(G6PDH) is the first enzyme of the oxidative limb of
the Pentose Phosphate Pathway (PPP) which supplies NADPH
for the reductive synthesis as well as ribose-5-phosphate,
a precursor for nucleotide synthesis. Recent evidence
indicates that the PPP in soybean nodules function as
a metabolic complex or metabolon. Cloning the
soybean gene was useful to begin investigating this
interaction. G6PDH has been previously cloned
from E.coli, cyanobacteria, rat, yeast, and potato and
these sequences were used to design PCR primers.
PCR reactions were attempted with total genomic DNA
and total RNA from soybean leaf, nodule and callus culture.
RNA extraction from potato leaf was used as a positive
control. Total RNAextracted from soybean callus
culture produced a clear, reproducible PCR product that
was consistent with the positive control. PolyA+
selected mRNA from soybean callus tissue was then used
to construct a cDNA library in lambda ZapII (Stratgene,
Lajolla CA). PCR was used to follow soybean G6PDH
during the library construction with samples of the
first strand, second strand, size fractioned cDNA, and
finally a portion of the finished lambda phage library
was used as template DNA. In each of these samples
it was possible to detect a soybean G6PDH PCR product.
Further experiments sought to isolate and sequence the
soybean G6PDH from this newly constructed library.