【作者】 王超；

【导师】 王广策；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2011， 博士

【摘要】 紫菜(Porphyra)属于红藻门,具有很高的经济和理论研究价值。与大多数高等植物中孢子体世代占优势的生活史类型不同,紫菜的生活史具有特殊性,即在整个生活史过程中,孢子体世代为微观阶段,配子体世代则为宏观阶段,配子体与孢子体都能独立进行光合作用。宏观阶段的紫菜配子体主要表现为生物量大,也就是其光合固碳能力强于微观孢子体世代。因此,对紫菜生活史两个不同世代的光合碳同化途径进行比较分析具有重要意义。Rubisco(核酮糖-1,5-二磷酸羧化酶/加氧酶)是光合碳同化途径CO2固定阶段的关键酶,也是Calvin循环正常进行的限速酶。为此,我们以紫菜为研究对象,在转录、翻译及酶活等不同水平上系统比较分析了光合碳同化途径中Rubisco在不同世代中的表达,发现紫菜单倍配子体世代中Rubisco的含量和活性都远大于二倍孢子体。为了进一步验证Rubisco的表达差异与倍性之间的协同关系,我们还对大型海藻中其它不同生活史类型的代表性物种(海带和红毛菜)不同世代间Rubisco在转录、翻译和酶活水平的表达进行了比较分析。结果表明,海藻主要生活史类型的代表性物种中Rubisco的含量和活性与其倍性相关,即单倍的配子体中Rubisco的含量和活性高于二倍孢子体,与生物量的大小无关。紫菜等红藻中Rubisco基因的位置与陆地高等植物明显不同,其大、小亚基的基因均位于叶绿体基因组,且大、小亚基基因之间存在一个高变区域—Rubisco spacer,是对红藻种质鉴定的理想区域。本论文将Rubisco spacer应用于紫菜物种的鉴定,先后对9种紫菜的丝状体进行了种质鉴定,同时发展了一种利用活体丝状体藻丝进行无需DNA提取的直接PCR扩增方法,为紫菜丝状体的快速种质鉴定奠定了基础。除了对紫菜中Rubisco在不同世代中的差异表达研究之外,本论文对Calvin循环中其它几种酶(包括PGK、GAPDH、TPI、FBPase、GPI、TKL和SBPase)基因在不同世代中的表达量在转录水平上进行了比较分析。通过real-time PCR的方法对各基因的表达进行绝对定量分析后发现,紫菜配子体世代中各基因的表达量均高于孢子体世代。所有的研究结果表明,紫菜Calvin循环中Rubisco等相关酶基因在配子体世代的表达量明显优于孢子体世代,这种表达差异与倍性直接相关。由此我们认为这种现象可能在其他生活史类型的大型海藻中也存在。更多还原

【Abstract】 Porphyra is a red algae genus and of great value on economic and theory research. In the special type of life history of porphyra, the sporophyte is microscopic phase while the gametophyte is the macro dominant phase, which is different from the sporophyte dominant life history in most of the higher plants. And both the gametophytes and the sporophytes of algae can photosynthesize and survive independently as autotrophic organisms. The macro phase gametophyte has a larger biomass than sporophyte, which displayed that the ability of photosynthetic carbon assimilation of gametophyte is greater than that of sporophyte. As a result, it is of great significance to have research on the related genes in carbon assimilation.Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase), a key enzyme of photosynthetic CO2 fixation, is also the rate-limiting enzyme in Calvin cycle. As a result, on the basis of Porphyra, systematic comparative study on the differenticial expression of Rubisco was carried out between gametophytes and sporophytes on the levels of transcription, translation and enzymic activity. Results indicated that the content and activity of Rubisco in haploid gametophyte were notably higher than that in diploid sporophyte. In order to verify the relationship of the expression level of Rubisco and the ploidy, the same comparative studies on the expression of Rubisco between gametophyte and sporophyte were carried out in Bangia fuscopurpurea and Laminaria japonica, which have representative life history in marine macroalgae. Results indicated that both content and activity of Rubisco were higher in algal gametophytes than in the sporophytes, which suggested that for the four algal species, the Rubisco content and the initial carboxylase activity were related to the ploidy of the generations rather than the accumulation of biomass.In rhodophyte (Porphyra et al.), unlike the land higher plants, the location of the Rubisco gene is different, that both the large subunit and small subunit of Rubisco are encoded in chloroplast genome, while a Rubisco spacer region with high variety exists, which is important for identification of red algae. In this study, Rubisco spacer region was applied in the identification of nine sprcies of Porphyra, and an improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was developed in rapid species identification, which is of great fundamental significance.Besides the research on the Rubisco expression in different generations of Porphyra, comparative studies on the genes related to carbon assimilation—containing PGK, GAPDH, TPI, FBPase, GPI, TKL and SBPase—were carried out for different generations of P. yezoensis on the level of gene expression using real-time PCR. Results indicated that the related genes expression level were higher in gametophyte than that in sporophyte.All the studies suggest that, the related expression level of genes in Calvin cycle (Rubisco et al.) were notablely higher in gametophyte than in sporophyte, which suggested that the expression the these genes were related to the ploidy of different generations of Porphyra, and this relationship maybe also exists in the other marine macroalgae with representative life history.更多还原