Cloning, Sequencing And Detecting By PCR-SSCP Of KAP Genes In Cashmere Goat

First, Cashmere goat DNA was extracted from the blood cell in this study,two pairs of primers, P1/P2 and P3/P4, were designed according to the KAP gene sequences reported previously in other species. The result showed that two DNA fragments, S1 and S2, were amplified by PCR. The DNA fragments was ligated by T4 polymerase into the Sacâ…¡and Not I site of the cloning plasmid pGM-T Vector. And then, by performed the following steps:,transfecting the competent TOP10, extracting the plasmid DNA from transfected cells, identifying restrictly the recombinant plasmids and sequence analysing, we obtained the Cashmere goat KAP coding sequences S1 and S2.S1 has 289 nucleotides, and S2 has 304 nucleotides. S2 and encodes a polypeptide containing 71 amino acids, respectively. Sequence Analysis indedicated that the S2 fragment contains the Specif N-terminal structural domain and carboxyl terminus structural domain that are highly conserved in the KAP6 gene family. Homogeneous analysis demonstrated that nucleotide sequences of Cashmere goat KAP gene between different species mammal are comparatively conservative.SNP (single nucleotide polymorphisms) is the most important research method in Molecular Genetics. First, the PCR-SSCP with about 300bp fragment have been established in the experiment. And then the present study selected KAP as a candidate gene to identify any difference in two gene fragments of KAP among Cashmere goat by PCR-SSCP. The results show that locus S2 in the genes (KAP6.1) which code the high-glycine-tyrosine keratin associated-protein of keratin associated-protein family is significantly differential with cashmere yield (P<0.05). Among the high-glycine-tyrosine keratin associated-protein, the AA and BB.genotypes of S2 are also significantly differential with cashmere yield (P<0.05). The AB and BB genotypes of S2 are also significantly differential with cashmere fineness trait (P<0.05).