Analysis of the Mutational Character of Chlamyolomonas Reinhardtii Nfr-4 Mutant Strain
|Keywords||Chlamydomonas reinhardtii mutant strain promoter mutation reverse phase-HPLC quantitative real-time PCR|
In order to examine the mutational characters of the Chlamydomonas reinhardtii mutant strain Nfr-4, the resistant ability of Nfr-4 to norflurazon were tested; reverse phase-HPLC analysis of the amount of phyteone, each chlorophyll and each carotene were carried out; the content test of total chlorophlls and total cocloured carotene were confirmed; the grouth characters of the two strains were determinated by the growth curve analysis; amplification and sequencing the promoter fragment of Pds gene and using the quantitative real-time PCR technology to analyze the amount of mRNA expressed by Pds gene were implemented, all these experiments were controlled by Chlamydomonas reinhardtii wild-type CC-137. The results were enumerated as follows:The norflurazon resistant experiment proved that the Chlamydomonas reinhardtii mutant strain Nfr-4 was more tolerant to norflurazon than Chlamydomonas reinhardtii wild-type CC-137. The concentration of norflurazon under which wild-type CC-137 could grow was 2.5μM, and for Nfr-4 was 5.0μM under mixed nutrition conditions. And the maximun norflurazon concentration that wild-type CC-137 could tolerant was 3.0μM when the media had acetate in it, on the contrast, the mutant strain Nfr-4 clould grow in 6.5μM norflurazon under the same condition.Reverse-phase-HPLC analysis revealed that the content of phytoene, which is the substrate of phytoene desaturase in the carotenoid biosynthesis pathway, was evidently higher than the mutant strain Nfr-4 on the one hand, and on the other hand, the amounts of all kinds of carotenoids and chlorophylls were changed, except forβ-carotene and antheraxanthin, the amounts of other carotenoids and chlorophylls were increased more or less, and take zeaxanthin for example, some carotenoids were aggrandized prominently. When we compared the amounts of total chlorophylls and total coloured carotenoids of Chlamydomonas reinhardtii mutant strain Nfr-4 with the wild-type CC-137, much more higher contents of these piments in Nfr-4 were found. And the increased content of total chlorophylls and total coloured carotenoids in Chlamydomonas reinhardtii mutant strain Nfr-4 would change its growth curve. These physiological changes were identical to the changes that were caused by the up-control mutation of Chlamydomonas reinhardtii Pds gene’s promoter reported in a published article. That was when compared with the wild-type CC-137: the mutant strain had the peculiarities of longer growth time and higher cell density.The total DNA were extracted from Chlamydomonas reinhardtii wild-type CC-137 and mutant strain Nfr-4 respectively, and the promoters of Pds gene were amplified from these total DNAs. And then the PCR products were purified and sequenced. The results revealed that there was a base mutation of C→T occurred in nt 428.The relative amounts of mRNA expressed by Pds gene in Nfr-4 and CC-137 were quantitatively analyzed by quantitative real-time PCR technology usingβ-actin as the inner control. It was revealed that the amounts of mRNA expressed by Pds gene in Nfr-4 were notablely higher than the wild-type CC-137 with the obtained data examined by grouped data t-test. And it proved that the C→T base mutation in the Pds promoter was likely the cause of boosting the expression of Pds gene in mutant strain Nfr-4.The experiments proved that the Chlamydomonas reinhardtii mutant strain Nfr-4 were a Pds gene promoter mutation strain. The relative report of the character of norflurazon resistance endowed by Pds gene promoter mutation was only reported in Cyanobacterium. And there was no Chlamydomonas reinhardtii Pds gene mutant strain reported so far except our reports. Phytoene dehydrogenized by the catalyzing of phytoene desaturase is a key speed-limiting step in carotenoid biosynthesis pathway, and the studies on Chlamydomonas reinhardtii Nfr-4 mutant strain will accelerate the research course of the significant function of gene expressional regulation in carotenoid biosynthesis pathway.