藓羽藻叶绿体基因组及其Rubisco的研究
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摘要
本文从不同的层次对藓羽藻叶绿体进行了研究，从其叶绿体基因组的大小测定、功能基因的克隆到基因编码产物的生理生化特性的研究，获得了藓羽藻叶绿体基因组和其编码的重要功能蛋白的信息，有助于海洋光合碳同化和海洋碳循环的研究，并为进一步研究打下坚实的基础。
用DNaseⅠ法和蔗糖密度梯度离心技术分离纯化藓羽藻叶绿体，并运用脉冲场电泳技术测定了叶绿体基因组的大小。结果表明藓羽藻叶绿体基因组大小约为150 kb。同时藓羽藻的叶绿体基因组与某些高等植物相似，存在叶绿体二聚体、三聚体、四聚体甚至多聚体的形式。
用高盐低pH法与氯化铯密度梯度离心相结合的方法纯化了藓羽藻叶绿体基因组DNA，并构建了其叶绿体基因组EcoRⅠ文库。
PCR扩增出藓羽藻Rubisco大亚基外显子基因，并克隆测序，用邻接法和最大简约法分别对藻类各门代表种的Rubisco大亚基外显子基因序列构建系统进化树，显示出明显的绿藻与非绿藻体系界限。绿藻体系中各种藻的亲缘关系在两种进化树中相近，而非绿藻体系各种藻在不同的进化树中亲缘关系不同。密码子偏爱性分析结果表明藓羽藻Rubisco大亚基外显子与光合细菌等原核生物同等序列的密码子偏爱性有较大差异。
采用硫酸铵分部沉淀与凝胶过滤的方法进行藓羽藻Rubisco的分离研究。结果表明，分离的藓羽藻Rubisco经SDS-PAGE检测呈两条清晰条带，分别为Rubisco大亚基与小亚基。藓羽藻Rubisco活力测定结果表明Rubisco分离过程中硫酸铵分部沉淀对Rubisco活力有较大影响；低温下活化对藓羽藻Rubisco活力测定效果较好，说明藓羽藻Rubisco相对陆地高等植物结构不稳定。
Study on the size of chloroplast genome and characterization of the Rubisco from Bryopsis hypnoides
Tian 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 DNaseⅠmethod 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 , EcoRⅠlibrary 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.
用DNaseⅠ法和蔗糖密度梯度离心技术分离纯化藓羽藻叶绿体，并运用脉冲场电泳技术测定了叶绿体基因组的大小。结果表明藓羽藻叶绿体基因组大小约为150 kb。同时藓羽藻的叶绿体基因组与某些高等植物相似，存在叶绿体二聚体、三聚体、四聚体甚至多聚体的形式。
用高盐低pH法与氯化铯密度梯度离心相结合的方法纯化了藓羽藻叶绿体基因组DNA，并构建了其叶绿体基因组EcoRⅠ文库。
PCR扩增出藓羽藻Rubisco大亚基外显子基因，并克隆测序，用邻接法和最大简约法分别对藻类各门代表种的Rubisco大亚基外显子基因序列构建系统进化树，显示出明显的绿藻与非绿藻体系界限。绿藻体系中各种藻的亲缘关系在两种进化树中相近，而非绿藻体系各种藻在不同的进化树中亲缘关系不同。密码子偏爱性分析结果表明藓羽藻Rubisco大亚基外显子与光合细菌等原核生物同等序列的密码子偏爱性有较大差异。
采用硫酸铵分部沉淀与凝胶过滤的方法进行藓羽藻Rubisco的分离研究。结果表明，分离的藓羽藻Rubisco经SDS-PAGE检测呈两条清晰条带，分别为Rubisco大亚基与小亚基。藓羽藻Rubisco活力测定结果表明Rubisco分离过程中硫酸铵分部沉淀对Rubisco活力有较大影响；低温下活化对藓羽藻Rubisco活力测定效果较好，说明藓羽藻Rubisco相对陆地高等植物结构不稳定。
Study on the size of chloroplast genome and characterization of the Rubisco from Bryopsis hypnoides
Tian 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 DNaseⅠmethod 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 , EcoRⅠlibrary 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.
引文
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