Isolation, Expression and Characterization of rbc L Gene from Ulva prolifera J. Agardh(Ulvophyceae, Chlorophyta)

【摘要】：Ulva prolifera is a typical green alga in subtidal areas and can grow tremendously fast. A highly efficient Rubisco enzyme which is encoded by Up Rbc L gene may contribute to the rapid growth. In this study, the full-length Up Rbc L open reading frame(ORF) was identified, which encoded a protein of 474 amino acids. Phylogenetic analysis of Up Rbc L sequences revealed that Chlorophyta had a closer genetic relationship with higher plants than with Rhodophyta and Phaeophyta. The two distinct residues(aa11 and aa91) were presumed to be unique for Rubisco catalytic activity. The predicted three-dimensional structure showed that one α/β-barrel existed in the C-terminal region, and the sites for Mg~(2+)coordination and CO_2 fixation were also located in this region. Gene expression profile indicated that Up Rbc L was expressed at a higher level under light exposure than in darkness. When the culture temperature reached 35℃, the expression level of Up Rbc L was 2.5-fold lower than at 15℃, and the carboxylase activity exhibited 13.8-fold decrease. Up Rbc L was heterologously expressed in E. coli and was purified by Ni~(2+) affinity chromatography. The physiological and biochemical characterization of recombinant Rubisco will be explored in the future.