Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication

BIOMAC-17227; No of Pages 11 International Journal of Biological Macromolecules xxx (xxxx) xxx

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication Hao Yan, Qingsong Liu, Feng Wen, Bingchuan Bai, Yuchan Wen, Wenwen Chen, Wei Lu, Ying Lin, Qingyou Xia, Genhong Wang ⁎ a b c

State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400716, China Chongqing Key Laboratory of Sericultural Science, Southwest University, Chongqing 400716, China Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing 400716, China

a r t i c l e

i n f o

Article history: Received 5 September 2020 Received in revised form 7 November 2020 Accepted 9 November 2020 Available online xxxx Keywords: Amylase Characterization Transposable element Artificial selection Bombyx mori

a b s t r a c t Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 °C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5′-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding. © 2020 Elsevier B.V. All rights reserved.

1. Introduction The silkworm, Bombyx mori (B. mori), is economically important for sericulture and was first domesticated from B. mandarina more than 5, 000 years ago [1,2]. Due to human selection, several phenotypic characteristics, including color, body size, egg laying, flying ability, cocoon weight, growth rate, and digestion efficiency have been greatly changed in B. mori [1,3]. The domesticated silkworm is visibly larger (Fig. 1A and B) and can produce 10 times more silk than the wild B. mandarina [3]. Previous reports showed that domestic plants and animals tend to have efficient nutritient utilization to adapt to human preferences [4,5]. Recently, a methionine-rich storage protein was reported to exhibit increased expression in the ova of domestic silkworms due to artificial selection, which might contribute to increased hatchability [6]. Amylases, which hydrolyze polysaccharide, such as starch, from food substrates into smaller sugars, play a major role in the digestive processes in animals and have been showed to be selected during domestication in several organisms [7–10]. High amylase activity conferring more efficient starch digestion in dogs is associated with a drastic ⁎ Corresponding author at: State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400716, China. E-mail address: [email protected] (G. Wang).

increase in the copy number of a pancreatic amylase, AMY2B, compared with that of wolves [8,9]. Aspergillus oryzae, a filamentous fungus domesticated from A. flavus, is used for making traditional oriental foods and beverages, and can secrete more starch-degrading enzymes than its ancestor. Hunter et al. [10] showed that ɑ-amylase underwent independent duplication in different A. oryzae strains, indicating that this was a human-favored domestication trait [10]. Different reports have shown that amylase activity varies significantly among silkworm genetic varieties and is correlated with some economic traits such as larval weight and survival rate [11–14]. Nipaporn et al. identified the first silkworm α-amylase (BmAmy) and investigated its tissue expression patterns [15]. However, the genetic basis of variation in amylase activity in different varieties still needed to be resolved. In this study, we identified one amylase gene, BmAmy1, the transcript abundance of which shows a dramatic difference between domesticated and wild silkworms. Our findings indicated that three kinds of fragmentary transposable element (TE) insertions upstream of the gene likely contributed to the increased BmAmy1 expression in different domesticated silkworm lineages. The activity of BmAmy1 could be largely inhibited by amylase inhibitors from mulberry. Furthermore, the growth of domesticated silkworm could be further enhanced by overexpressing BmAmy1 using piggBac-based transgenic technology.

https://doi.org/10.1016/j.ijbiomac.2020.11.064 0141-8130/© 2020 Elsevier B.V. All rights reserved.

Please cite this article as: H. Yan, Q. Liu, F. Wen, et al., Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domesti..., , https://doi.org/10.1016/j.ijbiomac.2020.11.064

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2. Materials and methods

2.5. Genomic DNA, total RNA extraction, cDNA synthesis and qRT-PCR

2.1. Silkworm strains, cell line and plant materials

Genomic DNA of both B. mori and B. mandarina was extracted from midgut (for analysis of the upstream region of the BmAmy1 gene) or individual pupae(for analysis of the insertion sites of transgenic silkworm) using an E.Z.N.A.® Tissue DNA Kit (OMEGA BIO-TEK, USA). Total RNA of silkworms and mulberry was isolated using TRIzol™ Reagent (Invitrogen™, USA) and then stored at −80 °C. First-strand cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANSGEN BIOTECH, Beijing, China) according to the manufacturer's protocol. Complete coding sequences of BmAmy1, MnAI1 and MnAI2 were amplified using the gene-specific primers BmAmy1-F/R, MnAI1-F/R and MnAI2-F/R, respectively (Supplemental Table S3). qRT-PCR was conducted with qTOWER2.2 (Analytikjena, Jena, Germany) using SYBR® Premix Ex Taq™ II (Takara, Japan) and performed in three independent replicates. Diluted cDNA (2 μL) was used as the template. Cycling conditions were as follows: 30 s at 95 °C, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s, then 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s. The silkworm sw22934 gene and a mulberry ribosomal protein gene (Morus024083) served as an internal controls to normalize the target gene expression data [18]. Relative gene expression level was defined according to the comparative Ct method [19]. All primers were designed using Primer Premier 6.0 software (Premier, Toronto, Canada) and listed in supporting information (Supplemental Table S3).

Individuals from 24 inbred and domesticated silkworm varieties (B. mori), and wild silkworm strains (B. mandarina) from mulberry fields in different Chinese geographic regions, were collected for analysis of the upstream region of BmAmy1. The information available for these strains is documented in Supplemental Table S1. A B. mori embryonic cell (BmE) line that was used for the dual-luciferase reporter assay was maintained at 25 °C in Grace insect medium (Gibco, Grand Island, USA) in the State Key Laboratory of Silkworm Genome Biology of China. The mulberry species, Morus notabilis Jialing 30, used to clone MnAI genes and carry out the bioassay, was kept in the Mulberry Germplasm Nursery of Southwest University. 2.2. Gene identification, multiple sequence alignments and phylogenetic analysis The BmAmy1 was identified by a homology search using BLASTP against the predicted protein sequences deposited in the Silkworm Genome Informatics Database (http://sgid.popgenetics.net/). Multiple sequence alignments were performed through MUSCLE (Align Codons). Then, phylogenetic trees were constructed using the Maximum Likelihood method with 1000 bootstrap replicates byMEGA 7.0 [16]. Analysis of the protein domains was performed using the online server SMART (http://smart.embl-heidelberg.de/), combined with the ExPASy Proteomics Server (http://www.expasy.org/). The GenBank accession numbers of protein sequences used in phylogenetic analysis are listed in Supplemental Table S2.

2.6. Vector construction In this study, three kinds of recombinant plasmids were constructed. They were vectors for dual-luciferase reporter assay, protein expression in P. pastoris, and BmAmy1 transgenic overexpression in B. mori, respectively. To construct dual-luciferase reporter assay vectors, the upstream regulatory regions of BmAmy1 from different domesticated silkworm strains and wild silkworms were amplified using the primer pairs BmAmy1-pro-F2 and BmAmy1-pro-R2. Primer design was according to the genomic sequence of Dazao (http://www.silkdb.org/silkdb/) and the reverse primer BmAmy1-pro-F2 was located on the first 23 bp of the BmAmy1 coding sequence. The amplified fragments were doubledigested with SacI and NheI and then ligated into the pGL3-Basic vector (Promega, USA) to construct recombinant plasmids. All constructed vectors were confirmed by DNA sequencing. In addition, to test the function of the insertion fragment, IS1, IS2 and IS3 were amplified separately and linked with Sac I and Nhe I restriction site at each end，and then added to the upstream of the BmSP95 [20] promoter in a reporter vector PGL3-SP95 vector (Supplemental Fig. S1A). To make constructs for protein expression in P. pastoris, the coding regions of BmAmy1, MnAI1, and MnAI2, without signal peptides and stop codons, were amplified and ligated into the pPICZα-A vector between the α-factor (allows for efficient secretion of most proteins from P. pastoris) and C-Myc epitopes (Supplemental Fig. S1B) with EcoR I and Not I. For constructing the BmAmy1 transgenic overexpression vector in B. mori, the midgut-specific promoter P3P + 5UI was used [21]. The BmAmy1 coding sequences were assembled into the intermediate plasmid pSL1180[P3P + 5UI-BmAmy1SV40] using BamHI and NotI. Then, the expression cassette of BmAmy1 was digested by AscI and sub-cloned into piggyBac[3 × P3-DsRed af] by T4 DNA ligase (NEB, UK) to produce the piggybac-based transgenic vector piggyBac[3 × P3-DsRed, P3P + 5UI-BmAmy1] (Supplemental Fig. S1C). All constructs were confirmed by DNA sequencing. A schematic of the three kinds of vectors is given in Supplemental Fig. S1.

2.3. Mulberry leaf treatments Mulberry (Jialing 30) seeds were surface-sterilized by 10% NaClO, and sown on sterilized plates with MS solid culture medium (PhytoTechnology Laboratories, Kansas, MO, USA) in a chamber at 25 °C with a 12-h photoperiod. The 75-day-old mulberry seedlings (about 15 cm in height) were used for silkworm treatment. All leaves selected for treatment were at similar positions. Silkworm treatment was according to the method of Appel [17], with small modifications. In brief, third instar (N = 2–3) silkworm larvae were allowed to feed for 30 min, causing ~20% leaf area removal on about two leaves of similar age from each plant. Silkworm larvae were removed when sufficient damage was achieved. After silkworm treatment, the plants were put back in the growth chamber. The mechanical wounding treatment using a sterile scissors was designed to approximate insect damage to plant tissues. Leaves were cut seven times along the veins, with the first time at the beginning of the silkworm treatment, thereafter leaves were damaged every 5 min. Control plants were jiggled with a sterile brush to simulate leaf movement caused by silkworm crawling or mechanical wounding. Damaged leaves were harvested for gene expression analysis at 2, 6, and 24 h after the start of silkworm feeding or mechanical wounding. Unwounded leaves, matched in both size and age to damaged or wounded leaves, were harvested separately. Leaves from 16 plants per treatment (silkworm, wounding and controls) at every time point were pooled for each of the three biological replicates. All leaf samples collected from each time point were immediately frozen and then stored at −80 °C until used for RNA extraction.

2.7. Transfection and dual-luciferase assay 2.4. Silkworm tissue sample collection The recombinant plasmids were co-transfected with the reference plasmid pRL-null (containing Renilla luciferase gene driven by Hsp70 promoter) into the B. mori embryonic cell line BmE using X-tremeGENE™ HP DNA Transfection Reagent (Roche). The transfection procedure was

Tissue samples from the silkworm head, fat body, silk gland, hemocyte, epidermis, midgut and Malpighian tubule were collected, immediately frozen and stored at −80 °C until RNA or DNA extraction. 2

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determined by incubating the reaction mixture at 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 70, and 80 °C for 30 min, followed by activity measurement. Optimal pH was determined using Britton-Robinson's buffer with pH set at 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The suppression of BmAmy1 by MnAI1 and MnAI2 was performed in a similar way at pH 7.0, except that enzyme (0.05 μg) and inhibitor (2.5 μg) were incubated for 30 min at 37 °C (total reaction system is 75 μL) before incubating with the substrate. BSA was used as a negative control for amylase inhibitor.

according to the method described in the manufacturer's protocol. Thirty-six hours after transfection, the cells were collected for luciferase assays and the activities of firefly and Renilla luciferase in the cell lysates were measured by Dual-Luciferase Reporter Assay System (Promega). 2.8. Expression and purification of BmAmy1, MnAI1 and MnAI2 The recombinant plasmids (pPICZαA-BmAmy1, pPICZαA-MnAI1 and pPICZαA-MnAI2) were linearized by SacI (NEB) digestion and transformed into P. pastoris strain X-33 (Invitrogen) by electroporation using a Gene Pulser (BIO-RAD Laboratories, Hercules, CA, USA). Screening of high-productivity recombinant P. pastoris and protein expression were done according to a previously published method [22,23] using a P. pastoris Expression Kit (Invitrogen). Time-course fermentation supernatants was analyzed by western blotting (Supplemental Fig. S2A), which was used to determine the optimal time for protein induction. About 1 L of fermentation supernatant was harvested for protein purification. First, the fermentation supernatant was adjusted to pH 7.0 with 1 M Tris (pH 8.0) and incubated with the Ni Sepharose excel (GE Healthcare, Buckinghamshire, UK) equilibrated with binding buffer (20 mM Tris-HCl and 200 mM NaCl at pH 7.0) for 3 h at 4 °C on a shaker at 80 rpm. Then, the mixture was loaded into a gravity flow column, releasing the flow-through. Thereafter, different imidazole concentrations of elution buffer (20 mM Tris-HCl, 200 mM NaCl and imidazole at pH 7.0) were used to purify the target protein. Finally, purified protein was further desalted and concentrated using a Millipore 30 kDa cutoff column (Merck, USA). Purified protein was quantified using a Modified Bradford Protein Assay Kit (Sangon Biotech, China) and BSA was used as a standard. The purified products were respectively analyzed using Coomassie Brilliant Blue staining and western blotting (Supplemental Fig. S2B). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (BioRad) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); then the PVDF membranes were sequentially incubated with primary antibody (rabbit anti-Myc) and secondary antibody (horse radish peroxidase -labeled Goat Anti-Rabbit IgG) after blocking. All antibodies were purchased from Thermo Fisher. Finally, the PVDF membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and observed in a ChemiScope 3300 mini (Clinx Science Instruments, Shanghai, China).

2.10. Germline transformation, screening and characterization of transgenic line A non-diapausing silkworm strain, D9L, was used for embryo microinjection. The BmAmy1 overexpression vector piggyBac[3 × P3-DsRed, P3P + 5UI-BmAmy1] and the plasmid pHA3PIG helper plasmid (stored in our laboratory) encoding piggyBac transposase were purified by QIAGEN Plasmid Mini Kit (Qiagen, Valencia, CA, USA). Then the two plasmids were mixed in equal amounts. We performed microinjection using a FemtoJet 5247 microinjector system (Eppendorf, Hamburg, Germany) [25]. The injected larvae were fed on fresh mulberry leaves in an incubator maintained at 25 °C under standard conditions [26]. When the G0 generation silkworms laid eggs and developed for 7 days, a SZX16 fluorescence stereomicroscope (Olympus, Tokyo, Japan), with excitation to measure red fluorescence, was used to screen the transgene-positive silkworms. Then, qRT-PCR was used to detect expression levels of BmAmy1 in the transgenic lines. Inverse PCR was used to investigate the insertion sites of transgenic strains. Genomic DNA was extracted using G1 pupae. Generally, genomic DNA was digested with HaeIII enzyme (NEB) and ligated with T4 ligase (NEB) [27]. Then, the ligation product was subjected to PCR using primers pBacL-F/R and pBacR-F/R. All PCR products (>500 bp) were cloned and sequenced to identify the insertion sites. Randomly selected 40 individuals (20 male and 20 female) from the fourth generation G4 with three replicates from the transgenic line were used to analyze their characteristics, including weight and cocoon shell ratio. An equal number of non-transgenic D9L individuals with the same breeding conditions were used as control. Weight of the individual larvae on day 4 of the fifth instar was recorded. Cocoon weight and cocoon shell weight were measured on day 4 of the pupal stage. The cocoon shell ratio is the percentage of cocoon shell weight to cocoon weight. The starch content in feces of transgenic silkworms was determined by anthrone‑sulfuric acid colorimetric method using a 620 nm wavelength, as described previously [28]. Starch content was measured in 0.1 g of dried feces of silkworms on day 4 of the fifth instar. The anthrone‑sulfuric acid used was prepared just before use. Each assay was performed with three biological replicates.

2.9. Measurement of BmAmy1 enzymatic activity and inhibitory effect of MnAI1 and MnAI2 Amylase activity was determined by measuring the amount of reducing sugars released after incubation with starch using 3,5dinitrosalicylic acid (DNS) colorimetry [24]. The DNS reacts with reducing sugar that is produced from starch by amylase to form a reddish −brown substance, which has an absorption peak at 540 nm. Amylase activity was calculated by measuring the increase rate of absorbance at 540 nm. A standard curve of absorbance against different amounts of glucose was constructed to enable calculation of the amount of reducing sugar released during the α-amylase assay. Serial dilutions of glucose (10 mg/mL) in the universal buffer (pH 7.0) were made to produce the following concentrations: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0.0078 mg/mL. Amylase dilution (75 μL) and substrate (75 μL, 1% soluble starch) were incubated at 40 °C for 5 min; then DNS (150 μL) was added, mixed and incubated at 90 °C for 10 min. Finally, the reaction solution (200 μL) was added to a 96-well plate, and absorbance at 540 nm was measured using a Multiskan™ FC Microplate Photometer (ThermoFisher). One amylase unit was defined as the amount of enzyme required to release 1 mg of reducing sugar per minute at 40 °C from the substrate (starch) under the given assay conditions. More detailed information is found in the α-Amylase Assay Kit instructions (Solarbio, China). Each assay was repeated at least three times. The effect of temperature on BmAmy1 activity was

2.11. Statistical analysis All experiments were performed with at least three replicates. The significance of differences between groups was determined by twotailed Student t-tests using GraphPad Prism 8.0 software. 3. Results 3.1. Identification of an α-amylase gene (BmAmy1) that is expressed abundantly in B. mori We cloned a BmAmy1 cDNA sequence containing a 1518-bp open reading frame (ORF) (Supplemental Table S4). The predicted amino acid sequence of BmAmy1 showed 54% identity with a previously reported silkworm α-amylase, BmAmy [15]. Seven conserved sequence regions, which are common to all animal α-amylases, were present in BmAmy1 (Supplemental Fig. S3). The catalytic amino acid residues (D212, E249 and D314) of a classical α-amylase were also conserved in BmAmy1. Three residues (R210, N312 and R346), which are involved in chloride binding, were found. R346 is a conserved site for chloride 3

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binding in α-amylases from mammals and non-lepidoptera insects [29]. In the case of lepidopteran α-amylases, a glutamine was usually present at that site. Phylogenetic analysis showed that BmAmy1 greatly differed from other lepidopteran α-amylases, although it was also clustered into the lepidopteran sub-family (Fig. 2A). Quantitative real-time PCR (qRT-PCR) was employed to analyze expression of BmAmy1. BmAmy1 was expressed predominantly in the midgut, with a relatively low expression level in other tissues, including the fat body and epidermis (Fig. 2B). The expression of BmAmy1 increased gradually during days 0–2 of the fifth instar, then maintained a high abundance on day 4 and decreased gradually from day 5 until the last day of the fifth instar (Fig. 2C). The expression pattern of BmAmy1 was consistent with the intake amount of mulberry leaves during the fifth instar. We also investigated expression of BmAmy1 in B. mandarina using qRT-PCR. Interestingly, there was almost no expression in the midgut of B. mandarina (Fig. 2D, left). This result was confirmed by gel electrophoresis of a PCR amplified BmAmy1 fragment (Fig. 2D, right). To verify that lack of detection of BmAmy1 expression in B. mandarina was not due to poorly-matched PCR primers, the corresponding exons of BmAmy1 were amplified using B. mandarina genomic DNA. This showed that all of the putative exons of BmAmy1 were present in the B. mandarina genome (Supplemental Fig. S4).

was downloaded from SGID (http://sgid.popgenetics.net/) and SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi), and confirmed by PCR amplification. Genomic sequences upstream of the start codon ATG were amplified and sequenced in individuals from 24 inbred and domesticated silkworm varieties, and B. mandarina collected from four geographical regions (Supplemental Table S1). Gel electrophoresis showed that 4 types of amplicons (V0-V3) were obtained (Fig. 3A and B). Based on BLAST analysis against the B. mori TE database (BmTEdb) [30], V1, V2 and V3 contained a fragment insertion (IS1-3) from helitron, mariner, and TE-1_BM TEs, respectively, compared with V0. The insertion sites for IS1, IS2 and IS3 were located 728, 750 and 666 bp upstream of the BmAmy1 start codon, respectively. Of the B. mandarina individuals investigated, 79% harbored amplicon V0, while 7% and 14% harbored V2, and both V0 and V2, respectively. Interestingly, all three amplicons with TE insertion (V1-V3) appeared in the 5′-upstream regulatory region of BmAmy1 in domesticated silkworm varieties, while V0, amplicon without TE insertion, was not found in the domesticated silkworm varieties investigated. Of the investigated domesticated silkworm varieties, 58% (Furong, Jingsong, Yun8 JPN, Yun9 JPN, Yun10 JPN, Su16, 54A, Xianghui, 7532, 794, 796, Mingzhu, Dongfei and 46) harbored a 338-bp IS1 in the upstream region of BmAmy1 (V1). The IS2 for varieties Dazao, 932, D9L and C108 was 599 bp in length. The region upstream of V3 for varieties Yanbai, Yun9 CHN, Yun10 CHN, 782, 532 and 732, contained a 1341-bp insertion (IS3). The sequence information for amplicons V0-V3 are listed in Supplemental Table S4. To determine whether the TE fragment insertions cause upregulation of BmAmy1 expression in B. mori, several luciferase reporter

3.2. TE fragment insertions in the 5′-upstream regulatory region are responsible for enhanced expression of BmAmy1 in B. mori In order to investigate the dramatically different BmAmy1 expression levels of B. mori and B. mandarina, the BmAmy1 genomic sequence

A B. mori

B. mandarina

B B. mori

B. mandarina

1 cm

1 cm

Fig. 1. Phenotype comparison of B. mori and its ancestor B. mandarina. (A) Comparison of larvae of B. mori and B. mandarina. (B) Comparison of cocoons of B. mori and B. mandarina. 4

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3.3. Enzyme activity of BmAmy1 and its interaction with amylase inhibitor from mulberry

gene systems containing the silkworm BmAmy1 upstream sequences, with or without TE fragment insertions, were constructed. The luciferase activities were examined at 36 h after transfection of a B. mori embryonic cell line. All three of the regulatory regions with TE insertions from B. mori caused higher expression levels than the regulatory region without TE insertion from B. mandarina. The regulatory regions with the TE insertions V1, V2 and V3 showed increases of 1.88-, 3.34and 6.63-fold, respectively, compared to the construct without an insertion (Fig. 3C). In an independent approach, we tested the regulatory effects of the three TE insertions by placing them in front of a silkworm serine protease (BmSP95) promoter [20] in the reporter vectors. A similar expression increase was detected using luciferase assays (Fig. 3D).

A



Two putative Kunitz-type α-amylase inhibitor genes from mulberry (MnAI1 for XP_010098224 and MnAI2 for XP_010098225), which were predicted by an automated computational analysis based on the mulberry genome sequences (https://morus.swu.edu.cn/morusdb/), were obtained from GenBank. Just like α-amylase inhibitors in other plants, the predicted MnAI1 and MnAI2 proteins had a conserved Kunitz motif and a reactive loop (Supplemental Fig. S5). To determine whether amylase from B. mori would interact with amylase inhibitor in the host plant, BmAmy1, MnAI2 and MnAI2 were independently expressed in Pichia pastoris and the proteins were purified (Supplemental Fig. S2).

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Fig. 2. Phylogenetic and expression analysis of BmAmy1. (A) A phylogenetic tree depicting the relationships of amylase genes at the amino acid level among different species. (B) Expression patterns of BmAmy1 in different tissue and organ samples on day 3 of fifth-instar larvae. (C) Expression profile of BmAmy1 in the midgut of fifth-instar larvae. (D) Comparison of BmAmy1 expression between B. mori and B. mandarina in midgut by quantitative real-time PCR (left) and gel electrophoresis of products from reactions of RT-PCR (right). Dip: Diptera; Hym:Hymenoptera; Ara: Arachnida; Col: Coleoptera; Lep: lepidoptera; Bla: Blattodea; Ver: Vertebrata. L5Dx: midgut collected from larvae on the Xth day of fifth instar. Furong, 932 and 782 are names of three domesticated silkworm strains. Error bars, SEM. 5

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(Fig. 4A and B). The highest enzymatic activity occurred at 35 °C and pH 9.0 (Fig. 4C and D). BmAmy1 activity could be reduced to 10.32% and 0.79% by MnAI1 and MnAI2, respectively, compared with BmAmy1 protein pre-incubated with an equal amount of bovine serum albumin (BSA), indicating that both MnAI1 and MnAI2 significantly inhibited BmAmy1 activity (Fig. 4E and F). We assessed the

Detailed information about vector construction, protein expression and purification are shown in the Materials and Methods. The optimum expression and purification conditions for the three proteins are shown in Supplemental Table S5. Enzyme activity assays and reference to calibration curve showed that around 0.52 mg of reducing sugars were produced within 1 min after starch was catalyzed by 1 mg of BmAmy1

A -1065 -728

-1249

BmoAmy1 genomic structure V1

-1507

BmoAmy1 genomic structure V2

BmoAmy1 genomic structure V3 -2307

-1348

-750

-666

-2006

183

158

172

116 190

174 132

201

162

30

183

158

172

116 190

174 132

201

162

30

183

158

172

116 190

174 132

201

162

30

183 158 -912

BmaAmy1 genomic structure V0

172

116

174 132 201 162

190

30

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(bp)

D

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-753 -728

(bp)

5000 3000 2000

5000 3000 2000 1000 750 500 250 100

1000 750 500 250 100 V0

V3

V2

V1

C

V0

-753 -728 -608

(-912)

pGL3

LUC

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Fig. 3. Different kinds of transposable element fragment insertions in the 5′-upstream regulatory region of BmAmy1 and their effects on gene expression. (A) Genomic structure of BmAmy1, showing the insertion positions of the transposable element fragments. Black rectangles indicate exons of BmAmy1, and black dotted lines between rectangles represent introns. The gray rectangle in front of the first exon indicates the predicted promoter region. Three kinds of insertions, IS1, IS2 and IS3, along with their location information in the promoter regions are shown with red, green and blue rectangles, respectively. (B) Genomic PCR showed that bands with four different sizes (V0, V1, V2 and V3) were obtained when amplifying the 5′-upstream regulatory region of BmAmy1 in individuals from different domesticated silkworm varieties and B. mandarina collected from different regions. (C) The relative luciferase expression activity of upstream regions of Amy1 genes from different silkworm strains. (D) Luciferase assay showing the effect of the three transposable element fragments on the promoter of BmSP95. Error bars, SEM; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test). 6

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Fig. 4. Characterization of the enzymatic properties of BmAmy1 and interaction with mulberry amylase inhibitor. (A) Glucose calibration curve for 3, 5-dinitrosalicylic acid (DNS) method. (B) Activity level of BmAmy1 compared with ɑ-amylase from Bacillus subtilis. (C and D) Effect of pH and temperature on BmAmy1 activity. (E) Colorimetric analysis of the activity of BmAmy1 and the inhibitory effect of amylase inhibitor MnAI1 and MnAI2 (F). Error bars, SEM; ***P < 0.001 (Student's t-test).

BmAmy1 from a midgut-specific promoter P3P [21]. Transgenic individuals were selected by using the 3 × P3 dsRed marker (Fig. 6A). One transgenic line, P3P: Amy1, was obtained. The results of inverse PCR showed that the transgenic insertion site was located at an intergenic region of chromosome 22 (Supplemental Table S4). qRT-PCR showed that BmAmy1 expression was significantly and exclusively upregulated (~10-fold) in the midgut of the transgenic line compared with control D9L (Fig. 6B). Both female and male transgenic silkworms grew larger than wild-type silkworms (Fig. 6D and G), with the average weight of BmAmy1-overexpressing transgenic silkworms on day 4 of the fifth instar being increased by 11.9% and 6.8% in females and males, respectively (Fig. 6E and F). The cocoon shell ratio increased by 12.7% and 5.7% in female and male transgenic silkworms, respectively (Fig. 6H and I). To determine whether the enhanced yield of transgenic silkworms was due to more efficient utilization of mulberry, the starch

expression of MnAI1 and MnAI2 in mulberry leaves in response to 30 min of artificial wounding or feeding B. mori larvae (Fig. 5A). Expression of MnAI1 and MnAI2 was moderately induced at 2 h after feeding, reached a high level at 6 h after feeding initiation, and returned to a baseline expression state after 24 h (Fig. 5B and C). Two marker genes were used as positive controls: pathogenesis-related gene (PR1) and β-1,3-glucanase(BGL2) [31]. Just like the expression pattern of PR1 and BGL2 (Supplemental Fig. S6), the expression responses of MnAI1 and MnAI2 were distinct from responses to wounding(Fig. 5B and C). 3.4. Growth of B. mori is promoted by transgenic-based overexpression of BmAmy1 To investigate the effects of a further increase in BmAmy1 activity, piggyBac-mediated germline transformation was used to overexpress 7

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and 17 wild germplasms, obvious improvement in nitrogen fixation capacity was found in domesticated soybeans [34]. In both dog and human, higher amylase level through copy number expansion has been detected to cope with an increasingly starch-rich diet during evolution [8,35–37]. Biochemical assays have shown that amylase activity varies among different B. mori breeds. Usually, the polyvoltine races exhibit high survival rate and adaptation to tropical climate zones, but with short and poor quality silk fibers; whereas the bivoltine races produce longer silk fibers and are adapted to temperate zones. The bivoltine races are susceptible to diseases if reared in tropical zones. Compared to the bivoltine races, the polyvoltine races had a higher activity of α-amylase in digestive fluid and this might be the reason for their adaptation to survive tropical conditions [15,38]. Higher amylase activity has also showed to be positively correlated with survival rate under unfavorable conditions and economic traits such as larva weight, effective rate of rearing by number, single cocoon weight and cocoon yield per 100 dfls [11–14]. However, little is known about the genetics underlying these variations in amylase activity in different silkworm breeds. We speculate that artificial selection on genes related to starch utilization also happened during silkworm domestication and

content of B. mori feces was measured and shown to be significantly lower in transgenic than in wild-type silkworms (Fig. 6C). 4. Discussion Domestic plants and animals are essential for modern human society. The trade of products from domesticated silkworm initiated the interaction between eastern and western civilizations. Sericulture calls for silkworm varieties that produce more silk, with less diet input. The most obvious changes in domesticated silkworm are larger body size and more silk produced compared with its ancestor B. mandarina. Previous studies found evidence for selection associated with the promotion of silk production during domestication [32,33]. However, whether domesticated silkworms enhance their diet utilization remained unknown. Our results indicated that artificial selection forced the early ancestors of domesticated silkworms to enhance their amylase expression for digesting starch more efficiently, relative to B. mandarina. Improvements related to nutritient utilization often occur during domestication of both plants and animals. By monitoring 31 cultivated

Fig. 6. Screening and characterization of transgenic silkworm line (P3P:Amy1). (A) Transgenic silkworms were identified by detecting DsRed expression in the compound eyes. (B) Realtime quantitative PCR (qRT-PCR) of BmAmy1 in multiple tissues of silkworm from P3P:Amy1 and wild-type (WT) silkworm. (C) Starch content in feces from P3P:Amy1 and WT silkworms. (D) Comparison of body size between P3P:Amy1 and WT larvae, including male (♂) and female (♀) individuals from each group. Body weight of female (E) and male (F)larvae from P3P: Amy1 and WT groups. (G) Comparison of cocoons made by female individuals from P3P:Amy1 (top) and WT (bottom) groups. Statistics of cocoon shell ratio between P3P:Amy1 and WT from female (H) and male (I) individuals, respectively. Error bars, SEM; *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). 8

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Conceptualization, Data curation, Funding acquisition,Writing – review & editing, Supervision.

improvement. On one hand, early ancestors of domesticated silkworms needed to adapt to new feeding conditions with abundant humansupplied mulberry leaves. On the other hand, as part of breeding programs, larger silkworm individuals were kept for further propagation. We discovered that a silkworm amylase, BmAmy1, is expressed abundantly in B. mori and hardly at all in B. mandarina. Three kinds of TE fragment insertions in the 5′-upstream regulatory region of BmAmy1, which were found in different silkworm inbred lineages, were shown to be responsible for the enhanced BmAmy1 expression, while the majority of B. mandarina individuals harbored no TE insertions. This demonstrated that BmAmy1 with TE insertion in the 5′-upstream regulatory region were artificially selected both during and after the domestication process. The improved silkworm strains may have been bred independently and descended from distinct ancestries. The interaction between amylases and amylase inhibitors is one example of the perpetual molecular arms race between insects and host plants. Plants produce defensive amylase inhibitors against insect amylases, while insects regulate their amylases to overcome the digestive inhibition from plants [39–42]. Five wheat α-amylase inhibitors of the structural 0.19 family exhibited intriguing inhibition differences against different α-amylases [42]. Some plant amylase inhibitors only inhibit insect α-amylases, and not mammalian α-amylases，which shows great potential for breeding insect-resistant transgenic plants [43]. Both mulberry α-amylase inhibitors, MnAI1 and MnAI2, showed significant inhibition of BmAmy1 and induced gene expression in response to silkworm feeding. So, the enhanced level of BmAmy1 in domesticated silkworms could also be one strategy for adapting to the nutritional condition of mulberry as the sole food supply during artificial selection. It is reported that chloride-activated α-amylases have three conserved amino acid residues (RNR or RNL) [29]. A glutamine (Q), instead of R or L, is usually present at the last site in lepidopteran α-amylases. Thus, most lepidopteran α-amylases are thought not to be activated by chloride [44]. Here, BmAmy1 was shown to have intact residues for chloride binding, which demonstrated that BmAmy1 differed from other lepidopteran α-amylases, as was also shown in the phylogenetic analysis. This was the first lepidopteran α-amylase found to have conserved chloride-binding residues. Domesticated plants and animals have a collection of human-favored traits. Various molecular mechanisms behind domestication have been gradually explored in plants and animals [4–6,32–34,45–48]. Recently, it was shown that domestication traits can be improved rapidly by gene engineering in plants [49–53]. Through editing loci important for yield and productivity using the CRISPR-Cas9 gene editing system, the morphology, size, number and nutritional value of fruits in engineered Solanum pimpinellifolium were modified, allowing the creation of new and genetically variable crops in a short period [49,50]. As traditional breeding methods often result in fitness penalties and reduction of genetic diversity, disease resistance and stress tolerance in domesticated plants and animals are generally reduced compared with their ancestors. Therefore, de novo introduction of domestication traits could become an alternative route for future breeding [54]. Here, we showed that further enhancement of BmAmy1 by piggyBac-based transgene could also benefit the growth of B. mori. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2020.11.064.

Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by grants from the State Key Program of National Natural Science of China (no.32030103 and 31530071) and Chongqing Research Program of Basic Research and Frontier Technology (Grant No. cstc2017jcyjAX0334). We thank professor Georg Jander for critical reading and comments of the manuscript. We thank colleagues in National Center for Silkworm Genetic Resources Preservation in Chinese Academy of Agricultural Science and Guangxi Sericulture Science and Technology Research Institute for supplying parts of the domesticated silkworm strains. References [1] Q. Xia, Y. Guo, Z. Zhang, D. Li, Z. Xuan, Z. Li, F. Dai, Y. Li, D. Cheng, R. Li, T. Cheng, T. Jiang, C. Becquet, X. Xu, C. Liu, X. Zha, W. Fan, Y. Lin, Y. Shen, L. Jiang, J. Jensen, I. Hellmann, S. Tang, P. Zhao, H. Xu, C. Yu, G. Zhang, J. Li, J. Cao, S. Liu, N. He, Y. Zhou, H. Liu, J. Zhao, C. Ye, Z. Du, G. Pan, A. Zhao, H. Shao, W. Zeng, P. Wu, C. Li, M. Pan, J. Li, X. Yin, D. Li, J. Wang, H. Zheng, W. Wang, X. Zhang, S. Li, H. Yang, C. Lu, R. Nielsen, Z. Zhou, J. Wang, Z. Xiang, J. Wang, Complete resequencing of 40 genomes reveals domestication events and genes in silkworm (Bombyx), Science 326 (5951) (2009) 433–436. [2] K.P. Arunkumar, M. Metta, J. Nagaraju, Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA, Mol. Phylogenet. Evol. 40 (2) (2006) 419–427. [3] S. Omura, Research on the behavior and ecological characteristics of the wild silkworm, Bombyx mandarina, Bulletin of Sericultural Experiment Station of Japan 13 (1950) 79–130. [4] M.B. Hufford, X. Xu, J. van Heerwaarden, T. Pyhajarvi, J.M. Chia, R.A. Cartwright, R.J. Elshire, J.C. Glaubitz, K.E. Guill, S.M. Kaeppler, J. Lai, P.L. Morrell, L.M. Shannon, C. Song, N.M. Springer, R.A. Swanson-Wagner, P. Tiffin, J. Wang, G. Zhang, J. Doebley, M.D. McMullen, D. Ware, E.S. Buckler, S. Yang, J. Ross-Ibarra, Comparative population genomics of maize domestication and improvement, Nat. Genet. 44 (7) (2012) 808–811. [5] P. Wiener, S. Wilkinson, Deciphering the genetic basis of animal domestication, Proc. R. Soc. B Biol. Sci. 278 (1722) (2011) 3161–3170. [6] Y.N. Zhu, L.Z. Wang, C.C. Li, Y. Cui, M. Wang, Y.J. Lin, R.P. Zhao, W. Wang, H. Xiang, Artificial selection on storage protein 1 possibly contributes to increase of hatchability during silkworm domestication, PLoS Genet. 15 (1) (2019) e1007616. [7] J.L. Da Lage, F. Maczkowiak, M.L. Cariou, Molecular characterization and evolution of the amylase multigene family of Drosophila ananassae, J. Mol. Evol. 51 (4) (2000) 391–403. [8] M. Arendt, T. Fall, K. Lindblad-Toh, E. Axelsson, Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes, Anim. Genet. 45 (5) (2014) 716–722. [9] A. Tonoike, Y. Hori, M. Inoue-Murayama, A. Konno, K. Fujita, M. Miyado, M. Fukami, M. Nagasawa, K. Mogi, T. Kikusui, Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds, Anim. Genet. 46 (5) (2015) 580–583. [10] A.J. Hunter, B. Jin, J.M. Kelly, Independent duplications of alpha-amylase in different strains of Aspergillus oryzae, Fungal genetics and biology: FG & B 48 (4) (2011) 438–444. [11] N.A. Ganie, A.S. Kamili, K.A. Sahaf, I. Murtaza, K.A. Dar, M.A. Malik, Comparative analysis of digestive amylase activity in some tropical and temperate breeds of mulberry silkworm, Bombyx mori L, Curr. Sci. 112 (3) (2017) 624–629. [12] N. Balachandran, C. Kamble, Evaluation and quantification of digestive amylase enzyme and its relation with productive and economic traits of multivoltine silkworm genetic resources of India, International Journal of Advanced Science and Research 1 (11) (2016) 01–08. [13] S.N. Chatterjee, C.G.P. Rao, G.K. Chatterjee, S.K. Ashwath, A.K. Patnaik, Correlation between yield and biochemical parameters in the mulberry silkworm,Bombyx mori L, Theor. Appl. Genet. 87 (3) (1993) 385–391. [14] S.N. Chatterjee, R.K. Datta, Hierarchical clustering of 54 races and strains of the mulberry silkworm, Bombyx mori L: significance of biochemical parameters, Theor. Appl. Genet. 85 (4) (1992) 394–402. [15] N. Ngernyuang, I. Kobayashi, A. Promboon, S. Ratanapo, T. Tamura, L. Ngernsiri, Cloning and expression analysis of the Bombyx mori α-amylase gene (Amy) from the indigenous Thai silkworm strain, Nanglai, J. Insect Sci. 11 (2011) 16. [16] S. Kumar, G. Stecher, M. Li, C. Knyaz, K. Tamura, MEGA X: molecular evolutionary genetics analysis across computing platforms, Mol. Biol. Evol. 35 (6) (2018) 1547–1549.

Author statement Hao Yan: Methodology, Investigation, Data curation, Writing – original draft. Qingsong Liu: Methodology, Investigation. Feng Wen: Methodology, Data curation, Validation. Bingchuan Bai: Methodology, Investigation. Yuchan Wen: Investigation. Wenwen Chen: Formal analysis. Wei Lu: Resources. Ying Lin: Resources. Qingyou Xia: Conceptualization, Supervision, Funding acquisition. Genhong Wang: 10

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[36]

[37] [38]

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