Study Of High Fecundity Related Genes Using Digital Gene Expression Tag Proifling In Small Tail Han Sheep

ObjectivesDigital Gene Expression Tag Profiling (DGE) technology was used to construct gene expressionprofile to screen differentially expressed genes, enriched GO and enriched pathways of the ovary tissuebetween FecB~BFecB~Band FecB~+FecB~+ewes of small Tail Han sheep and Moutain sheep during theoestrum and investigate the molecular mechanism related to fecundity difference between them.Methods1.152Small Tail Han sheep around the age of2were blood sampled for detection of the FecBmutation(A746G) in the BMPRIB gene by PCR-RFLP in Qingdao Ao-Te sheep breeding farm inShengdong province.2.5FecB~BFecB~Band5FecB~+FecB~+ewes of small Tail Han sheep and5Moutain sheep, who werein good condition, have been chosen to synchronize estrus. After estrus was diagnosed, all ewes havereached spontaneous estrus after an oestrous cycle (15-22d), then they were sacrificed between4and5h after spontaneous estrus was diagnosed. Whole ovaries were excised and samples were collected withbetter ovulation points on the surfaces of the ovaries. All samples were immediately removed inRNAlater protection reagent,4℃for one night, then stored at-80℃for total RNA extraction.3. Total RNA was extracted from ovaries in three groups (BB Han ewes,++Han ewes and Mountainewes) and then purified. After extracting the total RNA from the samples, mRNA was enriched and thenestablished a sequencing library. And the library products were ready for sequencing analysis viaIllumina HiSeq2000. DGE technology was used for sequencing data of the three groups.Differentially expressed genes were screened between every two groups of them (FecB~BFecB~BHansheep vs FecB~+FecB~+Han sheep; FecB~BFecB~BHan sheep vs Mountain sheep; FecB~+FecB~+vs Mountainsheep). Enriched GO (Gene Ontology) and enriched pathway analysis were conducted on differentiallyexpressed genes using a free web-based molecular annotation system DAVID.4.10genes were chosen for fluorescence quantitative PCR detection, whether they were inaccordance with solexa sequencing result.Results1. Genomic DNA was extracted from whole blood, and primers amplified a140-bp band. Afterdigestion with Ava II, the BB animals had a110-bp band, the B+animals had140-and110-bp bands,and the++animals had a140-bp band. And the result for detection of the FecB mutation (A746G) in theBMPRIB gene by PCR-RFLP of152Small Tail Han sheep is:73BB animals;66B+animals;13++animals.2.5FecB~BFecB~Band5FecB~+FecB~+ewes of small Tail Han sheep and5Moutain sheep havechosen to synchronize estrus. After estrus was diagnosed, all ewes have reached spontaneous estrus afteran oestrous cycle (15-22d), then they were sacrificed between4and5h after spontaneous estrus wasdiagnosed. After estrus synchronization, for the time from estrus synchronization to spontaneous estrus,the oestrous cycle of the small Tail Han sheep is shorter than the Moutain sheep’s, which the former is17-20d and the latter is20-22d.3. Firstly, we applied a series of de novo assembly pipeline to these sequencing reads. De novoassembly was carried out by Inchworm nested in Trinity software packages. We obtained22,507contigs for FecB~BFecB~BHan sheep,20,553contigs for FecB~+FecB~+Han sheep and23,965contigs for the Mountain sheep, respectively. The average length of assembly contigs is~300bp. In this work, weidentified25,552sheep contigs, including25,216new contigs (97%) for which no sequencesinformation was previously available. And all the contigs can be blasted with6000genes of cows.Secondly, DGE technology was used for sequencing data of the three groups (FecB~BFecB~BHan sheep,FecB~+FecB~+Han sheep and Mountain sheep). Differentially expressed genes were screened andenriched GO and enriched pathway analysis were conducted on differentially expressed genes using afree web-based molecular annotation system DAVID between every two groups of them:1）FecB~BFecB~BHan sheep vs FecB~+FecB~+Han sheep, we totally identified1,092(500genes up-regulated and592genesdown-regulated) differentially expressed genes. The genes including STAR, FLCN, TGFI1, INHA,INHBA and gap junction, GnRH signaling pathway and Notch signaling pathway may were consideredas the most important genes and pathways involved in fecundity differences between FecB~BFecB~BandFecB~+FecB~+ewes of small Tail Han sheep;2）FecB~BFecB~BHan sheep vs Mountain sheep, we totallyidentified1,273(426genes up-regulated and847genes down-regulated) differentially expressed genes.The genes including CTSB, INHBA, INHBB, INHA, FOXJ3, FOXO3, IGF-1R, STAT3, jagged1, notch2and the pathways including lysosome, ribosome, TGF-beta signaling pathway, Insulin/IGFpathway-protein kinase B signaling cascade, cholesterol biosynthesis and Notch signaling pathway maywere considered as the most important genes and pathways involved in fecundity differences betweenFecB~BFecB~BHan sheep and Mountain sheep;3）FecB~+FecB~+vs Mountain sheep, we totally identified928(323genes up-regulated and605genes down-regulated) differentially expressed genes. The genesincluding CTSB, INHA, INHBB, FOXO3, FOXJ3and the pathways including lysosome, ECM-receptorinteraction and TGF-beta signaling pathway may were considered as the most important genes andpathways involved in fecundity differences between FecB~+FecB~+Han sheep and Mountain sheep.4.10genes were chosen for fluorescence quantitative PCR detection, and they were in accordancewith solexa sequencing result.Conclusion1. we applied a series of de novo assembly pipeline to sequencing reads. De novo assembly wascarried out by Inchworm nested in Trinity software packages. We obtained22,507contigs forFecB~BFecB~BHan sheep,20,553contigs for FecB~+FecB~+Han sheep and23,965contigs for theMountain sheep, respectively. The average length of assembly contigs is~300bp.The result indicatedthat de novo assembly of the single–end sequencing data using trinity algorithm was very successful inthe non-model organisms.2. DGE technology was used for sequencing data of the three groups, differentially expressedgenes were screened and enriched GO and enriched Pathway analysis were conducted on differentiallyexpressed genes between every two groups of them, which have laid a foundation to investigate themolecular mechanism related to fecundity difference between three groups, and have provided aguidance for the next step experiments.