Cloning and sequence analysis of Gpdh in Callosobruchus chinensis (Coleoptera: Bruchidae)

Abstract

The Sn-Glycerol-3-phosphate dehydrogenase (GPDH: NAD + 2-oxidoreductase, EC 1.1.1.8) gene of C. chinensis was cloned and its nucleotide sequence was analyzed. The gene was obtained by screening a genomic library with Drosophila melanogaster Gpdh and PCR amplification. The 5,126 bp gene obtained is comprised of one 5′ untranslated region, eight exons, seven introns, and three 3′ untranslated regions. Comparison of Gpdh of D. melanogaster with that of C. chinensis showed a 89.9% identity in the coding region, 70% in the intron, 79% in the entire nucleotide sequence, and 83.2% in the deduced amino acid sequence. The transcription initiation site is located 33 nucleotides upstream of the initiation codon, and the sequence analysis of the promoter region showed TATA and CAAT boxes at the 5′ end. The stop codon (TAA) and polyadenylation signal (AATAAA) are located at the 3′ end of each of the exons 6 to 8. These findings show that GPDH isozymes in C. chinensis are produced by the alternative processing of 3′ exons. The occurrence of the three transcripts was proven by RT-PCR using synthetic oligonucleotides complementary to the predicted unique 3′ regions. Compared to the D. melanogaster GPDH isozymes, GPDH-1, -2, and -3, C. chinensis GPDH showed 83.6%, 83%, and 84% identities, respectively.