used as primers for the reference gene sw22934. Fluorescence signals were monitored during the annealing and extension steps, followed by melting curve analysis. Ct values were determined using 7000 System SDS Software (Applied Biosystems, Foster City, CA, USA), and relative expression levels were calculated based on the 2^−ΔΔCt^ method.

2.7. Data Analysis

All data are presented as the means ± standard error (SE). The statistical significance of differences between groups was evaluated using a two-tailed Student’s t-test in GraphPad Prism (version 8.0). Significance levels were defined as ** p < 0.001, * p < 0.01, and p < 0.05.

3. Results

3.1. Semi-Quantitative Validation of Salivary Gland-Specific Genes

The salivary gland is a key component of the silkworm’s digestive system, and plays important roles in the initial processing of ingested food (Figure 1a). With reference to B. mori tissue expression microarray data and salivary gland transcriptome datasets [10], we selected the following nine candidate genes showing high levels of expression predominantly within the salivary gland: BGIBMGA010988, BGIBMGA012697, BGIBMGA00778, BGIBMGA012195, BGIBMGA009101, BGIBMGA011595, BGIBMGA003769, BGIBMGA001026, and BGIBMGA009568 (Data S1). To validate the tissue-specific expression patterns of these genes, we performed semi-quantitative PCR using BmActin3 as an internal reference (Table 1). Based on an examination of the expression of the nine genes among 12 different tissue types (Figure 1b), we identified a cholinesterase-encoding gene, BGIBMGA010988 (BmCe), showing strong highly specific expression in the salivary glands, with only minimal expression detected in the anterior midgut. Given this pronounced tissue specificity, BGIBMGA010988 was selected for subsequent promoter cloning and functional analysis. To further characterize the developmental expression profile of BmCe, we conducted qRT-PCR analysis using salivary glands collected from wild-type silkworms throughout progression of the fifth instar, which accordingly revealed an increase in expression from day 0, reaching peak levels on day 2, and thereafter gradually declining (Figure 1c). Having established this temporal expression pattern, we selected fifth instar day 3 as the optimal time point for evaluating BmCeP-driven overexpression in transgenic lines.

3.2. Construction of Salivary Gland-Specific Promoter Vector

To predict potential cis-regulatory elements within the BmCeP promoter region, we screened the JASPAR database [24] (Data S2) and thereby identified a number of canonical promoter-associated motifs, including a TATA box, a GGAT box, and predicted binding sites for transcription factors such as GATA and C2H2, which are assumed to be involved in the regulation of downstream gene expression (Figure 2). The BmCeP promoter fragment was PCR-amplified and used to replace the A4 promoter in the vector pSL1180[Hr3-A4-DsRed], thereby generating the recombinant plasmid pSL1180[Hr3-BmCeP-DsRed] (Figure 3a). Double digestion of the recombinant plasmid with BamHI and SalI yielded a fragment of approximately 2000 bp (Figure 3b), consistent with the expected size of the BmCeP promoter. Sequence analysis confirmed an absence of mutations, indicating successful vector construction. Subsequently, pSL1180[Hr3-BmCeP-DsRed] and piggyBac[3×P3-EGFP] were digested with AscI, with ligation of the recovered fragments yielding the final recombinant vector piggyBac-3×P3-EGFP[Hr3-BmCeP-DsRed]. Single digestion of the vector with AscI generated a band of approximately 4800 bp (Figure 3c), corresponding to the expected size of the Hr3-BmCeP-DsRed cassette. Sequence verification confirmed construct integrity, and the validated vector was used for subsequent germline transformation.

3.3. Microinjection and Fluorescence Screening of Transgenic Silkworms

The recombinant vector and helper plasmid encoding the piggyBac transposase were mixed in equimolar ratios and microinjected into the eggs of D9L strain silkworms. Between days 5 and 6 of F_1_ embryonic development, individuals were screened using a fluorescence microscope. Given that the 3×P3 promoter specifically drives EGFP expression in the eyes and nervous system, transgenic silkworms were readily identified by the presence of strong green fluorescence in the eyes (Figure 4).

3.4. qRT-PCR Validation of BmCeP Promoter Activity

Compared with the wild-type larvae, transgenic individuals were found to be characterized by markedly higher levels in red fluorescent protein (RFP) gene expression. Similarly, we detected a significant upregulation of DsRed expression in transgenic larvae, whereas DsRed transcripts were undetectable in wild-type samples. Moreover, in most of the assessed tissues, the expression of DsRed remained extremely low or undetectable, indicating a clear salivary gland-predominant pattern of expression. These findings thus enabled us to confirm the efficacy of the candidate promoter with respect to salivary gland-predominant expression (Figure 5). Consistent with the findings of semi-quantitative PCR analysis, these findings revealed that the BmCeP promoter contains cis-regulatory elements with the capacity to drive tissue-specific transcription in the salivary glands.

4. Discussion

The primary functions of animal salivary glands include moistening food, maintaining oral homeostasis, and facilitating digestion via the secretion of digestive enzymes [25], such as α-amylase [26]. Beyond digestion, salivary glands also play roles in the excretion of metabolic waste and the regulation of trace element homeostasis, and also contribute to systemic metabolism and fluid balance, thereby influencing carbohydrate, protein, and lipid metabolism [27]. In addition, Chibly et al. have demonstrated that salivary glands secrete bioactive molecules, including epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1), which contribute to protecting and regulating intestinal physiology [28]. In insects, salivary glands play essential roles in insect–plant interactions. For example, during feeding, insect saliva is released onto the host leaf surface, potentially inducing plant physiological responses, modulating plant defense signaling, and contributing to the establishment of a stable feeding association between the insect and its host plant. In addition, by synthesizing cis-vaccenic acid, salivary glands have also been implicated in the regulation of satiety in B. mori [10]. Given these multiple and often underappreciated physiological roles, in-depth analyses of salivary gland function are particularly valuable for economically important species such as B. mori. In this regard, tissue-specific promoters that drive gene expression exclusively or predominantly in particular organs are of considerable value from the perspectives of both basic research and applied biotechnology. By facilitating spatially precise control over transgene expression, such tissue-specific promoters can serve as highly effective tools for optimizing traits in animals and plants, including the enhancement of stress resistance and improving growth performance [29], as well as the modification of metabolic pathways [30]. In the present study, we identified BmCeP, a salivary gland-predominant promoter, which we believe to have potential applicability for advancing functional studies on silkworm salivary glands.

Establishing gene expression profiles in multiple tissue types provides a valuable initial basis for identifying genes with tissue-specific activity and their corresponding promoters. In 2007, genome-wide microarray analysis examined gene expression in ten silkworm tissues on day three of the fifth instar, including hemocytes, fat body, silk glands, head, integument, Malpighian tubules, midgut, ovaries, and testes [19]. Given that a portion of the salivary gland is anatomically located within the head, head-expressed genes were initially screened. More recently, a genome-wide expression profile of silkworm salivary glands was generated based on RNA-seq data [10]. Subsequently, by integrating these two datasets, we succeeded in identifying nine candidate salivary gland-specific genes, with semi-quantitative RT-PCR validation confirming BGIBMGA010988 (BmCe) to be a gene with highly specific salivary gland expression.

Having cloned the sequence of the candidate promoter BmCeP, further sequence analysis revealed a number of canonical regulatory elements, including a TATA box, a GGAT box, and predicted binding sites for GATA, C2H2, and bZIP transcription factors. The promoter was sub-cloned into the piggyBac[3×P3-EGFP] vector to drive DsRed expression, and the recombinant construct was microinjected into D9L strain eggs to generate transgenic silkworms. Notably, although red fluorescence was visually detected in the salivary glands of both wild-type and transgenic larvae, qRT-PCR analysis confirmed that DsRed transcripts were detected only in transgenic individuals and were predominantly enriched in the salivary glands. Previous studies have reported that multiple endogenous proteins of varying molecular sizes show red fluorescence in the silkworm midgut [31,32,33,34]. It is thus conceivable that similar proteins characterized by red autofluorescence are present in silkworm salivary glands. Furthermore, given that the activity of the BmCeP promoter is not particularly high, the detection of background red autofluorescence in the salivary glands of wild-type silkworms makes it difficult to detect significant differences between transgenic and non-transgenic silkworms regarding red fluorescent protein expression via fluorescence microscopy. These findings further indicate that RFP might not be an optimal reporter for salivary gland-specific studies in silkworms, despite its utility at the transcript level.

5. Conclusions

In this study, we sought to address the current lack of characterized salivary gland-specific promoters in B. mori, and accordingly identified BmCeP as a salivary gland-predominant promoter. As the first such promoter detected in B. mori, BmCeP can activate genes primarily expressed in salivary glands, thereby facilitating assessment of the expression and function of salivary gland-associated genes. Although the precise regulatory elements controlling this specific expression pattern have yet to be established, and given that its expression is not particularly strong, we believe BmCeP could serve as a valuable molecular tool for functional studies of the salivary gland, a key organ playing multiple roles in nutrient utilization and the regulation of feeding in silkworms.