Molecular Evolution Analysis Of PiRNA Pathway Genes In Vertebrates

piRNA pathway is a novel small RNA pathway, which is essential for gametogenesis, transposon silencing and genome integrity in diverse organisms. Mobile elements can disrupt gene functions. In the germline, transposon activities may lead to apoptosis, interfere transmitting of genetic information to offspring. The diversity of transposable elements is much higher in teleost fish than in mammalian genomes. Mobile sequences undergo a higher turnover in teleost fish genomes. As a class of genes which act as guardians of the genome, protect it from invasive transposable elements in the germline, piRNA pathway genes are subjected to strong selective pressure in evolution. Most of teleosts reproduce by external fertilization. In order to ensure sufficient fertilized eggs for species survival, germ cells of the teleosts undergo continuous mitosis and meiosis during adulthood. Thus, gametogenesis is very active so as to maintain sustained reproductive capacity, which gives rise to higher risk of transposon jumping in genome at the same time. It is thought that there is a coevolutionary relationship between piRNA machinery and transposable elements.Herein,18genes from29vertebrates representing mammals and teleosts were obtained from public databases. Nine miRNA/piRNA pathway genes were cloned in the rice field eel(Monopterus albus), which belongs to Synbranchiformes, Teleostei taxonomically. Based on analyses of selective pressure on miRNA/piRNA genes across30vertebrate species, accelerated evolution of piRNA genes was observed in the teleost lineage. In additon, episodic adaptive evolution of piRNA genes on the ancestral branch of teleosts was detected, suggesting adaptive evolution of piRNA pathway genes. Furthermore, faster evolution of piRNA pathway genes in the rice field eel than other fishes, and high expression level of piRNA pathway genes in three gonads of the rice field eel suggested an important role in gametogenesis and sexual differentiation during the sex reversal.1. Cloning of Ago and piRNA pathway genes from the rice field eelUsing degenerate PCR and RACE,9rice field eel genes, including Agol?Ago2? Ago3a?Ago3b?Ago4?Piwill?Piwil2?Tdrdl and Prmt5, were identified. Ago family genes, core components involved in miRNA/siRNA pathway, were used as control genes in evolution analysis. Phylogenetic analysis showed that these genes were clustered into relative lineages. 2. Expression analysis of Ago and piRNA pathway genes in the rice field eelUsing semi-quantitative RT-PCR and real-time PCR, transcripts of Ago genes were detected in all adult tissues examined, while the expression of piRNA pathway genes were mainly expressed in three types of gonads. High expression levels of piRNA pathway genes in three types of gonads in the rice field eel suggested a potential role in gametogenesis and sexual differentiation during sex reversal. By transfection of GFP taged Ago and Piwi genes, these gene were mainly expressed in the cytoplasm.3. Accelerated evolution of piRNA pathway genes in the teleost lineageUsing distance analysis with the modified Nei-Gojobori method in MEGA, much higher dN/dS values of12piRNA genes except Henl were observed in teleost fishes than in mammals. Furthermore, branch model analysis confirm the results obtained from distance analysis.4. Accelerated evolution of piRNA pathway genes in the rice field eelBranch model analysis of piRNA pathway genes were performed between gonochoristic fishes and hermaphroditic fish rice field eel. The results showed piRNA pathway genes in the rice field eel evolved faster than other fishes, suggesting that reproductive genes in the rice field eel may be subjected by stronger selective pressure than other fishes.5. Adaptive evolution of piRNA pathway genes in the teleost lineageUsing the branch-site model implemented in PAML, many positively selected sites in functional domains of piRNA pathway genes were detected. As for Piwi genes, many positively selected sites were distributed in PAZ-MID domain. The PAZ-MID domain is structural domain for piRNA2'-O-methylated3'-end recognition, consistent with the idea that selection is strongest at the interface between target RNA molecules and silencing machinery.