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Mechanisms of signal transduction which controls carbon-dioxide fixation

控制二氧化碳固定的信号转导机制

基本信息

批准号：
09660357
负责人：
FUKUZAWA Hideya
金额：
$ 2.11万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Several chimeric constructs were generated by fusing series of 5'-upstream region of CAHl to the arylsulfatase reporter gene (ars) driven by βィイD22ィエD2-tublin minimal promoter. Some Chlamydomonas cells transformed with the chimeric constructs expressed the reporter gene in response to changes in the external COィイD22ィエD2 concentration. The 5'-upstream region is divided into two parts ; a part functions as a silencer in high-COィイD22ィエD2 condition and the other part functions as an enhancer in low-COィイD22ィエD2 condition.Seven high-COィイD22ィエD2-requiring mutants, which cannot grow as wild type Chlamydomonas does in air level of COィイD22ィエD2(0.04%), were isolated by gene tagging. It was indicated that these mutants carried lesions in genes related to the carbon concentrating mechanism (CCM) or the signal transduction pathway between sensing changes of environmental COィイD22ィエD2 concentration and induction of CCM-related genes. By using an indexed PAC genomic library, two mutants were complemented. The mutation of a mutant C1, which accumulate less inorganic carbon (Ci) into the cell, was complemented by a PAC clone containing 20-kb of genomic DNA. This suggests that Ci-transport related genes could be encoded within this DNA fragment. Another mutant C16, which does not induce CCM-related genes in low-COィイD22ィエD2, was transformed by a DNA fragment which correspond to the flanking sequences of the NIT1 insertional locus and the transformants recovered the induction of CCM-related genes. The cloned DNA fragment encoded a gene which does not show significant homology with any previously known genes.

通过将CAH 1的一系列5 '-上游区域融合到由β-微管蛋白D2-微管蛋白最小启动子驱动的芳基硫酸酯酶报告基因（ars）来产生几种嵌合构建体。一些用嵌合构建体转化的衣藻细胞表达报告基因，以响应外部COイD22 D2浓度的变化。利用基因标签技术分离到7株高CO浓度的衣原体D22-D22-D22需要型突变株，这些突变株不能像野生型衣原体那样在空气中生长，但在高CO浓度的D22-D22环境中却能生长。这些突变体可能存在与碳浓缩机制（CCM）相关的基因或感受环境CO_2浓度变化与CCM相关基因诱导之间的信号转导途径的损伤。通过使用索引的PAC基因组文库，两个突变体被互补。突变的突变体C1，其中积累较少的无机碳（Ci）到细胞中，由PAC克隆含有20-kb的基因组DNA的补充。这表明Ci-转运相关基因可能编码在该DNA片段内。另一个突变体C16在低CO_2条件下不诱导CCM相关基因的表达，用与NIT 1插入位点侧翼序列相对应的DNA片段转化C16，转化子恢复了CCM相关基因的诱导。克隆的DNA片段编码的基因与任何先前已知的基因没有显示出显著的同源性。