Cloning, Bioinformatic Analysis And Polymorphism Analysis Of KRT-IF Gene In Gansu Alpine Fine-Wool Sheep

This study taked Gansu Alpine Fine Wool Sheep as a research object,with the technology of clone, bioinformatic analysis,PCR-SSCP and HRM,cloned and determined the KRT-IF 35 of Gansu Alpine Fine Wool Sheep,the correlation was analyzed between mutation locus of KRT-IF 35,KRT-IF 31 and wool traits.Experimental results are as follows:1.A 4414bp KRT-IF 35 gene was amplified by PCR technique from the genome of Gansu Alpine Fine Wool Sheep(NCBI No:GU456629),the coding region is 1751bp, contained 455 amino acids.It's a hydrophilic protein,has two transmembrane regions,the main secondary structure is helix and there is 1 structure domain.2.The results of PCR-SSCP and DNA sequencing showed:there is a novel C to T mutation in KRT-IF 35 CDS area 145bp. A pair alleles A and B and three genotype AA, BB, AB were dectected; A allele is the advantage allele of Fine strains and Super fine strains and AA genotype is the advantage genotype of Fine strains and Super fine strains, the fineness of AA, AB genotype significantly different, the fineness discrete of AA, BB genotype significant different, the bending of AA, BB genotype significant different. Tests of Hardy-Weinberg equilibrium confirmed that three strains was in equilibrium (P>0.05). So this loucs maybe the molecular genetic markers linked with the major gene that affect wool fineness,bending or fineness discrete or control factor.3.The results of HRM and DNA sequencing showed:there is a novel A to G mutation in KRT-IF 31 CDS area 166bp,a pair alleles C and D and three genotypes CC,CD and DD were dectected. C allele is the advantage allele of Fine strains and Super fine strains;CC genotype is the advantage genotype of Fine strains and Super fine strains, CD genotype is the advantage genotype of Mutton-wool strains, tests of Hardy-Weinberg equilibrium confirmed that Fine strains was in equilibrium(P>0.05). CC, CD genotype significantly different, the fineness of CC, DD genotype significantly different. Therefore this Locus may be concerned with the wool fineness.4. The results of HRM and DNA sequencing showed:there is a novel T to C mutation in KRT-IF 31 CDS area 191bp, a pair alleles E and F and three genotypes EE,EF and FF were dectected. E allele is the advantage allele of Fine strains,Super fine strains and Mutton-wool strains, EE genotype is the advantage genotype of Fine strains and Mutton-wool strains and EF genotype is the advantage genotype of Super fine strains. Tests of Hardy-Weinberg equilibrium confirmed that Fine strains was in equilibrium (P>0.05). EE, EF and EF, FF genotype significantly different, the fineness discrete of EE, EF genotype significant different. Therefore this Locus may be concerned with the wool fineness and fineness discrete.