Determination of expression levels of chlamydomonas reinhardtii arylsulfatase genes by semi-quantitative PCR and QPCR methods

Arylsulfatases are enzymes that hydrolyze sulfate from other molecules. These enzymes are found in microorganisms and animals, but it is argued in the literature that there is no arylsulfatase enzyme in plants and that there is no sulfatase activity in plants. Mutations in the arylsulfatase genes in humans cause different diseases. These data show that arylsulfatases are important enzymes in the fields of biotechnology, medicine and agriculture. In the study, 19 arylsulfatase (ARS) genes in the genome of the model organism Chlamydomonas reinhardtii were determined in the Phytozome database, and their gene and protein sequences were obtained. In the literature, although there are studies on ARS1 and ARS2 genes; this thesis is the first functional study on 17 other gene models. Domain, motif and molecular phylogeny analyzes of possible protein sequences were performed with bioinformatics tools. Bioinformatics studies have supported that the gene models are arylsulfatase proteins. RT-PCR analyzes were performed to determine whether there were changes in the transcription levels of these genes in sulfate deficiency. For RT-PCR analyses, C. reinhardtii cells were first grown in normal medium (TAP) and then transferred to sulfate-free medium (TAP-S) and samples were taken from cultures at certain time intervals. RNA was isolated from the samples taken, then RNAs were converted into cDNA and PCR reactions were established with cDNAs. Results showed increased transcription in sulfate-free media in ARS6, ARS7, ARS11, ARS12, ARS13, ARS17 and ARS19; however, this increase is not as high as the increase in the positive control gene (ARS2). Interestingly, the increase in other genes was seen at the 5th hour of sulfate starvation, while the increase in the ARS17 gene was seen at the 24th hour. Phylogenetic tree was drawn using ARS protein sequences from C. reinhardtii and other organisms. In the tree, C. reinhardtii proteins are in close branches with Volvox carteri proteins while they were clustered in a separate group with Homo sapiens and bacterial species. As a result, 19 ARS genes were found in the C. reinhardtii genome. These results will shed light on future applications.