RIBULOSE-1, 5-BISPHOSPHATE CARBOXYLASE SMALL SUBUNIT GENES IN CASSAVA

Abstract

The aim of this project was to characterize the Ribulose-1,5-bisphosphate carboxylase small subunit (RbcS) genes the of cassava by investigating the sequence of its cDNA. Messenger RNA from leaves of cassava cultivars was successfully isolated. The cDNA was used for the construction of ?gt 10 cDNA libraries. About 500 potential RbcS clones were found to hybridize with the rubber 3' end RbcS cDNA probe (Hev23). When these clones were rescreened with the rubber 5' end RbcS cDNA probe (Hev78), only eleven clones were detected. Seven full-length cDNAs, two incomplete cDNAs, one 5' end cDNA and eight 3' end cDNAs from Buloh and Brazil cultivars of cassava were sequenced. The RbcS cDNAs of cassava are grouped into three subfamilies based on nucleotide (nt) and deduced amino acid (aa) sequence homology. While the transit peptide of RbcS precursor for all the subfamilies consists of 59aa, the mature RbcS belonging to Subfamilies I, II and III consist of 125aa, 123aa and 117aa, respectively. Members of Subfamily I differ from members of Subfamily II in 9 to 11aa in the transit peptide and 17aa in the mature small subunit. There is only a limited homology in the 3' non-coding trailer regions of the members between Subfamilies I and II. Only a single member of Subfamily III was isolated. Members of Subfamilies II and III are very similar, even within the 3' non-coding trailer region. Starting from position 28 to 39 of the mature small subunit of Subfamilies I and II, 12aa are missing in the mature small subunit of Subfamily III. The 12aa are replaced by 6 different amino acids. Although the change occurs in a non-conserved region of RbcS, no similar finding has ever been reported in other RbcS proteins. A long cDNA of Subfamily I was sequenced. The 5' and 3' sequences flanking the coding region are 266nt and 280nt long, respectively. This clone also has a poly A tract of 70nt. The size of this clone is 1163nt. From the cDNA sequences of this clone and others, several interesting features of the cassava Rbcs cDNA structure were found. When the deduced RbcS precursor protein sequences of cassava were compared with those of other plants, they were found to be most similar to the protein from rubber (Hevea brasiliensis). Clone R314 of Subfamily I and clone R37 of Subfamily II have amino acid homologies of 80.9% and 83.0% with respect to the rubber RbcS precursor, respectively. Amino acid homology between R314 and R37 is 84.1%. These results suggest that the subfamily classification described here for RbcS may be a common feature of the Euphorbiaceae family in which cassava and rubber belong.