Cloning And Characterization Of RbcS Genes And Activity Analysis Of Its Promoter From Chlorella Pyrenoidosa

Chlorella, a common unicelluLar green alga, is good material in the study of photosynthesis and the occurrence of chloroplast. RbcS is the small subuint of ribuLose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme in the Calvin cycle. In this paper, a fuLl-length cDNA and two fuLl-length DNA sequences were obtained and analyzed from Chlorella pyrenoidosa 820, respectively. Then the reporter gene of green fluorescent protein (GFP) was used to detect the promoter activities of rbcS2 gene in the transgenic alga Dunaliella salina. Firstly, sequences of 245bp were cloned,based on one pair of degenerate primers (RF/RR) which was designed according to the conserved regions of rbcS genes from other green algae. Then, combined the partial rbcS fragments with 5'-RACE and 3'-RACE resuLts, the fuLl-length rbcS sequence was obtained. Sequences analysis showed that the fuLl-length rbcS cDNA of C. pyrenoidosa 820 was 907 bp, which encoded 44 amino acids of transit peptide and 140 amino acids of mature peptide. Structural analysis displayed the Kozak consensus sequence (A/GXXATGG) around the start codon ATG, and a 5-bp TGTAA tailing signal in the 3'untranslated region. The highly conserved rbcS sequence of YYDGRYWTMWKLPMFG in higher plants was also found in the Chlorella pyrenoidosa with only two amino acids differences. Similarity analysis showed that rbcS cDNA of Chlorella pyrenoidosa 820 shared wide and moderate similarities with other green algae and high plants.Secondly, 3 pieces of rbcS DNA fragments (named rbcS1, rbcS2 and rbcS3, respectively) were obtained by the degenerate primers (RF/RR) from C. pyrenoidosa 820. And two fuLl-length DNA sequences of rbcS1 and rbcS2 were obtained using the genomic walking techniques. Sequences analysis of the two rbcS DNAs showed that rbcS2 DNA was accordant to the known rbcS cDNA sequence. Several conserved promoter motifs such as TATA-like box, CAAT-like box and so on were found in the 5'upstream of rbcS2, however, the motifs were not found in rbcS1 sequence.Thirdly, 4 pieces of different length of promoter fragments (361 bp,528 bp,907 bp and 1302 bp, respectively) of rbcS2 were amplified and ligated to promoterless pAcGFP1-1 vector. Then the constructed GFP vectors were introduced into Dunaliella salina to detect the promoter activities through electroporation method. Observation under the fluorescence microscope indicated that the transgenic algae appeared at the third day and reached the peak at the sixth day. The resuLts showed that the 4 pieces of promoters couLd successfuLly drive the expression of GFP in D. salina, and the expression level showed no significant differences between the four different promoters. The research proved that the rbcS2 promoter region of Chlorella pyrenoidosa 820 had the activity.