Study On The Size Of Chloroplast Genome And Characterization Of The Rubisco From Bryopsis Hypnoides

Study on the size of chloroplast genome and characterization of the Rubisco from Bryopsis hypnoidesTian chao (Marine Biology)Directed by Prof. Wang Guangce Chloroplast from Bryopsis hypnoides was studied on different aspects in this dissertation. With researches carried on the size of B. hypnoides chloroplast genome, cloning of functional genes and physiological characteristics of functional protein, some information of the chloroplast genome and vital functional protein encoded by the genome were abtained. These results will contribute to the study of photosynthesis and carbon cycle in marine and be convenient to the further research. Chloroplasts from B. hypnoides were isolated and purified by DNaseâ… method and sucrose density gradient centrifugation, and the size of the chloroplast genome was determined by pulse field gel electrophoresis. The results showed that the size of B. hypnoides chloroplast genome was about 150 kb. PFGE analysis of B. hypnoides revealed that there were dimeric, trimeric, tetrameric and even larger multimetic cpDNA forms in chloroplasts.Chloroplast genome DNA of B. hypnoides was purified by high ionic buffer and low pH method combined with CsCl density gradient centrifugation , EcoRâ… library of the chloroplast genome DNA was also constructed in the experiment.RbcL exon of B. hypnoides was amplified by PCR and its sequence was determined, Neighbor-joining method and Maximum Parsimony method were used respectively to build phylogenetic tree according to rbcL exon sequence. Two kinds of trees showed clear that there were two groups among those species, green lineage and non-green lineage. And the relationships of algae in green lineage were similar in two trees but that in non-green lineage were not consistent. Analysis of codon preference indicated codon preference of rbcL exon of B. hypnoides distinctly differed from the relevant sequence of photosynthetic bacteria. Rubisco from B. hypnoides was isolated by ammonium sulfate fractional precipitation and gel filtration method. SDS-PAGE showed that isolated Rubisco from B. hypnoides was separated into two clear bands which represented the large subunit and small subunit of rubisco respectively. Activity of Rubisco from B. hypnoides was determined during the isolation process and the result indicated that ammonium sulfate had strongly affected the activity. Pre-incubation in ice would be helpful to enhance the activity that implied rubisco from B. hypnoides was less stable than that from higher plants.