| Blunt snout bream(Megalobrama amblycephala),belonging to Cypriniformes,Cyprinid genus Megalobrama,is also known as Wuchang fish,which has the advantages of taste,small head,high meat content,good shape,and moderate size.It is one of the important farming species of freshwater fish in China.However,due to the narrow natural distribution,weak genetic diversity,and lack of germplasm resources protection,M.amblycephala germplasm resources are under the threat of recession,with the decrease of growth rate and early maturation.let-7 miRNAs are the important members among all the miRNAs families,which had been identified in mammals,molluscs,fish species,and so on.As a critical regulator of gene expression,let-7 family miRNAs have been reported to be involved in multiple physiological processes,including growth and gonadal development in animals.In the present study,the evolution of let-7 miRNAs was systematically investigated based on the published fish small RNA data.Using M.amblycephala as our research object,we studied the expression regulation of ten let-7miRNAs memmbers in the development of gonad and growth.The results obtained from this study could lay the basic information to explore the let-7 miRNA regulation function in fish growth and gonadal development.The main results are as follows:1.All the sequences of let-7 miRNAs members exist in fish species,as well as in five out-group species and two species considered to be the ancestors of fish,were collected in this study.Comparative genomic analysis of let-7 miRNAs members in fish species indicated that let-7 miRNA is a sequence conserved family in fish,while different species have the variable gene copy numbers.Among the ten members including let-7a/b/c/d/e/f/g/h/i/j,the let-7a precursor sequence was more similar to ancestral sequences,whereas other let-7 miRNA members were separate from the late differentiation of let-7a.The predicted target genes of let-7 miRNAs were mostly involved in biological process,especially developmental process and growth through Gene Ontology(GO)enrichment analysis.The results of KEGG pathway enrichment analysis had showed that the predicted target genes were widely exist in oocyte meiosis pathway.All of these results provided a theoretical basis that the let-7 miRNAs have an important role in the growth and gonad development of M.amblycephala.2.In order to identify the possibly different functions of these ten miRNAs in fish growth development,their expression levels were quantified in adult males and females of M.amblycephala,as well as in 3-,6-,and 12-months-old individuals with relatively slow-and fast-growth rates.These ten miRNAs had similar tissue expression patterns between males and females,with higher expression levels in the brain and pituitary than that in other tissues(P<0.05).Among these miRNAs,the relative expression level of let-7a was the highest among almost all the tested tissues,followed by let-7b,let-7d and let-7c/e/f/g/h/i/j.As to the groups with different growth rates,the expression levels of let-7 miRNAs in pituitary and brain from the slow-growth group were always significantly higher than that in the fast-growth group(P<0.05).The expressions of let-7a/b/c/d/e in 3 months from the slow-growth group were always significantly higher than that in the fast-growth group(P<0.05).These results suggest that let-7 miRNA members could play an important role in the regulation of growth development in M.amblycephala through negatively regulating expression of their target genes.3.We had found that the let-7a/b/d miRNA were mainly expressed in different gonadal developmental stages by analysis the expression patterns of let-7 miRNA family members during the different stages of gonadal development.let-7a/b/d/f/h expression gradually increased in the first five periods,but dropped significantly at the phase VI.The expression of let-7a/b/e/f in pituitary gland increased in the first four periods,and then decreased in the last two periods.These expression patterns were consistent with the gonad development in fish,indicating that the let-7 miRNAs may regulate gonad development in both sex hormones and their target genes levels.It is worth noting that the let-7 miRNAs expression in the different gonad development periods of M.amblycephala brain and pituitary tissue were exactly the opposite.Among all the let-7 miRNAs,let-7a/b had the highest expression and showed regular expression in ovary,brain and pituitary of M.amblycephala in different gonad periods,suggesting that let-7a/b may play an important role in M.amblycephala gonad development process. |
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