Cloning of the Metacase Type I Gene of Chlamydomonas reinhardtii and Its Involvement in the Regulation of Programmed Cell Death

Abstract:

The metacaspase is a cysteine protease with the substrate of arginine/lysine, specifically found in protozoa, fungi, and plants. The metacaspase is generally believed to be closely related to the caspase of the metazoan. Previous studies have shown that metacaspases can be divided into type I and typeⅡ according to the differences in their structural characteristics, and both types have been found to be involved in the regulation and the control of programmed cell death in various plants and protozoa. Based on the information of the sequence of the Chlamydomonas reinhardtii type I metacaspase cDNA, obtained from the Genbank database (Genbank NO. XM_001696904), this study was able to use the method of two-step nested PCR cloning to retrive the Open Reading Frame (ORF) of the sequence of the C. reinhardtii type I metacaspase cDNA. The ORF sequence, mentioned above, was named as the CrMC1 in this study. The length of the CrMC1 ORF, in its entirety, is 987 bp, from which can be inferred that there are 405 amino acids encoded in this sequence. By the method of homologous sequence comparative analysis with the already known type I metacaspase protein, it was then found that the CrMC1 has conservative p20, p10, linker domain, and arginine and cysteine active center sites. Furthermore, this study is also able to show that, during the H2O2 induced programmed cell death of C. reinhardtii, the gene transcripts of the CrMC1 was increased at a statistically significant level (P<0.05). The gene transcripts of the CrMC1 then reached its peak after 2 h. Eventually, the gene transcripts of the CrMC1 decreased to the same level as it was in the control group at 3 h. In conclusion, the results as discussed above have indicated the following: the C. reinhardtii CrMC1 was involved in the regulation and the control of the H2O2 induced programmed cell death of C. reinhardtii.