权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of genes for calcification and photosynthesis of coccolithophorids

颗石藻钙化和光合作用基因分析

基本信息

批准号：
07836013
负责人：
ITO Shoko
金额：
$ 1.47万
依托单位：
Tokyo University of Pharmacy and Life Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07836013/
关键词：
coccolithophorids calcification photosynthesis gene

项目摘要

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.

1.我们用鉴别筛选的方法分离出了一个基因的部分cDNA片段，该基因在球粒孢胸膜裂菌的球粒携带细胞中特异性表达。Northern分析显示，2.0kb mRNA在含球粒细胞中积累，而在裸细胞中没有。这表明该基因可能参与了球粒的形成。目前我们正在尝试分离完整的cDNA克隆，以分析基因产物的结构和功能。为了将与钙化有关的基因引入裸突变株，我们尝试建立转化系统。首先，我们以衣藻为微藻的模式生物，利用电穿孔法改进转化。通过调整电穿孔溶液的渗透压（TAP培养基+40 mM蔗糖）并在电镀前加入玉米淀粉，实现了高效转化（8 × 10^4个转化体/马克杯DNA，效率约为玻璃珠法的100倍）。然后，利用球孢体Pleurochrysis确定筛选转化子的条件。在所测试剂中，zeocin对菌落形成的抑制效果最好。利用ble基因（zeocin耐药基因），目前正在研究胸膜裂电穿孔的条件。为了阐明球石类动物的系统发育关系，测定了23种球石类动物核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）大亚基rbcL基因的核苷酸序列。利用简约、邻域连接和最大似然分析构建了分子系统发育树。这些树表明：(1)球石属属单系群，(2)单系群包括一个由Emiliania、Gephyrocapsa和Helicosphaera组成的分支和一个其他球石属的分支，(3)一个非球石属等裂体与Emiliania和Gephyrocapsa密切相关。