To clone the Bombyx xanthine dehydrogenase (XDH) gene
as a dominant marker for silkworm
transgenesis, we performed nested reverse transcriptase–polymerase
chain reaction (RT-PCR) using
embryonic mRNA and primers designed from the conserved region of Drosophila and rat XDH
genes. Sequencing of amplified 180 bp fragments showed that two different
sequences were present
in the fragments. Since both possessed striking similarity to XDH genes
of other organisms, we
considered these to be portions of silkworm XDH genes and designated them
BmXDH1 and
BmXDH2. Subsequently we cloned separately the entire region of the two
cDNAs by PCR using
phage DNA of an embryonic cDNA library and sequenced them. The two cDNAs
were around
4 kb in size and possessed complete open reading frames. The deduced amino
acid sequences of
the two BmXDHs were very similar to each other and to those of other organisms.
The expression
pattern of wild-type larvae basically followed the tissue specificity
of the enzyme and no significant
difference was observed between the two XDH genes. The expression of both
genes was detected in
the XDH-deficient mutants, oq and og, but non-synonymous
substitutions were specifically
detected in the BmXDH1 of the oq mutant. In addition, a length
polymorphism of the second
intron of the BmXDH1 co-segregated with the oq translucent
phenotype, suggesting that deficiency
in BmXDH1 is the cause of the oq translucent phenotype.