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

1. 在莱因哈特衣藻中克隆了7个磷酸转运体基因同源物（CrPTA1、CrPTA2、CrPTA3、CrPTA4、CrPTB1、CrPTB2、CrPTB3）和1个磷酸抑制磷酸酶基因（CrAP）。在这些转运蛋白同源基因中，至少有CrPTA1和CrPTA3补充了酵母磷酸转运蛋白缺乏突变体的磷酸摄取表型。Southern blotting分析表明，衣藻中不存在CrPTA同源基因。利用实时RT-PCR检测这些基因在磷酸盐限制条件下的转录调控。结果表明，在磷酸盐限制条件下，CrPTA1和CrPTA3的转录受到显著抑制。同时，在相同条件下，CrPTB2和CrAP的表达也被显著诱导。利用在向缺磷细胞补充Pi时，细胞的相对密度在Pi重吸收过程中迅速增加的事实，对Pi摄取诱导突变体进行了广泛的筛选。诱变细胞通过插入诱变产生。采用Percoll等密度梯度离心分离不同相对密度的细胞。从大约100万个独立突变体中分离出16个独立的pi摄取诱导突变体。利用PSR1基因进行遗传互补测试，发现16个突变体中至少有10个是新类别突变体，这表明它们携带了一个未知的诱导磷酸吸收的调控基因突变。

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.