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Molecular genetic study of acclimation mechanism to phosphate deprivation stress in photosynthetic euaryote.

光合真生物缺磷胁迫适应机制的分子遗传学研究。

基本信息

批准号：
12640637
负责人：
KOSUKE Shimogawara
金额：
$ 2.18万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

1. Seven phosphate transporter gene homologues (CrPTA1, CrPTA2, CrPTA3, CrPTA4, CrPTB1, CrPTB2, CrPTB3) and one phosphate repressible phosphatase gene (CrAP) were cloned in Chlamydomonas reinhardtii. Among these transporter homologue genes, at least CrPTA1 and CrPTA3 complemented the phosphate uptake phenotype of yeast phosphate transporter deficiency mutant. Southern blotting analysis revealed that there are no more CrPTA homologue gene in Chlamydomonas. Transcriptional regulation of these genes under phosphate limitation condition were examined by real-time RT-PCR. It was revealed that transcription of CrPTA1 and CrPTA3 were dramatically repressed under phosphate limitation condition. Meanwhile that of CrPTB2 and CrAP were drastically induced under the same condition.2. Extensive screening of mutant of Pi-uptake induction were performed by using the fact that relative density of the cells rapidly increase during reabsorption of Pi when Pi was resupplied to P-starved cells. The mutagenized cells were generated by insertional mutagenesis. The cells with different relative density were separated by isopycnic density gradient centrifugation using Percoll. Sixteen independent mutants of Pi-uptake induction were isolated from about one million of independent mutants. Genetic complementation test using PSR1 gene, which was previously characterized as a gene responsible for phosphate starvation acclimation, revealed that at least ten mutants out of sixteen were new class of mutants, and thus suggested to carry a mutation on a regulatory gene of Pi uptake induction previously unknown.

1.克隆了莱茵衣藻（Chlamydiumreinhardtii）的7个磷酸盐转运蛋白基因（CrPTA 1、CrPTA 2、CrPTA 3、CrPTA 4、CrPTB 1、CrPTB 2、CrPTB 3）和1个磷酸盐阻遏型磷酸酶基因（CrAP）。在这些转运蛋白同源基因中，至少CrPTA 1和CrPTA 3互补了酵母磷酸盐转运蛋白缺陷突变体的磷酸盐摄取表型。Southern杂交分析表明，衣原体中已不存在CrPTA同源基因。通过实时荧光定量RT-PCR检测这些基因在磷限制条件下的转录调控。结果表明，在磷酸限制条件下，CrPTA 1和CrPTA 3的转录受到显著抑制。同时，CrPTB 2和CrAP在相同条件下也被显著诱导.利用磷饥饿细胞再补充磷后，磷重吸收过程中细胞相对密度迅速增加的事实，对磷摄取诱导突变体进行了广泛的筛选。通过插入诱变产生诱变的细胞。采用Percoll等密度梯度离心法分离不同相对密度的细胞。从约100万个独立突变体中分离出16个Pi摄取诱导的独立突变体。利用PSR 1基因进行的遗传互补试验表明，在16个突变体中，至少有10个是新的突变体类型，这表明PSR 1基因是一个未知的磷吸收诱导调控基因。