| MicroRNAs (miRNAs) as a length of about22nucleotides highly conservedendogenous non-coding small RNA are newly discovered in recent years. As animportant component of the biological gene regulatory networks, they are prevalent inthe metazoan genome. More and more evidence shows that miRNAs play animportant role in animal development and human diseases. They are widely involvedin early embryonic development, organ formation, cell proliferation anddifferentiation, apoptosis and other physiological processes, they also have a certaininfluence on cancer.miRNA clusters refer to the phenomenon of the close distribution of miRNAgenes in the chromosomes. As a common phenomenon in animals, miRNA clustersplay an important role in the regulation of network systems. With more complexregulation styles, they are more efficient than single miRNA in regulation andfunction of gene expression. Relative to a single miRNA, the expression, function andevolution of miRNA clusters is still in its infancy. Therefore, it is necessary to studythe existing miRNA gene cluster in-depth. At the same time, to study a number ofhighly conserved miRNA clusters will help us understand the evolution anddevelopment of the animals and the occurrence of the miRNA.As a highly conserved miRNA cluster in deuterostomes, miR-183cluster encodethree miRNAs including miR-182, miR-96and miR-183in vertebrate, two miRNAsincluding miR-96and miR-183in amphioxus. MiR-216cluster, which is a specificmiRNA cluster in chordate and does not exist in invertebrate, includes miR-216andmiR-217. This thesis makes the classic model animal-the amphioxus as the studyobject. The miRNA members of miR-183cluster and miR-216cluster were identifiedto clarify the tissue distribution of these miRNAs and the expression pattern in the embryo. Combined with the expression patterns in other animals, we can infer theirpossible functions and the meaning in the origin and evolution of vertebrate.In this study, miR-183cluster and miR-216cluster were identified fromamphioxus (Branchiostoma belcheri tsingtauense). Meanwhile, their spatial andtemporal expression patterns in tissues and embryos were analyzed.First, according to the mature sequences and primary sequences of amphioxus(Branchiostoma floridae) miR-183cluster and miR-216cluster, primers for miR-183cluster and miR-216cluster of amphioxus (Branchiostoma belcheri tsingtauense)were designed and these miRNAs were idenitfied from amphioxus (Branchiostomabelcheri tsingtauense). Analysis of available miRNA and genomic sequence datasuggests that miR-183and miR-216cluster members are present throughoutdeuterostomes.Secondly, expression patterns of miR-183cluster and miR-216cluster indifferent tissues of the amphioxus adult were studied using the real-time quantitativePCR technology. The results show that miRNAs of miR-183cluster arepredominant-expressed in the dorsal tubular nerve cord, weak expression in thehepatic caecum and notochord, lower expression in the remaining other tissues;miRNAs of miR-216cluster are predominant-expressed in the gills, followed by thehepatic caecum, low expression in the remaining other tissues.Finally, the expression of miR-183cluster in the tissues and embryos were alsostudied using in situ hybridization. Whole-mount in situ hybridization shows thatmiRNAs of miR-183cluster does not express in early embryos and one-day old larvae,while it expresses in the dorsal tubular nerve cord of the two-days old larvae. Slices insitu hybridization demonstrates that miR-183cluster expresses in the dorsal tubularnerve cord, hepatic caecum and gills.Based on these results, the following conclusions can be drawn: miRNAs ofmiR-183cluster and miR-216cluster have shown tissue-specific expression. Theformer is mainly expressed in the dorsal tubular nerve cord, whereas the latter islargely expressed in the gills and hepatic caecum. The miR-183cluster may beinvolved in the development and function of amphioxus sensory nerve organs, while miR-216cluster may be related to the origin of the vertebrate pancreas. |
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