CDNA cloning and expression of the large subunit of juvenile hormone-regulated vitellogenin from Lymantria dispar

Abstract

The 5.4 kb RNA enriched in mRNA coding for the large subunits of vitellogenin (VgL mRNA) in the gypsy moth ( Lymantria dispar) was purified, and used to construct a cDNA library. One of the clones, pVL-80, had sequences representing an internal region of VgL mRNA. The cDNA representing the 5′-terminal region of VgL mRNA was isolated by 5′-RACE (Rapid Amplification of cDNA Ends), and found to contain sequences encoding the N-terminal amino acid sequence of Vg 190. The insert in pVL-80 was 822 bp long and had an open reading frame (ORF) extending beyond the insert in both directions. In vitro expression of the ORF produced a 32 kDa protein which reacted with Vg 190 antiserum. Southern analysis of gypsy moth genomic DNA suggested that a single copy of the VgL gene is present per genome. The pVL-80 was demonstrated to be useful as a specific and sensitive probe to investigate developmental and juvenile hormone regulation of VgL mRNA in the fat body.