Journal of Insect Biotechnology and Sericology 83, 59-70 (2014) The complete nucleotide sequence of the Eri-silkworm (Samia cynthia ricini) fibroin gene Hideki Sezutsu and Kenji Yukuhiro＊ National Institute of Agrobiologicl Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634 Japan (Received July 14, 2014; Accepted December 19, 2014) We examined 8,640 bp of the Samia cynthia ricini (Scr) fibroin gene together with the 5’ and 3’ flanking se- quences and deduced the Scr-fibroin amino acid sequence. A large region of the Scr-fibroin amino acid se- quence consists of repetitive arrays, which include a common polyalanine block and one of four variable nonpolyalanine blocks. These four types of nonpolyalanine blocks were rich in glycine (Gly) residues, differing from saturniid fibroins in which at least one type of nonpolyalanine block is Gly poor. The presence of abundant Gly residues increases the GC content of the Scr-fibroin gene. However, preferential use of GGA and GGU iso- codons for Gly decreases the GC content. The amino acid sequences of C-terminal regions among saturniid fi- broins were conserved, but were considerably diversified from non-saturniid fibroins. Three conserved Cys residues in the C-terminal region, which are conserved in Antheraea pernyi and A. yamamai fibroins, contribute to S-S bond formation between the fibroin homodimers, although another three cysteine residues conserved be- tween C-terminal regions of Bombyx mori and Galleria mellonella fibroin heavy chain contribute to formation of disulfide bonds between fibroin heavy chain (fhc) and fibroin light chain (flc). Key words: Samia cynthia ricini, fibroin, repetitive motifs, codon usage bias ability likely contributes to differences in silk properties INTRODUCTION among saturniid species. Several species of silkmoths in Saturniidae have been We report here the full length sequence of the S. c. ric- utilized in non-mulberry sericulture in Asian countries, in- ini fibroin (Scr-fibroin) gene. We analyzed its encoded cluding Japan. Antheraea yamamai is commonly used in amino acid sequence and confirmed 93 repetitive motifs, Japan, while A. pernyi is mostly cultured in China. On the which consist of a PAB coupled to an NPAB. We further other hand, varieties of saturniid silkmoths are used in In- classified 86 out of the 93 motifs into four categories ac- dia. The most abundant silk production derives from the cording to differences in the NPAB amino acid sequence. Eri silkmoth, Samia cynthia ricini. The four types of NPAB sequences were rich in glycine Saturniid silk consists of polymerized fibroin homodi- (Gly), which increased the GC content among the first mers (Tamura and Kubota, 1989; Tanaka and Mizuno, and second codon positions. In contrast, the sum of fre- 2001). This is in contrast to the domestic silkmoth, Bom- quencies of the T- and A-ending Gly isocodons (GGU and byx mori, in which three significantly different compo- GGA) was relatively high; therefore, the GC content of nents, namely fibroin heavy chain (fhc), fibroin light chain the entire gene was not strongly inflated. In this report, (flc) and P25, have been observed (Tanaka and Mizuno, we also discuss the evolutionary significance of the C-ter- 2001). Furthermore, saturniid fibroins consist mainly of minal region of Scr-fibroin coupled with two Antheraea repetitive elements, each of which contains a polyalanine fibroins (A. pernyi and A. yamamai) by comparing the B. block (PAB) and a nonpolyalanine block (NPAB) (Sezutsu mori and Galleria mellonella fibroin heavy chains (Zhou and Yukuhiro, 2000; Sezutsu et al., 2008a, b, 2010). The et al., 2001; Zŭrovec and Sehnal, 2002). Bombyx counterpart to saturniid fibroin is the fibroin heavy chain, which completely lacks PABs (Zhou et al., 2000). MATERIALS AND METHODS In addition, the fibroin components of non-saturniid spe- cies of Lepidoptera show similar tendencies to those of Insects Bombyx (Zŭrovec and Sehnal, 2002; Fedič et al., 2003; S. c. ricini larvae were the kind gifts from Hitoshi Yonemura and Sehnal, 2006). Saitou (Kyoto Institute of Technology). In saturniid fibroins, PABs are conserved and NPAB se- quences are highly variable among species relative to the Genomic DNA preparation and library construc- non-repetitive region of fibroin sequences (Sezutsu and tion Yukuhiro, 2000; Sezutsu et al., 2008a, b, 2010). This vari- We prepared S. c. ricini genomic DNA from the silk glands of a final instar larva according to the method and *To whom correspondence should be addressed. constructed a genomic DNA library using a similar method Fax: +81-29-838-6263. Tel: +81-29-838-6285. as that were described by Sezutsu and Yukuhiro (2000). Email: [email protected] 60 Sezutsu and Yukuhiro Preparation of a genomic clone inclusive of the S. Structure of S.c. ricini fibroin gene c. ricini fibroin gene We successfully subcloned and sequenced three frag- We screened the genomic clones containing the Scr- ments covering a 9.6-kb region inclusive of the 8.8-kb fibroin gene using the A. yamamai fibroin DNA sequence Scr-fibroin gene plus its 5’- and 3’-flanking sequences of as a probe (Tamura et al., 1987). We obtained a clone in- 0.7 kb and 0.1 kb, respectively (Fig. 1). The 9.6-kb se- cluding a 12.9-kb DNA fragment of this gene (Fig. 1), quence was deposited into DDBJ as accession number which was named Scr4. We next isolated three fragments AB971865. from a 9.6-kb fragment inclusive of the entire Scr-fibroin Similar to other saturniid fibroin genes, the Scr-fibroin gene using NotI, BamHI, and EcoRI digestion (Fig. 1). gene was found to consist of two exons with different The three fragments were subcloned into the plasmid vec- lengths, interrupted by a 131-bp intron. Overall, the cod- tor Bluescript SK+ (Stratagene). To determine the nucleo- ing region of the Scr-fibroin gene was 8,640 bp in length. tide sequence of this 9.6-kb fragment, we prepared a The second exon mainly consisted of repetitive sequences, series of deletion derivatives of the three subclones ac- and both terminal regions comprised unique nucleotide se- cording to Sezutsu and Yukuhiro (2000) and Sezutsu et al. quences. (2010). DNA sequencing and editing were performed ac- cording to Sezutsu et al. (2010). Conservation of intronic sequence length between saturniid fibroin genes and non-saturniid species Analytical tools The Scr-fibroin gene contains a short intron, which is a DNA sequence analyses were performed using Genetyx nearly equal length as those of the four other saturniid version 10.0 (GENETYX CORPORATION) as described species: A. pernyi (Sezutsu and Yukuhiro, 2000), A. yama- by Sezutsu and Yukuhiro (2000). MEGA (Tamura et al., mai (Hwang et al., 2001 and Sezutsu et al., 2010), Satur- 2011) and DNAsp V5.1 (Librado and Rozas, 2009) were nia japonica (Sezutsu et al., 2008a) and Rhodinia fugax used for sequence alignment and haplotype classification, (Sezutsu et al., 2008b). The intronic sequence of the A. respectively. MEGA (Tamura et al., 2011) was also used yamamai fibroin gene (Hwang et al., 2001; Sezutsu et al., to reconstruct the phylogenetic relationship. The signal 2010) was slightly longer due to insertion of a retroposon. peptide cleavage site was predicted using SignalP 4.1 Much longer introns were observed in non-saturniid fibro- (Petersen et al., 2011). in or fibroin heavy chain (fhc) genes, such as the B. mori fhc gene, which contains a 1-kb intron. Despite the conserved intronic sequence lengths, the RESULTS nucleotide sequences were less conserved among these six We isolated and sequenced S.c. ricini fibroin gene. The introns. Functional constraints may restrict intron size in structure of the gene was consistent with Antheraea per- the saturniid fibroin gene. nyi (Ap) and A. yamamai (Ay) fibroin genes (Sezutsu and Yukuhiro, 2000; Hwang et al., 2001; Sezutsu et al., Scr-fibroin amino acid sequence 2008b). We then analyzed the deduced amino acid se- The first exon was shorter and encoded 14 amino acid quence and revealed a specific repetitive structure. residues, while the longer second exon encoded 2,866 amino acid residues (Fig. 2). In total, Scr-fibroin consisted Fig. 1. Schematic expression of the Scr4 clone comprising the S. c. ricini (Scr) fibroin gene. Underlined fragments were subcloned and sequenced. The sequences of NotI -EcoRI fragments were deposited in the database (AB971865). Fibroin gene of Eri-silkmoth 61 of 2,880 amino acid residues, among which three amino quence except for deletion of a single amino acid residue acid residues (Ala, Gly, and Ser) accounted for approxi- (Fig. 4a). These amino acid sequences shared two con- mately 84% of the total residues (Fig. 3). Because Scr- served cysteine (Cys or C) residues at identical positions, fibroin encodes the large amount of Ala and Gly, the GC suggestive of functional significance (Fig. 4a). Note that content of the coding region was up to 63.6% (Fig. 3). We non-saturniid moths show strong similarity to the Scr- describe a details of this issue below. fibroin as well as other saturniini fibroins (Fig. 4a). Two trichopteran fibroin genes encode similar amino The amino acid sequence encoded by the first acid sequences from their first exons, wherein amino acid exon substitutions were observed at one of the two conserved The 14-amino acid sequence of the first exon is similar Cys residues (Fig. 4a) (Yonemura and Sehnal, 2006; Sehnal to those of saturniid fibroin genes, including those from A. and Sutherland, 2008). Since Trichoptera is a sister taxon pernyi (Sezutsu and Yukuhiro, 2000), A. yamamai (Hwang of Lepidoptera, the fibroin gene prototype was likely pres- et al., 2001; Sezutsu et al., 2010) and R. fugax (Sezutsu ent in a common ancestor of the two taxa. et al., 2008a) (Fig.