Isolation and characterization of a soybean (Glycine max) lipoxygenase-3 gene

Abstract

cDNA and genomic DNA clones for a soybean (Glycine max) seed lipoxygenase-3 (lox-3) isozyme have been isolated and characterized by molecular cloning and sequence analysis. The cDNA clone p3H9 was identified as encoding a lipoxygenase isozyme based on hybridization selection and immunoprecipitation assays. The insert from p3H9 was used to screen 700,000 recombinants in a phage genomic library. Two overlapping genomic clones were isolated. After localizing the mRNA coding region to the midportion of one of these clones, nucleotide sequence analysis was performed. The structure of the gene was deduced following the additional cloning and sequencing of nearly full length cDNAs. Primer extension and S1 protection experiments were used to define the 5′ boundary of the gene. The gene is encoded by nine exons interrupted by eight introns. All exon-intron junctions conform to the GT-AG rule. The deduced amino acid sequence consists of 857 amino acids with a molecular weight of 96,663 daltons. The 5′ untranslated segment contains approximately 46 nucleotides before the first methionine (ATG) codon. A typical TATA box occurs some 30 nucleotides upstream of the message start site while a CAAG sequence occurs in place of a CAAT box some 80 nucleotides upstream of the start of transcription. Four polyadenylation signals are present in the 3′ untranslated region. At least two of these are used, giving rise to cDNA clones bearing 3′ untranslated segments of 148 and 196 nucleotides. To identify which member of the lox gene family had been isolated, a gene-specific probe from the 3′ untranslated region of p3H9 was hybridized to dot blots of soybean embryo RNAs derived from lox-1,-2, or-3 null mutants. No hybridization to RNA from the lox-3 null mutant was seen, while the remaining mutant RNAs showed no reduction in hybridization. Further confirmation that the gene encodes a lox-3 isozyme was obtained by a comparison of deduced and determined amino acid sequence data. An analysis of the lox-3 sequence and a comparison to other available soybean lox sequences is also presented.