Journal of Insect Biotechnology and Sericology 77, 35-44 (2008) Purification and cDNA Cloning of Vitellogenin of the Wild Silkworm, Saturnia japonica (Lepidoptera: Saturniidae) Yan Meng1,+, Chao Liang Liu2, Kunihiro Shiomi1, Masao Nakagaki1 and Zenta Kajiura1,＊ 1 Laboratory of Silkworm Genetics and Pathology, Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1, Ueda, Nagano, 386-8567, Japan, and 2 Life Science School, Anhui Agricultural University, Changjiang West Road 130, Hefei, Anhui, 230036, China (Received January 5, 2007; Accepted September 21, 2007) We purified the major yolk protein, vitellin, from Saturnia japonica, by column chromatographies. SDS-PAGE and immunoblot analysis of the S. japonica vitellin (SjVn) showed that SjVn consisted of only a large subunit with a molecular size of approximately 200 kDa. We then cloned and sequenced cDNA of the S. japonica vitellogenin (SjVg), a SjVn precursor. The SjVg cDNA was 5731 nucleotides long and encoded 1776 amino acids for the en- tire subunit. The molecular weight of the predicted polypeptide was 200,000. Consensus motifs, such as GL/ICG (at the amino acid position 1592) and DGGR (located 17 residues upstream from the GL/ICG motif) were found in the deduced amino acid sequence. There is no RXRR motif, which is a cleavage site between the small and large subunits. Two polyserine regions were found in the deduced amino acid sequence. Key words: Saturnia japonica, vitellogenin, vitellin, RXRR motif, polyserine Generally, biosynthesis of Vg in insects is transcription- INTRODUCTION ally regulated in tissue-, stage-, and sex-specific manners Vitellogenesis, through which yolk proteins are accu- (Dhadialla and Raikhel, 1990; Yano et al., 1994b; Liu et mulated in oocytes for subsequent utilization, is a crucial al., 2001). Vg synthesis is suppressed by a juvenile hor- part of embryogenesis for oviparous animals including in- mone in Lymantria dispar and Locusta migratoria (Wyatt, sects. Vitellogenin (Vg), the precursor of the major yolk 1988; Davis et al., 1990; Hiremanth and Jones, 1992). protein, is extraovarially synthesized by metabolic tissues, Characterization of a variety of insects’ Vg has shown such as the liver in vertebrate animals, the intestine in that in most species a primary Vg gene product with a nematodes, and the fat body in insects (Wahli et al., 1981; molecular mass of approximately 200 kDa is generated by Wang and Williams, 1982; Spieth et al., 1985; Wheeler proteolytic cleavage of large (140-190 kDa) and small and Kawooya, 1990). After being secreted into hemo- (40-60 kDa) polypeptides in the fat body and subsequent- lymph, Vg is transported to the developing oocytes by ly secreted into the hemolymph. However, we discovered selective receptor-mediated endocytosis and deposited as that the vitellogenin of the wild silkmoth, Antheraea per- vitellin (Vn). cDNA cloning and isolation of a number of nyi, with a molecular mass of 200 kDa, is secreted with- Vg genes from insects, vertebrates, and nematodes indi- out cleavage (Yokoyama et al., 1993; Liu et al., 2001). cated that they share several conserved features and are Vitellogenins secreted without cleavage have also been re- derived from a common ancestral gene (Nardelli et al., ported in higher hymenopteran insects of the suborder Ap- 1987; Trewitt et al., 1992; Yano et al., 1994a, b; Romans ocrita, the honeybee, Apis mellifera, the parasitoid wasps, et al., 1995; Chen et al., 1997). For most insect species, Pimpla nipponica, with a molecular mass of around 180 Vgs and Vns are large molecular phospholipoglycopro- kDa, and Encarsia formosa (Nose et al., 1997; Piulachs, teins, post-translationally processed by cleavage, lipida- et al., 2003; Donnell, 2004). tion, glycosylation, and phosphorylation, providing amino The Japanese wild silkworm family, Saturniidae, is di- acids, lipids, carbohydrates, phosphates, and other nutri- vided into two subfamilies, Saturniinae and Agliinae; and ents to the developing embryo (Kunkel and Nordin, 1985; eight genera, Actias, Antheraea, Attacus, Loepa, Rhodnia, Byrne et al., 1989; Kanost et al., 1990; Raikhel and Samia, Saturnia, and Aglia (Jinbo, 2005). We have stud- Dhadialla, 1992). ied variations of these species’ vitellogenins, and deter- mined six full cDNA sequences of vitellogenins from *To whom correspondence should be addressed. Antheraea yamamai, A. pernyi, Samia cynthia ricini, S. Fax: +81-268-21-5331. Tel: +81-268-21-5337. cynthia pryeri, Bombyx mandarina (both Japan and Chi- Email: [email protected] na). We then reported on the purification, cDNA se- +Present address: Department of Agricultural and Environmental quencing, and mRNA expression of Antheraea pernyi Biology, Graduate School of Agricultural and Life Science, The vitellogenin (ApVg) (Liu et al., 2001), genetic variations University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, in Bombyx mandarina vitellogenin (BmaVg) of Japanese Japan. populations (Meng et al., 2006a), and gene organization 36 Meng et al. of Antheraea yamamai vitellogenin (AyVg) (Meng et al., ovaries, so we started the purification from approximate 2006b). Our results indicated that ApVg has only a large 350 mg vitellin in crude extraction, and obtained 56 mg subunit and no RXRR motif in the deduced amino acid purified vitellin. sequence, that stage- and sex-specific synthesis of ApVg is regulated at the level of RNA, that there are nine Bma- Gel electrophoresis Vgs, a major vitellogenin and eight variations, and that SDS-PAGE was performed according to the method of AyVg consists of seven exons and six introns. Laemmli (1970). Molecular sizes were estimated using Our preliminary experiment shows that two Vgs of Sa- high-molecular-weight calibration kits (Amersham Biosci- turnia japonica and Rhodinia fugax have only a large sub- ences). We used 6% stacking gel and 10% separating gel. unit, as well as ApVg. In this report we describe the purification and the primary structure of Saturnia japonica Immunoblot analysis vitellogenin (SjVg) in which no RXRR motif was found. We used antiserum raised against the vitellin of Anther- We discuss the relationship of vitellogenins from five Sa- aea pernyi (Liu et al., 2000). Immunoblotting was per- turniinae insects, S. japonica, A. yamamai, A. pernyi, S. formed according to the manual of ECL Plus Western cynthia ricini, and S. cynthia pryeri, and three Bombyci- Blotting Detection Reagents (Amersham Biosciences) dae insects, two B. mandarina and B. mori. with some modifications. After SDS-PAGE the gel was equilibrated in transfer buffer [100 mM Tris, 192 mM gly- cine, and 20% methanol (v/v)] for 15 min and electropho- MATERIALS AND METHODS retically transferred to polyvinilidenfluoride (PVDF) Experimental animals membrane (Immobilon™ -PSQ, Millipore) at 2 mA/cm2 for Wild silkworms of the species Saturnia japonica are 3 h. The blot was blocked with 5% non-fat dried milk in agricultural pests, because their larvae consume the leaves PBST [137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, of chestnut trees and walnut trees. Larvae of Saturnia ja- and 1.8 mM KH2PO4, pH 7.4 containing 0.1% Tween-20 ponica were collected at the Tokida campus of Shinshu (v/v)] for 1 h and then incubated for 1 h with the first an- University and reared on the leaves of the Japanese chest- tibody, a 1:5000 dilution of anti-ApVn serum in PBST. nut, Castanea crenata, or the Japanese walnut, Juglans The blot was rinsed 3 times briefly and then washed 1 × sieblodiana, at 24 ± 2°C in the rearing room. 15 min and 3 × 5 min with PBST. Then, a PVDF filter was reacted with the second antibody, peroxidase-conju- Preparation of tissues gated goat anti-mouse IgG 1:10000 diluted in PBST, for The mature ovaries of adult S. japonica were dissected another 1 h. The bound antibodies were exposed to Hyper and homogenized with 10 vol. of 10 mM phosphate-buffer film™ ECL (Amersham Biosciences). saline pH 7.0 containing 0.75% NaCl and 1 mM phenylm- etylsulfonylfluoride after weighing. The homogenates Total RNA preparation were centrifuged at 10,000 g for 10 min. The supernatants The fat bodies of female larvae of Saturnia japonica at were stored at −20°C until use (Kajiura and Yamashita, larval-pupal metamorphosis were dissected and rinsed 1989). with ice-cold 10 mM phosphate buffer pH 7.0 containing 0.75% NaCl and then quickly frozen by liquid nitrogen. Purification of vitellin The tissues were stored at −80°C until use. Total RNA of To purify SjVn, we referred to the methods of Yokoya- fat bodies was extracted using ISOGEN (Wako) according ma et al. (1994), Kajiura et al. (1998), and Liu et al. (2001). to the protocol of the supplier. SjVn was purified from 4.5 gram of mature ovaries by column chromatography using anion-exchange chromatog- Reverse transcription/polymerase chain reaction raphy with DE52 (Whatman International Ltd. Madison, (RT-PCR) England) and hydrophobic column chromatography with First-strand cDNA was synthesized with Superscript™ I butyl-cellulofine (Seikagaku Kogyo, Tokyo, Japan). In the RNaseH− reverse transcriptase (Gibco BRL) and EcoRI- purification procedures, the presence of SjVn in the frac- NotI-oligo dT18 (ENdT) synthetic primer, using 1.5 μg of tions (5 ml each) obtained at each step was checked by the total RNA prepared from the fat bodies of the spin- SDS polyacrylamide gel electrophoresis (SDS-PAGE). ning larvae. Sense and antisense primers were designed The amount of protein was measured using bovine serum on the basis of the highly conserved vitellogenin cDNA albumin as a standard (Bradford, 1976). The amount of sequences among known wild silkworms. PCR was per- vitellin in the ovaries was calculated by the scanning den- formed under standard conditions with ExTaq DNA poly- sitometer CS9000 (SHIMADZU). The calculated percent- merase (TaKaRa) using the designed primers and the age of vitellin was about 70% of total proteins in the ENdT primer. Rapid amplification of cDNA ends (RACE) Vitellogenin of Saturnia japonica 37 was used to amplify SjVg cDNA ends (Frohman et al., access at http://www.cbs.dtu.dk/services/.