5 Small-Tail Han Sheep Reproduction And Disease Resistance Relational Genes Cloning And Comparative Genomics Analysis

As an important renewable resource in the world, sheep holds the important station in agricultural production. In case of The Human, Chicken, Cow and Pig Genome Initial Sequencing already having been completed, the research on sheep genome presents obvious laggard status. Based on tools of comparative genomics, the information from model organisms can be used to confer quantities, positions, function, and expression model and species evolution of genes in domestic animal and poultry variety. So important economical character candidate genes can be appraised, cloned and gotten characteristic. We attempt to induct the tools of comparative genomics on function genome study of Chinese local sheep variety to clone sheep reproduction and the disease resistance correlation genes, in order to provide the theory basis for local sheep variety breeding or resources conservation, and instruct the sheep variety molecular breeding and resources protection work-Sheep YB-1, sheep and goat ISG15, TLR9, MSY2 and CIB1 were cloned, and analyzed by methods of comparative genomics. The results as followed:Small Tail Han Sheep YB-1 mRNA was 1449bp in length (GenBank No. EF488163). ORF consists of 867 nucleotides, which codes 288 amino acids. Sheep YB-1 is composed of typical CSD motif. The comparison of amino acid sequence revealed the sequence identities over 95%. Sheep YB-1 is TATA-less promoter. GATA-1, GATA-2, GATA-3 and AML-la distribute on 5'-UTR. Species potential transcription factors binding sites Analysis of YB-1 5'-UTR indicated that, the complexity of regulation on YB-1 expression and direction of species evolution had some kind of relativity. Phylogenetic relationship of 43 species based on YB-1 and its ortholog uses amino acid sequences was parallel to species biological evolution direction. And species protein 3-D structures were also conservative. Because YB-1 untranslated regions of species played the different role in gene regulation and expression, untranslated regions showed different evolution direction.YB-1 recombinant prokaryotic expression was constructed to obtain the antiserum or for the further YB-1 protein function research.We got 75 non-redundant alternative splicing (AS) production, which 26 were from ovary, and 49 from testicle. The molecular mechanisms of YB-1 AS included exon skip, alternative 5' or 3' splicing and intron retention. 34 non-redundant AS sequences could code in excess of 20 amino acids. According to blastp results, the protein production can be grouped in six. The first group of AS(1 from ovary, and 9 from testicle) consisting of 10 sequences, could code various length of YB-1 C motif. The second group (4 from ovary, and 13 from testicle) consisting of 17 sequences, could code the similar sequence of another YB-1 AS in Canis familiaris. The third group (consisting of 3 sequences) and the fourth groups (consisting of 4 sequences) were both from testicle, and the function of protein production was unclear. 12 AA on the fifth group(l from testicle) shared 85% similar to YB-1 C motif. The sixth group was from ovary, and the function of protein production was unclear. There were a lot of ncRNA in sheep ovary (21/49) and testicle (22/26). Predicted 7 RNA secondary structure of YB-1 AS productiom length less than 150bp present the structures just like "stem bubbles". With base numbers of AS production increase, the structures appeared like "the cross".ISG15 full length of Small Tail Han Sheep and Jining black goat is 1033bp and 911bp, respectively, constitutes by two exons. The sheep ISG15 mRNA sequence had 94% identity with the cow ISG15 mRNA, and chimp 77%, human 77%, rhesus 77%, house mouse 77%. The sheep ISG15 protein sequence had 87% identity with the cow, and chimp 66%, human 66%, rhesus 67%, house mouse 63%. ISG15 contains two domains with structural homology close to ubiquitin, and species protein 3-D structures were also conservative. There existed a "G" insertion mutation after 904bp, and a "G" deletion mutation appeared 13bp behind. ORF of Small Tail Han Sheep ISG15 enlarged into 525bp, which coded 174aa. The 3'-UTR shortened into 19bp. ORF of Jining black goat ISG15 was 402bp, which coded 174aa. The 3'-UTR shortened into 19bp. ISG15 is TATA-less promoter. Expect that DPE of dog ISG15 appeared at +41~+45bp, the others appeared at +23～+27 showing as (GTGA) AGATG. Because of the mutations, ISG15 C-terminal of Small Tail Han Sheep and Jining black goat did not present LRLRGG motif.TLR9 shows a single copy in sheep genome. Gene cloning product was 4192bp, consisted of 5'-UTR(115 bp), 2 exons and 3'-UTR(44bp). After gene predicted, The sheep TLR9 mRNA sequence had 96% identity with the cow TLR9 mRNA, and pig 87%, horse 86%, cat 85%, dog 85%, chimp 83%,, rhesus 83%, human 83%, rat 78%, house mouse 77%, and short-tailed opossum 70%. The sheep TLR9 protein sequence had 95% identity with the cow TLR9protein, and pig 85%, horse 83%, cat 84%, dog 83%, chimp 79%,, rhesus 79%, human 79%, rat 72%, house mouse 73%, and short-tailed opossum 60%. The difference on the quantity, type and the relative position of TLR9 protein motif caused rhesus and house mouse location diversity between TLR9 Phylogenetic relationship and cluster analysis on TLR9 conserved domain.MSY2 shows a single copy in sheep genome. Gene clone product was 4927bp, consisted of exon 2 to exon 8, and 3'-UTR (493bp). During the Spermatogenesis and Ovogenesis, MSY2 possibly plays the vital role on gene transcription, RNA stability and RNA translation regulation.CIB1 genome cloning production was 3Kb in length, consisted of 5'-UTR (50bp), 7 exons and 3'-UTR (189bp), while the testicle cDNA cloning product was 799bp, which coded 191aa. The sheep CIB1 mRNA sequence had 98% identity with the cow CIB1 mRNA; and dog 95%; human, rhesus, house mouse and rat 93%. CIB1 of species is composed of typical EF-hand motif. The protein sequence had 97% identity with the cow CIB1 protein; dog and rhesus 95%; human 88%, house mouse and rat 85%.