Preliminary Study On The Function Of MiR-21 And MiR-200b In Grass Carp Under The Condition Of GCRV Infection

Ctenopharyngodon idella,as the largest freshwater fish in China,has made great contributions to China’s aquaculture industry.However,grass carp are susceptible to Grass carp reovirus(GCRV),it causes haemorrhagic disease of grass carp,which seriously restricts the healthy development of grass carp breeding industry and causes great economic losses.miRNA widely exists in organisms and participates in various physiological reactions.It also plays an important role in the process of virus response.This study used high-throughput sequencing technology to explore the potential functional miRNA molecules of grass carp in the process of coping with GCRV,and preliminarily explored the functional roles of miR-21 and mir-200 b,so as to lay a foundation for analyzing the functional mechanism of miRNA in the process of grass carp coping with GCRV.The main research results are as follows:1.Comparative analysis of head kidney miRNA transcriptome before and after GCRV infectionThrough Illumina high-throughput sequencing technology,high-quality grass carp head kidney miRNA transcriptome before and after GCRV infection were obtained.The results of Fast Qc,Pearson correlation analysis and PCA analysis show that the miRNA transcriptome data has good accuracy and high reliability.956 and 1005 miRNAs were identified in WT group and GCRV group respectively,of which 82 and 118 were unique to WT group and GCRV group.Through difference analysis,3 highly significantly up-regulated and 29 highly significantly down regulated miRNAs were screened.Targetscan and Miranda were used to predict the target genes of these 32 DE miRNAs,and then Go and KEGG enrichment were performed for the predicted target genes.The enrichment results showed that these genes were closely related to immune response.Combined with the sequencing results and relevant literature reports,we excavated seven miRNAs that play potentially important functions in immune regulation,such as miR-21,mir-100,miR-155,mir-722,miR-122,mir-194 a and mir-200 b.A large number of studies showed that miR-21 and mir-200 b played an important role in the diseases of many species.Therefore,these two miRNAs were selected for further research in this study,in order to provide research ideas for the research of disease resistance related molecules and the breeding of disease resistant grass carp.2.Cloning and preliminary study on the function of miR-21Cid-mir-21 was cloned by PCR,with a length of 87 nt,which had a typical stem-loop structure.Then,homology alignment and phylogenetic tree of miR-21 precursor and its mature sequence were carried out.Homology alignment reveals that the precursor sequence of miR-21 has great differences between different species,but both ends have very conservative regions.The consistency of miR-21 mature sequence in different species reached 93.48%,indicating that miR-21 has high conservatism in the process of biological evolution.miR-21 can be detected in 12 tissues of healthy grass carp,including liver,spleen,kidney,head kidney,skin,muscle,gill,intestine,brain,heart,hypothalamus and pituitary,and has high expression in immune tissues.After GCRV infected CIK,the expression of miR-21 showed a downward trend as a whole.The GCRV infection experiment after overexpression of miR-21 showed that miR-21 could inhibit virus replication in the early stage of GCRV infection.Subsequently,the CIK transcriptome overexpressing miR-21 was constructed and sequenced.Through differential analysis,it was found that there were 654 differential genes after overexpression of miR-21,including 225 up-regulated genes and 432 down-regulated genes.The results showed that GO was enriched in immune related GO terms such as phagosome-lysosome fusion involved in apoptotic cell clearance and transferrin receptor binding;KEGG was enriched into immune related pathways such as ubiquitin mediated proteolysis,thyroid cancer and HIF-1 signaling pathway.The above results show that miR-21 can effectively inhibit GCRV replication in grass carp cells and participate in the immune regulation pathway in cells.3.Cloning and preliminary study on the function of miR-200bCid-mir-200 b with a length of 87 nt was cloned by PCR.Rnafold prediction results shows that it has a typical stem-loop structure.The results of homology comparison and phylogenetic tree showed that mir-200 b was highly conservative in the process of evolution,and the consistency of miR-200 b mature sequence among species was as high as 97.44%.The expression characteristics of miR-200 b in grass carp tissues showed that miR-200 b was widely distributed in grass carp tissues and had a high expression level in immune tissues.After GCRV infection of CIK,miR-200 b was slightly up-regulated as a whole.The GCRV infection experiment after overexpression of miR-200 b showed that miR-200 b could promote virus replication.Subsequently,the transcriptome of CIK after overexpression of miR-200 b was sequenced.A total of 383 differential genes were obtained,including 118 up-regulated genes and 269 down-regulated genes.Then,GO and KEGG enrichment analysis was carried out for all differential genes.The enrichment results included many immune related GO term and KEGG pathways,such as phagosome and lysosome fusion,assembly and maturation,apoptosis,herpes simplex virus type I infection and autophagy.The above results suggest that miR-200 b can promote virus proliferation by regulating relevant immune pathways in grass carp cells.In conclusion,this study screened seven potential functional miRNA molecules by highthroughput sequencing of grass carp head kidney before and after GCRV infection,and selected miR-21 and mir-200 b as further research objects in combination with literature research.It is found that miR-21 and miR-200 b are highly conservative in the process of evolution,widely exist in grass carp tissues and have a high expression level in immune tissues.GCRV infection CIK can cause changes in the expression of miR-21 and miR-200 b,and can significantly affect the proliferation of GCRV.Overexpression of miR-21 and miR-200 b in CIK can change the expression of many immune related genes.The above results show that miR-21 and miR-200 b play an important role in the response of grass carp CIK to GCRV,which provides an important theoretical basis for the study of the function of miRNA in the immune regulation of aquatic animal virus,but its specific regulation mechanism needs to be further studied.