Transcriptome Analysis Of Small-tailed Han Sheep And Dorperrs Biceps Brachii And Structure Characteristics Of Myosin Light Chain Gene Families

Sheep is the main source of mutton. With rising of the price, improvement of muttonproduction and quality has become an important issue.An investigation of gene expression inovine muscle among different breeds would significantly promote our understanding ofmuscle growth and development.RNA-seq is a recently developed analytical approach for transcriptome via high-throughputsequencing.In this experiment, two cDNA libraries were constructed from biceps brachii ofDorper (DP)and Small-tailed Han sheep (SH), whose growth performance were significantlydifferent.The two libraries were sequenced using Illumina HiSeq2000sequencing platform.After mapped to the O. aries genome and reference genes, characteristics of the twotranscriptome were analyzed by bioinformatics analysis, including gene expression,annotation, differentially expressed genes (DEGs), alternative splicing (AS), novel transcriptunits (nTUs), SNP, SSR, etc.12genes were tested to verify the reliability of sequencing databy qRT-PCR. Then taken muscle of SH and DP as experimental materials, the cDNAs ofMYL1, MYL2, MYL3and MYL4were cloned using RACE methods. Additionally, structure,characteristics and mRNA expressing profile of these genes were analyzed. The main resultswere showed as follows:(1) For the SH and DP libraries, a total of50,264,608and52,794,216clean reads wereobtained, respectively. Approximately two-thirds of these reads could be mapped to O.ariesgenome. Among them,42.77%and33.10%reads could be aligned to20,236reference genes,respectively. Only about1%of these genes belong to high-expressed genes (RPKM≥1000),while the majority of them were low-expressed genes (RPKM≤1000).(2) Up to1,300significantly differential expressed genes (DEGs) with FDR≤0.001andabsolute value of log2Ratio≥1were found between the two libraries (554were up-regulatedand746down-regulated in the SH). When annotated to Gene Ontology (GO) database,1,066,1,137and1,067genes were found in biological processes, cellular components andmolecular functions items, respectively. After annotated to Kyoto Encyclopedia of Genes andGenomes (KEGG) pathway,1,152genes were found in240pathways. Among which,metabolic and actin cytoskeleton items were the main pathways with114and21genes,respectively. Combined annotated results of GO and KEGG, a total of31DEGs wereidentifiedbe related to muscle cell development, differentiation and skeletal muscle growth.After analysis of protein interaction network, COL4A1and ITGA8had numerous interactionswith other proteins but myosin, troponin, IGFBP5, TGFBR3and VEGF on the other way. (3)40481and38851cSNPs were exploited in SH and DP libraries. All of these cSNPswere mainly distributed in chromosome1,2and3.4,721SSRs were observed in the twolibraries.Among them, Aâ†'G and(AC)nwas the main type.(4)37.72%and39.03%genes had gone alternative splicing (AS) in the SH and DPlibraries, respectively. A3SS was the main type. According to sequenced clean reads,6,989reference genes were extended and optimized. And a total of123,678novel transcript unitswith average343bp length were discovered in the two libraries.(5) The cDNA sequences of MYL1, MYL2, MYL3and MYL4were cloned and submittedto NCBI (GenBank accession number are KJ700419, KJ710701, KJ710702, KJ710703,KJ710704, KJ710705, KJ710706and KJ768855). MYL1had two cDNA sequences withdifferent5′ends. Homology of the five cDNA sequences were higher between SH and DPthan SH (or DP) and predicted sequences published in NCBI. The difference mainly focusedon5′and3′tail ends.(6) Each of MYL1, MYL2, MYL3and MYL4gene had5exons. Many binding sites ofMEF2, MyoD and MyoG were found in upstream of their start codons.Thismeans thattranscriptional expression of MYLs might be regulated by these transcription factors.(7) One or two different amino acids were found between MYL1a, MYL1b, MYL2, MYL3MYL4protein and predicted sequences in NCBI. All of these proteins were acidic andhydrophilic with many glycosylating and phosphorylating sites. But they had no signalpeptide. Nearly half percent of their secondary structures was α-helix. And human MYL2protein was the best tertiary structure templatewith symmetric barrelfor the five types ofpredicted proteins. According to clustering results, sheep and goat, human and chimpanzee,squirrel monkeys and macaques, mice and sewer rat, cattle and yak, cat and dog were similar,while zebrafish and xenopus laevis were far from above vertebrates.(8) MYL1gene mainly expressed in longissimus of sheep. It had two kinds of alternativesplicing isoforms, MYL1aand MYL1b. MYL2, MYL3and MYL4gene were expressed inheart.In conclusion, RNA-Seq analysis of two transcriptome libraries in this study could afford afoundmantal databases for further selecting genes related to skeletal muscle. Meanwhile,characteristic analysis of cDNAs and proteinssequences about MYLs offered a foundation forstudy of theirfunction and regulated mechanism in the future.