Cloning and characterization of a gene encoding small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Dunaliella parva

Ribulose-l,5-bisphosphate carboxylase/oxygenase(Rubisco)is a key enzyme in photosynthesis,and its small subunit gene rbcS can affect the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme.We cloned and described the rbcS gene and promoter from Dunaliella parva. Based on the highly conserved nucleotide regions of known rbcS,a pair of specific primers were synthesized to amplify 380 bp cDNA and 1 841 bp DNA sequence in Dunaliella parva.Then the 5′ genomic DNA and 3′ cDNA sequences were cloned by Genome walking and rapid amplification of cDNA ends(RACE)technology.Based on the sequences of the 5′- and 3′-termini,primers were synthesized to obtain the full-length genomic DNA and cDNA.The full-length rbcS cDNA contained 570 bp open reading frame(ORF)(GenBank accession no.HQ315783),294 bp of 3′-noncoding region.The full--length rbcS genomic DNA was 2 031 bp.In addition,a 476 bp promoter was obtained.Similarity analysis revealed that the highest identity was found between D.parva and Dunaliella salina.The D.parva rbcS also showed wide similarity with other green algae.This study laid foundation for further research on the function analysis and overexpression of rbcS genes,genetic manipulation of Rubisco.In addition,high levels of expression have made the promoter of the rbcS gene an attractive candidate to drive expression of transgene.Therefore the D.parva rbcS promoter after optimization should facilitate the development of transformation system of halotolerant algae and efficient expression of transgene.