Molecular cloning and expression analysis of RbcL cDNA from the bloom-forming green alga Chaetomorpha valida (Cladophorales, Chlorophyta)

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a crucial enzyme catalyzing the CO2-fixing reaction in the initial step of the Calvin cycle. In this study, the full-length cDNA of Rubisco large subunit (RbcL) from the bloom-forming green alga Chaetomorpha valida (designated as CvRbcL) was isolated based on homologous cloning and the rapid amplification of cDNA ends (RACE) (accession: KF514135). It was 1,966-bp long, with an open reading frame of 1,431 bp without intron. The deduced 476 amino acids gave a predicted molecular weight of 52.38 kDa and theoretical isoelectric point of 6.15. Three catalytic regions and a CO2 activator region were identified in the CvRbcL, which highly conserved with those of other green algae and higher plants. Homologous analysis indicated that CvRbcL shared 52-86 % similarities with other 13 known algal and higher plant RbcLs sequences. Phylogenetic analysis showed that it had closer relationship with those of green algae and higher plants but was comparatively far from the red and brown algae clade. Real-time PCR detection revealed that the CvRbcL expression were dramatically upregulated by light and strongly suppressed in the dark. Temperature also markedly affected the CvRbcL transcription. A relatively high transcript level appeared between 15-25 A degrees C and reached the maximum at 20 A degrees C. This indirectly reflected that highly expressed Rubisco was involved with CO2 metabolism of C. valida under favorable natural environments, and might contribute to its fast growth and bloom formation. In vitro expression of CvRbcL showed that one distinct band existed at similar to 55 kDa, and western blot detection proved that it was positive to the anti-His antibody with high specificity. Our study is the first to characterize RbcL gene from Chaetomorpha species providing more knowledge of RbcL properties and facilitating further exploration of Rubisco in green macroalgae. Our results also help to decipher Rubisco molecular functions in the rapid growth period of C. valida and provide clues to develop forecasts of the seasonal C. valida bloom.