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Study On Cloning And Expression Of BMPR-IB, FSHβ Gene And Differentially Expressed Genes Of Ovary In Mongolian Sheep

Posted on:2011-01-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:X L HeFull Text:PDF
GTID:1103360305973586Subject:Animal breeding and genetics and breeding
Abstract/Summary:PDF Full Text Request
The reproduction traits of ovine are generally divided into conception rate, prolificacy and lambs survival, it is directly related to production cost and efficiency of sheep industry and it has a great economic significance. The Mongolian Sheep is one of the ancient indigenous sheep breeds in our country. Ujumuqin sheep is a good group of Mongolian Sheep through a long breeding under the local ecological environment and it has many fine characteristics, it is especially famous for its high disease resistance, dressing percentage and reproductive performance. In order to reveal its high reproductive performance and to make an effective utilization of the Mongolian Sheep precious resource, this research used Ujumuqin sheep as the experiment material. First, as candidate genes for prolificacy, the BMPR-IB and FSHβgene were cloned, sequence analyzed and differentially expressed in ovary, uterus, oviduct, hypothalamus, pituitary tissues of normal and estrus physiological stage of single Mongolian Sheep, late pregnancy physiological stage of biparous Mongolian Sheep by relative quantitative Real-time Quantitative PCR (RQ-PCR). Secondly, differentially expressed genes between ovaries from single and biparous Mongolian sheep were selected by suppression subtractive hybridization (SSH) technique and some differentially expressed genes were further cloned and verified by silicon cloning and RT-PCR. Given all the above research, important theoretical basis were provided for improving fecundity and breeding new varieties of meat sheep in our country, and the following mainly results were obtained:(1) The BMPR-IB and FSHβgene cDNA sequence were amplified from Mongolian Sheep ovary tissues by RT-PCR respectively. The full-length of Mongolian Sheep BMP-IB gene is 1567bp, including complete 1509bp open reading frame (ORF) and encoding 502 amino acids. The bases were"A"and"C"in the 746 and 1113 sites of coding region, not occurred the same mutations (A→G) and (C→A) of BMPR-IB gene in Booroola Sheep by the sequence analysis of single and biparous Mongolian Sheep BMPR-IB gene. The full-length of Mongolian Sheep FSHβgene is 1021bp, including complete 390bp ORF and encoding 129 amino acids.(2) Theβ-Actin, BMPR-IB and FSHβgene RQ-PCR reaction systems were established by relative quantitative double standard curve method. Calculated with ovary tissue quantitative result of normal physiological stage of single Mongolian Sheep, the FSHβ gene expression was the highest in hypothalamus and the lowest in oviduct of estrus physiological stage of single Mongolian Sheep, the BMPR-IB gene expression was the highest in ovary and lowest in uterus of estrus physiological stage of single Mongolian Sheep. For the ovary tissue, the expression of FSHβgene in estrus physiological stage was 1.31 times than that in normal physiological stage and biparous sheep was 0.70 times than that in single sheep; the expression of BMPR-IB gene in estrus physiological stage was 2.65 times than that in normal physiological stage and biparous sheep was 2.16 times than that in single sheep. The results proved that the BMPR-IB gene was the mainly prolificacy candidate gene of Mongolian Sheep. Although the FSHβgene have an important role in reproduction, but it's not the prolificacy candidate gene of Mongolian Sheep and may be linkage with the prolificacy major genes or QTL.(3) Forward and reverse subtracted cDNA libraries between ovaries from single and biparous Mongolian sheep were constructed by SSH technology, and 768 masculine clones from the two subtracted cDNA libraries were obtained. The Length of inserted fragments were between 150~1000bp. Combined with dot blot technology 373 differentially clones were obtained and 185 genes sequence, 10 ESTs, 4 unknown ESTs were obtained from the differentially clones by sequencing and homology blast.(4) According to the analysis of the differentially expressed genes function, genes obtained from the experiment were mainly grouped into the following groups: cell/body defense or immune, 14 genes, metabolism, 53 genes, transporters, 20 genes, modified nucleic acid, 12 genes, cell development, 18 genes, signaling transduction, 12 genes, cell structure, 9 genes and the unknown or unclassed, 47 genes. The metabolism genes was the biggest clusters and the percentage was 28.65%. There were 12 differentially expressed genes consistent with the reproduction traits related to the genes that had been reported by comparing and screening, and the gene were ITGB1,BMP6,MHC-Ⅰ,ACVRL1,IGFBP5,SPON1,FABP5,ADAMTS1,FAM46A,THY1,ARP3 and Id3.(5) The UniGene number Oar.7453 and Oar.5068 were obtained by homology blastn in the ovine EST database using the differentially expressed ADAMTS1 and Id3 gene fragment obtained in SSH as seed sequence, the length of Mongolian Sheep ADAMTS1 and Id3 gene silicon cloning cDNA sequence were 2418bp and 1099bp respectively by sequence assembly. The length of Mongolian Sheep ADAMTS1 and Id3 gene were 2420bp and 1045bp respectively by RT-PCR of experimental cloning, the homology were 99.4% and 99.3% respectively with the silicon cloning sequence. The sequence has been registered in the GenBank and the accession were GU437212 and GU437213.
Keywords/Search Tags:Mongolian Sheep, Bone morphogenetic protein, Follicle stimulating hormone, Real-time quantitative PCR, Suppression subtractive hybridization, Differentially expressed, Silicon cloning
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