Short duplication in a cDNA clone of the rbcL gene from Picea abies.

Abstract

The plastidic rbcL gene encodes the LSU of Rubisco (EC 4.1.1.39), the enzyme that catalyzes CO, fixation during photosynthesis (Hallick and Bottomley, 1983). In higher plants the enzyme structure is commonly given as a hexadecameric structure composed of eight LSUs and eight small subunits. Nucleotide sequence data from the rbcL gene have been used extensively in studies of plant phylogeny and molecular evolution (Morden and Golden, 1991; Pasternak and Glick, 1992). To investigate the expression of the rbcL gene in damaged and undamaged Norway spruce trees (Picea abies), we have isolated a rbcL cDNA clone via reverse transcriptasePCR (Table I). Using the proofreading ability of the DNA polymerase from the hyperthermophilic archaebacterium Pyrococcus furiosus (Pfu DNA polymerase) we have minimized the number of mutations obtained from amplifications performed with Taq DNA polymerase (Lundberg et al., 1991). The primer sequences were determined by sequence alignment of coniferous rbcL genes using the EMBL data library. The clone was sequenced by the dideoxynucleotide chain termination method using deletion subclones and additional primers. Sequence analysis of the cDNA clone revealed an ORF encoding a 475-amino acid polypeptide, highly homologous to the deduced protein sequences from the two conifers Pinus thunbergii and Pseudotsuga menziesii (Hipkins et al., 1990; Mukai et al., 1991). Furthermore, nucleic acid sequence comparison with the rbcL genes of these two species revealed no possible RNA editing site. In addition to the whole coding region (1425 bp) the sequence contains 28 bp of the 5’ leader including the possible ribosome binding site, 38 bp of the 3’ trailer, and the two PCR primer sites. Moreover, a short, 19-bp duplication was found at the 3’ end of the ORF, which seems to be a specific feature of the genus Picea. This duplication causes a frameshift that creates a new stop codon. It was also found in the North American species Picea pungens (Doerksen et al., 19911, but not in other coniferous or angiosperm species.