Study Of Screening And Function Of MicroRNAs Related To MSTN In Carassius Auratus In Qihe River

Carassius auratus in Qihe River,a variety of Carassius auratus gibelio,its natural gynogenetic.Wide and chick back is the most important feature different from other varieties of Carassius auratus gibelio.In recent years,the research on breeding,disease immunization,nutrition and other aspects of feeding Carassius auratus in Qihe River have been carried out more and more,but the basic research related to its growth traits is relatively deficient and the mechanism of muscle growth is still unclear.Myostatin(MSTN)is a negative regulator whose main function is to regulate animal skeletal muscle growth and development.microRNAs(miRNAs)are a class of non-coding single-stranded small RNAs and its roles and functions have been increasingly explored in recent years.The development of miRNAs technology has developed a new way for us to explore the complexity of generosity of Carassius auratus in Qihe River.We extracted the total RNA of Carassius auratus in Qihe River and obtained the 3’UTR sequence of MSTN gene through cloning.According to the 3’UTR sequence of MSTN,we predicted the possible miRNAs through the TargetScans software,and ultimately selected the miR-181 a and miR-181 b as candidate miRNAs after the verification of RNAhybrid software.Using the total muscle DNA as template,the full length of pri-miR-181 a and pri-miR-181 were obtained.Eukaryotic expression vector and miRNA lentiH1 vector were constructed to analyze the relationship between MSTN and miR-181 a,miR-181 b at the molecular level and organizational positioning level by fluorescence detection and fluorescence in situ hybridization,respectively.The expression of miR-181 a,miR-181 b and MSTN expression in eight organizations and different stages of embryonic development were analyzed in Carassius auratus in Qihe River by qRT-PCR.Therefore,our results are expected to reveal the multi-level network of regulation pattern of muscle growth related genes,and then to elucidate the muscle growth mechanism of Carassius auratus in Qihe River.1 3’UTR cloning of MSTN of Carassius auratus in Qihe RiverThe total RNA was extracted from the muscle of Carassius auratus in Qihe River for cloning 3’UTR of MSTN.After cloning and sequencing,we obtained the 865 bp sequence of 3’UTR of MSTN.Blasting in the NCBI database demonstrated that it has a high similarity with that of Cypriniformes,especially Labeo.Rohita(91%,KR052242.1),Cyprinus carpio(92 %,LN590705.1),and Carassius auratus in Qihe river(88%,KC851952.1).2 Prediction,verification and cloning of the possible miRNAs associating with MSTN of Carassius auratus in Qihe RiverAccording to the 3’UTR sequence of MSTN,the possible miRNAs were predicted using RegRNA,Pictar,TargetScan,miRWalk,miRDB and other softwares,and further examined through RNAhybrid software Finally,miR-181 a and miR-181 b were selected as candidate miRNAs may associated with MSTN.And pri-miR-181 a and pri-miR-181 b were cloned using the extracted DNA.3 Construction of eukaryotic expression vector and dual luciferase reporter assay for miR-181 a,miR-181 b and MSTNIn this experiment,the wild type MSTN 3’ UTR and the mutated MSTN 3’ UTR were relatively connected into pmirGLO,a eukaryotic expression vector.The pri-miR-181 a and pri-miR-181 b were connected into Lenti H1 vector.The pmirGLO and Lenti H1 vectors carried targeted DNA fragments were co-transfected into 293 T cells.The comparative study of fluorescence detection showed that the two miRNAs both had a certain degree of inhibition to MSTN.Moreover,it was also found that these two miRNAs may have other binding sites with MSTN to inhibit its expression.4 Expressing localization of MSTN and miR-181 a in muscle tissueThe back and abdominal muscles of Carassius auratus in Qihe River were paraffin sectioned.And we detected the expression of MSTN and miR-181 a in the muscle tissue using probes.Experimental results showed that,MSTN had a higher expression in muscle fibers,while miR-181 a expressed higher in sarcolemma and also expressed in connective tissue.In addition,the expression of miR-181 a is lower in back muscles than in abdomen whereas significantly higher in abdominal muscle fibers than in the back.This showed that miR-181 a has a stronger inhibition of MSTN in the back muscles than the abdominal.5 The specific expression of miR-181a/b and MSTN of Carassius auratus in Qihe RiverqRT-PCR results showed that the expressions of MSTN of Carassius auratus in Qihe river were significantly different in different tissues,with the highest in heart and brain,then muscle,kidney the least.In different stages of embryonic development,MSTN had a significantly higher expression at the stage before the gastrula stage,about 20 days after born.miR-181 a had a significantly higher expression in muscle and gill than other tissues,and also had a high expression before the gastrula stage and 10 days after hatching.miR-181 b had a higher expression in brain,muscles and intestines,and its expression was significantly higher before t the gastrula stage and 10 days after hatching than other periods.