A Gene Encoding Acetyl-Coenzyme A Carboxylase from Brassica napus

ACCase (EC 6.4.1.2) is one of the key regulatory enzymes in fatty acid biosynthesis catalyzing the formation of malonyl-COA from acetyl-COA and bicarbonate in an ATPdependent reaction providing the substrate for fatty acid synthesis. ACCase from plants is proposed to be a dimer consisting of identical subunits of larger than 200 kD (Egli et al., 1993; Gomicki and Haselkom, 1993). To date the sequence of a plant ACCase gene has not been reported. Here we describe the sequence of an ACCase gene from rapeseed (Brassica napus) (Table I). Based on regions conserved between ACCase from chicken and Escherichia coli BC (Kondo et al., 1991), a specific hybridization probe was generated by PCR from cDNAs synthesized from poly(A+) RNA of immature seeds of B. napus. Degenerate oligonucleotide primers for PCR were deduced from amino acids 305 to 312 and 384 to 391 of chicken ACCase and resulted in amplification of a 260-bp fragment covering the exon sequences in the region of nucleotides 4300 to 4813 of the rapeseed sequence reported here. This fragment encodes 84 amino acids showing 88.4 and 65.1% similarity to ACCase of chicken and BC from E. coli, respectively, and was used as a probe for the isolation of clones from a rapeseed genomic library constructed in A-FIX I1 (Stratagene, La Jolla, CA). Fragments of two overlapping genomic clones were subcloned and sequenced. A comparison with protein sequences of the ACCase from chicken, yeast, Cyclotella cypt ica , and E. coli allowed us to postulate the presence and location of 31 introns. Potential promoter elements were localized at position 2283 to 2286 (CAAT box) and 2416 to 2419 (TATA box). A putative ATG start codon is located at position 2506 to 2508. The Lys residue in the common motif for biotinyla-