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Genome-wide studies on the mechanisms of the stress perception by sensors for low-temperature and osmotic stress in cyanobacterium

蓝藻低温和渗透胁迫传感器应激感知机制的全基因组研究

基本信息

批准号：
14540606
负责人：
MIKAMI Koji
金额：
$ 2.3万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Histidine kinase, Hik33, functions as both low-temperature sensor and osmosensor in Synechocystis sp. PCC 6803. To elucidate the molecular mechanisms of stress perception by Hik33, the study was focused on the identification of stress-sensing domains by domain-swap experiments.(1)Making domain-swap constructsDomain swapping was designed to use Hik31 that is not involved in the perception of cold and osmotic stress. According to the tight relation between stress perception and physical state of membranes, transmembrane and/or periplasmic regions of Hik33 were swapped with corresponding regions of Hik31. Then, to produce plasmids for genome-integration, resultant chimeric genes sandwiched between 5' and 3' franking regions of the hik33 gene:(2)Production of a deletion mutant of an ORF of the hik33 geneTo avoid the possibility of the homologous recombination between the coding regions of Hik33 between genome and plasmid that sometimes disrupt the ORF, a deletion mutant of the whole ORF of the hik33 gene was made. Although Synechocystis contains over 10 copies of the genome per cell, segregation of deletion mutation was completed for all of genomes.(3)Introduction of the chimeric genes into genomes of the hik33 deletion mutantHomologous recombination at 5' and 3' franking regions of the hik33gene was successfully operated for all of domain-swap constructs.(4) Complementation test of domain-swapped genesTo identify the regions of stress sensing in Hik33, checking of the responsibility of domain-swap mutants obtained was scheduled. As an initial test, wild-type hik33 gene was introduced into the deletion mutant by homologous recombination and analyzed stress-inducible genes by DNA microarray experiments. Unfortunately, deletion of the hik33 gene was not complemented completely by the wild-type hik33 gene. Thus, experimental systems should be developed as soon as possible.

组氨酸激酶Hik 33在集胞藻PCC 6803中既是低温传感器又是温度传感器。为了阐明Hik 33感受压力的分子机制，本研究通过结构域交换实验对Hik 33的压力感受结构域进行了鉴定。（1）构建结构域交换构建结构域交换是利用不参与冷应激和渗透应激感知的Hik 31来设计的。根据应激感知和膜的物理状态之间的紧密关系，Hik 33的跨膜和/或周质区域与Hik 31的相应区域交换。然后，为了产生用于基因组整合的质粒，将所得嵌合基因夹在hik 33基因的5 ′和3 ′侧翼区之间：（2）hik 33基因ORF的缺失突变体的产生为了避免有时破坏ORF的在基因组和质粒之间的Hik 33编码区之间的同源重组的可能性，制备了hik 33基因的整个ORF的缺失突变体。虽然集胞藻每个细胞含有超过10个拷贝的基因组，但所有基因组都完成了缺失突变的分离。（3）hik 33缺失突变体基因组中嵌合基因的导入hik 33基因的5'和3'侧翼区的同源重组对所有结构域交换构建体都成功地进行了。(4)为了鉴定Hik 33中的胁迫感受区域，计划检查获得的结构域交换突变体的责任。作为初步试验，通过同源重组将野生型hik 33基因导入缺失突变体中，并通过DNA微阵列实验分析胁迫诱导基因。不幸的是，野生型hik 33基因不能完全补充hik 33基因的缺失。因此，应尽快开发实验系统。