畠山 正統 神戸大学, 理学部, 助手 (50281142)
TAKEDA Makio Kobe University Graduate School of Science and Technology, Professor, 自然科学研究科, 教授 (20171647)
NAITO Tikahiko Kobe University Faculty of Agriculture, Professor, 農学部, 教授 (70031226)
|Budget Amount *help
¥13,400,000 (Direct Cost : ¥13,400,000)
Fiscal Year 2000 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1999 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1998 : ¥9,200,000 (Direct Cost : ¥9,200,000)
Most oviparous animals, both invertebrates and vertebrates, employ vitellin (Vn) as the major egg yolk protein. In most insects, the precursor, vitellogenin (Vg), is synthesized in the fat body cells, cleaved to subunits and secreted into the hemolymph. Vg is then sequestered to and accumu-lated in the developing oocytes. Recent studies have indicated that Vgs are evolutionarilly well conserved. Earlier we proposed that Vg might be used as a molecular marker to construct an insect phylogenetic tree. Prior to the present project, there were only six species, all holometabolous insects, in which complete Vg cDNA clones were obtained.
In the present project, we thus attempted to obtain and analyze complete Vg cDNAs from hemimetabolous insects. We cloned complete Vg cDNAs from Graptopsaltria nigrofuscata (Homoptera), Plautia stall (Heteroptera) and Periplaneta americana (Dictyoptera). The deduced amino acid sequences were aligned with the known insect Vg sequences. It became apparent that there existed a highly conserved GL/ICG motif and up to nine cysteine residues that follow at conserved locations near the C-termini. Based on these characteristics we developed a simple and rapid method to clone insect Vg cDNAs. Adaptor-ligated double-strand cDNA libraries were prepared and subjected to PCR using primers for the motif and the adaptor.
The new method was applied for cloning the Vg cDNAs from Ephemera japonica (Ephemeroptera), Oyamia seminigra (Plecoptera), and Gryllus bimaculatus (Orthoptera).
A molecular phylogenetic (neighbor-joining) tree, taking the most primitive and the only paleopteran species, E.japonica, as an outgroup, seems to agree at least in its basic form with the accepted phylogenetic tree based on comparative morphology.