| The Siniperca chuatsi, commonly known as the mandarin fish, belonging to Perciformes,Sinipercinae,Siniperca. It is a valuable economic fish in China. In 2014, the culture production has reached 293,853 tons in China. Guangdong, Jiangxi and Anhui are the major farming provinces. Increasing stocking density lead to deterioration of ecological environment, and long-term artificial breeding lacking selective breeding result in a decline in the growth rate and anti-disease ability. Therefore, it is necessary to establish a selective breeding program for the mandarin fish in order to improve the economical traits of the fish and to sustain the development of the mandarin fish industry. Skeletal muscle is the major part of the fish, but also the important indicators of growth and development of fish. Postnatal muscle growing due to the processes of fibers hyperplasia and hypertrophy. Skeletal muscle differentiation and growth were exactly controlled by growth factors, regulatory proteins, the miRNA transcription factors, MAPK signaling pathway and Wnt signaling pathway.MicroRNAs(miRNAs) are small non-coding RNA molecules that approximately 22-nt-long, they are endogenous posttranscriptional regulators of target genes and involved many biological processes in organisms. In this study, muscle of S. chuatsi in two group having significant difference in the body growth were investigated. Growth-related mi RNAs were selected initially by Illumina HiSeq deep sequencing technology, bioinformatics and molecular biology methods. This study can provide candidated genes in the molecular breeding of the mandarin fish. The main results are as follows:1. Identification of the conserved miRNAs and statistic analysis of their base edit. microRNA transcriptome sequencing of Slow-growthing-group, Fast-growthing-group were performed using HiSeq high-throughput technology, and 11241781,11410753 clean reads were obtained, respectively. Comparising with miRBase database, a total of 433 conserved miRNAs were identified in these two groups. 413 miRNAs and 410 miRNAs were found in fast group and slow group, espectively. Moreover, we statisticaly analyzed the sequence variants and seed edits of the miRNAs. The result show that G-to-U and U-to-C, were the most abundant substitution types, while C-to-U and C-to-A were found with low frequences. miRNA editing not only increased the functional diversity of miRNA transcriptome, but aslo is evolutionarily stable feature of transcriptomes. Analysis of these data could rich transcriptome information of the mandarin fish.2. Identification and q-PCR validation of the differentially expressed miRNAs. 8 differential miRNAs between fast-growing group and slow-growing group were identified using Expdiff method and verified by Real-time quantitative reverse transcription PCR. Four miRNAs, miR-122, miR-192, miR-200b-5p and miR-451 were significantly highly expressed in slow-growing group, while miR-10b-3p, miR-499-5p, let-7j-5p, miR-204-3p were significantly highly expressed in fast-growing group. The results of qRT-PCR showed that: miR-122, miR-192, miR-451 had higher expression levels in Slow-growing-group(p <0.05), and was consistent with the sequencing results, while miR-200b-5p has significantly lower expression level; let-7j-5p had higher expression level in Fast-growing-group(p <0.05), while miR-499-5p(p <0.05) and miR-10b-3p(p <0.01) had lower expression levels. No significant difference of miR-204-3p between the two groups was found. The study of differential miRNAs lays the foundation for further understanding of growth and development of S. chuatsi.3. Prediction of the target genes of the differential miRNAs and analysis of KEGG Pathway. Take the intersection results predicted by the RNAhybrid and Targetscan software, 36 065 miRNA- target gene mRNA were obtained. Analysis of genetic polymorphism of miRNA-target gene interactions can provide valuable information for molecular breeding of mandarin fish. 307 signaling pathways involved in growth, metabolism and development were obtained by KEGG. Among them, the Wnt signaling pathway is an important signaling pathways in skeletal muscle of fish.4. q-PCR analysis of the expression pattern in embryo of miR-192. Differential miR-192 expression pattern in embryo of Siniperca chuatsi was analyzed by qRT-PCR, and the results showed that MiR-192 can be detected in unfertilized eggs suggested that it is a maternally expressed gene. The expression of this gene show a trend of decline at the beginning of fertilzed and then rised up from the zygote stage to mouth-open period. Highly expressions were observed in unfertilized egg, fertilized egg and multicellular stage, then expression level was sharply declined in blastocyst stage, and lowest expression levels were found from gastrula stage to muscular effect stage. However steeply rising of expressions were observed from pre-hatching stage to mouth-open stage. The results suggested that miR-192 may play a great role in differentiation and development of S. chuatsi’s embryo. |
PDF Full Text Request