Introduction Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue8-9.Sept2000:

Insect Biochemistry and Molecular Biology 30 2000 829–837 www.elsevier.comlocateibmb Molecular cloning of a female-specific cDNA with unique repeat sequences from the fat body of the adult locust, Locusta migratoria Qili Feng a, b , Subba R. Palli b , Tim R. Ladd b , Sardar S. Sohi b , Arthur Retnakaran b , Kenneth G. Davey a, a Department of Biology, York University, 4700 Keele Street, North York, Ontario M3J 1P3, Canada b Great Lakes Forestry Centre, Canadian Forest Service, PO Box 490, 1219 Queen Street East, Sault Ste. Marie, Ontario P6A 5M7, Canada Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000 Abstract A cDNA clone encoding a 25-kDa protein 25K was isolated from a cDNA library made from RNA isolated from the adult fat body and ovaries of the locust, Locusta migratoria. The longest open reading frame of this cDNA clone encodes a 225-amino acid polypeptide, the N-terminal end of which was similar to the 21-kDa and 19-kDa juvenile hormone induced proteins identified in the locust hemolymph, but the C-terminal end was different. The C-terminal end of the 25K cDNA contained seven unique repeat elements of 10 amino acids each, most of which are polar residues. Expression of the 25K mRNA was tissue-, development- and sex-specific. A 1.2-kb mRNA was detected using the 25K cDNA as a probe only in the fat body of adult females. The mRNA started to appear at day 4 after the insect molted to the adult and rapidly increased by day 6. The mRNA was absent in the ovarian follicle cells and fat body of adult males. In vitro transcription and translation of the 25K cDNA produced a protein that migrated around 32 kDa on sodium dodecyl sulfate polyacrylamide gels. The 25K cDNA was expressed in a baculovirus expression system and the protein produced also migrated around 32 kDa.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Hemolymph; Fat body; Follicle cell; Reproductive maturation; Repeat element; Baculovirus expression system

1. Introduction

Juvenile hormone JH plays an important role in the reproductive physiology of adult insects Wyatt and Davey, 1996. During reproductive development in adult Locusta migratoria, JH induces the synthesis of several proteins, including vitellogenins Vgs Dhadialla et al., 1987; Locke et al., 1987, a hexameric storage protein Wyatt et al., 1992, a 19-kDa hemolymph protein 19K Kanost et al., 1988, and a 21-kDa hemolymph protein 21K Zhang et al., 1993. Most of these proteins are female-specific, synthesized in the fat body, transported in the hemolymph and finally taken up into the developing oocytes Wyatt and Davey, 1996. The syn- Corresponding author. Tel.: + 1-416-736-2100; fax: + 1-416-736- 5698. E-mail address: daveyyorku.ca K.G. Davey. 0965-174800 - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 5 5 - 2 thesis of these proteins is stimulated by an increase in JH titer, which occurs at about 1 week after adult ecdysis Zhang et al., 1993. JH not only induces the synthesis of these proteins but it is also known to control the entry of these proteins into the follicle. It acts via a putative JH membrane receptor JHMR on the follicular epithelium to cause large spaces to appear between the cells through which the proteins in the hemolymph gain access to the oocyte sur- face Davey et al., 1993; Sevala et al., 1995. In an attempt to clone the gene encoding the JHMR, a locust cDNA library was constructed with mRNA isolated from the ovary and fat body, and screened with antibodies raised against the partially purified putative JHMR from the follicle cell membranes. Among the cDNA clones identified during the screening process, one clone enco- ded a protein that was similar to the 21K and 19K pro- teins described earlier. In this paper we report the characterization of this cDNA and expression of its 830 Q. Feng et al. Insect Biochemistry and Molecular Biology 30 2000 829–837 mRNA. The N-terminal end of the deduced amino acid sequence of this cDNA shows similarity with the amino acid sequence of the 21-kDa and 19-kDa proteins ident- ified from the locust Kanost et al., 1988; Zhang et al., 1993, but the amino acids at the C-terminal end are different. Expression of the mRNA for this protein is tissue-, development- and sex-specific.

2. Materials and methods