Maize acetyl-CoA carboxylase (ACCase) is a multifunctional, biotinylated protein that provides malonyl-CoA for fatty acid synthesis and elongation, and for synthesis of secondary metabolites. Fatty acid synthesis is proportional to and rate-limited by ACCase activity in seeds and leaves. Knowledge of how ACCase is regulated may be useful for increasing kernel oil content, especially in combination with the ability to alter relative amounts of specific fatty acid components.
Most ACCase activity in maize leaves and in developing endosperm tissue and embryos is encoded by the Acc1 gene (Somers et al., Plant Physiol. 101:1097, 1993). Acc1 is a semi-dominant nuclear gene, mutant forms of which confer tolerance to certain herbicides (Marshall et al., Theor. Appl. Genet. 83:435, 1992). Both wildtype and mutant Acc1 maize leaves contain two forms of ACCase activity (I and II) that differ in charge, size, cellular location, immunoreactivity with ACCase I antiserum, and herbicide inhibition. The predominant form, ACCase I, is plastid-localized and its inhibition by herbicides is altered by mutations in the Acc1 gene (Egli et al., Plant Physiol. 101:499, 1993; Egli et al., MNL 66:94, 1992).
Antiserum to SDS-denatured ACCase I (Egli et al., 1993) was used to screen a -gt11 cDNA expression library derived from oligo-dT-primed A188 seedling mRNA. Seven incomplete cDNAs of 1.2 to 5.44 kb were obtained (type A cDNA); partial sequencing and restriction mapping indicated they were identical and were significantly similar to known ACCases. A 2 kb EcoRI subclone of ACCase type A cDNA hybridized to an 8.25 kb mRNA, which is large enough to encode the 227 kD ACCase I polypeptide plus an expected plastid transit peptide (about 6.3 kb).
A W22 genomic library (Clontech) was probed with the 2 kb EcoRI ACCase type A cDNA subclone to obtain additional 5' coding sequence. Two different 15 kb genomic clones (types A and B) were identified. A 2.7 kb coding sequence from genomic clone type A was 100% identical to type A ACCase cDNA; the genomic clone also extends 5' from the cDNA and should contain the remaining coding sequence plus the promoter. A partial sequence (750 nt) of the type B genomic clone was 96% identical to ACCase type A cDNA. The genomic type B clone extends 3' from the gene and may lack the 5' end. The cDNA library was rescreened and a low frequency of clones corresponding to type B were obtained. We are continuing to sequence type A and B cDNA and genomic clones to obtain gene-specific probes that may be useful for mapping and for mRNA expression analyses.
PCR primers derived from the 5' end of type A cDNA clones and from genomic type A coding sequence were used to amplify A188 cDNA. Sequence analysis of these PCR clones is in progress. Currently available cDNA clones cover 87% of the expected ACCase coding sequence. Additional 5' coding sequence will be obtained from RACE-PCR products and genomic clone A. Peptide sequence comparisons to yeast, rat and chicken ACCase indicate they are colinear with maize ACCase cDNA. High sequence identities in the biotin carboxylase (> 33% for 57 aa), biotin binding (48% for 40 aa), and transcarboxylase domains (51% for 602 aa) were observed. The typical eukaryotic ACCase biotin binding site (VMKM) was located approximately 4.36 kb 5' of the C-terminus of the maize ACCase type A coding sequence.
Recombinant inbred maize lines from Tx303 x CO159 were used for mapping. Blots of HindIII-digested parent and progeny DNA were hybridized with a 1.2 kb EcoRI type A cDNA subclone that contained about 50% non-coding 3' sequence. The location of the only polymorphic band was mapped (Mapmaker, Kosambi mapping function) to chromosome 2S, between umc131 (4.6 cM) and umc2b (10.4 cM). Two other monomorphic bands were also present and could not be mapped in this population. We are currently investigating whether herbicide tolerance conferred by the mutant Acc1 gene cosegregates with RFLPs identified with ACCase cDNA probes.
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