Analysis of genes for calcification and photosynthesis of coccolithophorids

1.利用差异筛选法，从一株侧金藻的细胞中分离到一个特异表达基因的部分cDNA片段。北方分析表明，2.0kb的mRNA在携带颗石的细胞中积累，而在裸细胞中不积累。这意味着该基因可能与颗石的形成有关。目前，我们正在尝试分离出完整的cDNA克隆，以分析基因产物的结构和功能。2.为了将钙化相关基因导入裸突变株，我们尝试建立转化体系。首先，我们改进了电穿孔转化方法，使用衣原体作为微藻的模式生物。通过调节电穿孔溶液（TAP培养基+40 mM蔗糖）的渗透压并在平板接种前加入玉米淀粉，实现了高效转化（8 × 10^4转化体/mug DNA，约为玻璃珠法的100倍）。然后，使用球石藻属侧金藻确定筛选转化体的条件。在所测试的试剂中，zeocin是抑制菌落形成最有效的。为了研究球石藻的系统发育关系，对23种Prymnesiophyta中的核酮糖-1，5-二磷酸羧化酶/加氧酶大亚基rbcL基因进行了序列测定。利用简约法、邻接法和最大似然法分析构建了分子系统树。这些树表明：（1）球石藻属属于一个单系群，（2）该单系群由Emiliania、Gephyrocapsa和Helicosphaera的一个分支和其他球石藻属的一个分支组成，（3）非球石藻类的等鞭金藻与Emiliania和Gephyrocapsa密切相关。

1.We have isolated a partial cDNA fragment of a gene which is expressed specifically in coccolith-bearing cells of a coccolithophorid Pleurochrysis, using differential screening method. Northern analysis showed that the 2.0kb mRNA accumulated in coccolith-bearing cells, but not in naked cells. This implies that the gene could be involved in coccolith formation. Now we are trying to isolate the complete cDNA clone to analyze the structure and function of the gene product.2.To introduce genes involved in calcification into naked mutant strains, we have attmpted to develop transformation system. First, we have improved transformation by electroporation method, using Chlamydomonas as a model organism of microalgae. Highly efficient transformation (8x10^4 transformants/mug DNA,about 100 fold as efficient as the glass beads method) was achieved, by adjusting osmotic pressure of the solution for electroporation (TAP medium+40 mM sucrose) and adding cornstarch before plating. Then, conditions for screening of transformants were determined, using the coccolithophorid Pleurochrysis. Among tested regents, zeocin was the most effective to inhibit colony formation. Using the ble gene (zeocin resistant gene), conditions for electroporation of Pleurochrysis are now under investigation.3.To clarify the phylogenetic relationships of coccolithphorids, the nucleotide sequences of rbcL genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were determined from 23 species of Prymnesiophyta. Molecular phylogenetic trees were constracted using parsimony, neighbor-joining, and maximum likelihood analyzes. These trees suggest that (1) coccolithophorids from a monophyletic group, (2) the monophyletic group is consising of a clade of Emiliania, Gephyrocapsa, and Helicosphaera and a clade of other coccolithophorids, and (3) a noncoccolithophorid Isochrysis is closely related to Emiliania and Gephyrocapsa.