| Grass carp (Ctenopharyngodon idella) is one of the essential freshwater fishes which occupy an important position in freshwater aquaculture in China. However, hemorrhage of grass carp caused by Grass carp reovirus (GCRV) is a thorny problem. It has restricted the sustainable healthy development of grass carp aquaculture. To solve this issue, we should reveal the molecular genetic regulation mechanism of the disease, which involves in the resistance mechanism of grass carp after infected GCRV, the regulation factors and target genes associated with the disease. Based on the knowledge, we then find an effective way to control and prevent the disease from the genetic perspective. In recent years, in the field of small RNA (microRNA) research has attracted more and more attention. Many researches have proved that miRNA is closely related with the biological growth, development, metabolism and the generation, diagnosis, treatment of the disease.In this study, we used high-throughput sequencing technology and bioinformation methods to identify and analyze normal and GCRV infected miRNAs in grass carp and barbel chub. Seven tissues of thymus, head kidney, mesonephros, gill, liver, spleen and muscle were collected. Based on the comparative analysis of the differential expression, microRNA target prediction and enrichment analyses were carried out for the mixing samples of the two fishes. We expect to find and reveal the differences in molecular regulation mechannism of disease resistance between grass carp and barbel chub. It can provide theoretical basis for the feasibility of cross breeding between the two fishes. The main results are as follows:1. miRNA Solexa sequencing of Grass carp and barbel chubTo identify miRNAs expressed in grass carp (cid) and barbel chub (scu), the small RNA libraries are analyzed by deep sequencing. A total of 8,834,873 valid reads (61.36% in raw reads) and 9,381,928 valid reads(77.50% in raw reads) are obtained,corresponding to 153,388 and 218,619 unique sequences which are the associated counts of the identical total unique sequences respectively from cid and scu libraries. Based on statistical analysis for the length of clean reads, majority of the reads are 20-24nt, which is identical to the classical size of miRNA’s definition.To further identify miRNAs in cid and scu,all valid reads are mapped to known miRNAs in the miRBase 21.0 database.929 precursor miRNAs and 1122 mature miRNAs sequences are identified. Blast analysis against all mature miRNAs of selected species in miRBase,revealed 545 miRNAs from cid and scu that have no homology with other species,also named predict candidate miRNA(PC miRNA).There are 81 miRNAs which have known precursor but 3’or 5’mature miRNAs havn’t been reported.A total of 626 unique novel miRNAs which accout for 55.79% of unique miRNAs are identified.The percentage of 14% sequences are completely consistent with the known fish miRNAs in total unique miRNAs,and 30.2% sequences are isomerize expression miRNAs.2. The analysis of differential miRNAs in cid and scuThe results of differential miRNAs analysis in cid and scu showed that there are 562 co-expressed and 560 differentially expressed sequences.Through the normalized analysis of the two species miRNAs expression quantity,the data of p-value>=0.05 and |logfoldchange|<=1 are removed which do not show significant difference.There are 558 miRNAs are significant differential between cid and scu.A great amount of differential miRNAs are significant difference(P<0.01). The expression levels of majority PC miRNAs are middle and low.When the distance of miRNAs are among 3000nt,cluster analysis have identified 84 miRNA clusters that contain 311 miRNAs.There are 13 miRNAs distribute in the same chromosome(scaffold_LG38).It construct a conserved miRNA clueter miR-17-92 in vertebrates.The identified cid and scu miRNAs have the highest homology with aebrafish.There are 277miRNAs sequences can comparable with the known miRNAs of zebrafish in miRBase database(21.0).128 kinds of conserved miRNAs families are identified,and 100 belong to the known miRNA families in the miRBase database(21.0).Ten kinds of let-7 miRNA families which cover most of the known let-7 families in the miRBase were identified.It is the highest conserved miRNA family in evolution. So far,Let-7h family is only find in fish,it imply let-7h may be the highly conserved miRNA family in fish.3. Q-PCR validation, target gene prediction and bioinformatics analyses of differential expression miRNATo verify results,15 significantly differential expression miRNAs in cid and scu were randomly selected for Q-PCR. Of them,11 miRNAs showed the similar differential expression levle with result of the high-throughput sequencing.The differential expression miRNA were predicted for target genes. Of 558 miRNAs, 413 miRNAs were anchored 8572 genes. From the predicted results,we found 13 and 30 target genes associataed with the grass carp hemorrhagic disease and immunity, respectively.From enrichment analyses, there were 16 significant KEGG pathways, of which seven pathways reached the most significant level of P<0.01. And there were 121 GO terms, of which 25 Go terms have the extremely difference (P<0.001).To sum up, when the GCRV challeged,the fish own immune responses are activated.The miRNA expression levels of the two species fish are differential.It explain that there is distinct differences in miRNA regulation mechnisms of grass carp and barbel chub after infected with GCRV. It may be the primary cause for the two species show different resistance after viral challeged. |
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