The Phylogenetic Digging, Expression Analysis And Function Verification Of Meiosis Specific Genes Of Phaeodactylum Tricornutum Bohlin

Sexual reproduction is unique to a wide range of eukaryotes, and meiosis is a hallmark of sexuality. Genetic recombination between homologous chromosomes is needed in most organisms for successful completion of the first meiotic division. We selected 24 genes from literatures, which encode important proteins functioning at different stages of meiosis. We determined th evolutionary relationship of five of six currently recognized eukaryotic supergroups through Bayesian phylogenetic analysis.Phaeodactylum tricornutum is an abundant unicellular diatom in aquatic habitats, and the genome of P.tricornutum has been sequenced. P. tricornutum has the ability of producing substantial amounts (e.g.20-50% dry cell weight) of triacylglycerols (TAG) and polyunsaturated fatty acids (PUFAs) as the storage lipids under photo-oxidative stress or other adverse environmental conditions. But at present the range of reproduction mode of P. tricornutum is unknown. Nineteen genes (Smcl, Smc2, Smc3, Smc4, Smc5, Hop1, Rad21, Spoil, Rad51, Rad50, Mnd1, Pms1,Mre11, Mlh1, Mlh3, Msh2, Msh4, Msh5 and Mer3, six of them are known to function only in meiosis in model organisms) are likely to exist in P. tricornutum. These genes encode proteins involving in sister chromatid cohesion, pairing of homologous chromosomes, synaptonemal complex formation, and interhomolog DNA strand exchange. These data suggest that the ancestor of P. tricornutum was capable of performing meiosis.Using RT-PCR, the expression of some meiosis specific genes (MND1, MSH4, MSH5, MER3, MRE11, MLH1) and two gene duplications (SPO11-1, SPO11-10, DMC1-2, DMC1-9, DMC1-22) were analyzed in P. tricornutum. All of the genes were transcribed in the life cycle of P. tricornutum.The SPO11 protein creates double-strand DNA breaks (DSBs) only during the early stages of meiosis. The DMC 1 protein catalyzes interhomologous DNA strand exchange also only during meiosis. To study the functions of these two important proteins, we have successfully cloned four genes encoding DMC1-2, DMC1-9, SPO11-1 and SPO11-10 from the genome of P. tricomutum. The complementation study was performed with the deletion strain of Saccharomyces cerevisiae in which the target genes (DMC1 or SPO11) were inactivated. Results indicated that SPO11-10 gene maintained its function in meiosis.Homologs of meiosis-specific genes were identified in several organisms previously considered to be asexual, indicating that they are derived from sexual lineages, and may have as-yet-unobserved meiosis. P. tricomutum has six of nine meiotic-specific genes and some of meiotic genes have been expressed during the life cycle, even more, SPO11 gene has maintained its function in meiosis. These data suggested that P. tricomutum had the potential of meiosis, or lost its sexuality not long ago.Our study built the meiotic gene function verification technique of marine microalgae in the deletion strain of S. cerevisiae, which may be used to detecting sexual reproduction in unicellular microalgae, including those presumed to be asexual, by searching for meiotic genes and testing their function.