| As a significant raw material of textile industry, wool is one of themost important products of graziery. The wool quality is mainly depended on itscurve and thickness characteristics. With the development of wool textile technologyand people’s pursuit of dresses which are natural, expensive, frivolous and soft, thewool which diameter below21.5μm is badly needed. In recent years, the wooloutput of our country has increased constantly, however, the quality of wool is varied,the high trait of wool is still not enough and the import of wool goods continues to goup year by year. Thus, it is imperative to culture new breeds of sheep which producehigh quantity and quality wool.For that reason, in order to provide theory of reference for the cultivate of finewool sheep, five Chinese sheep breed （Chinese merino sheep breed, hu sheep breed,Romney sheep breed, Chinese merino×hu sheep breed and Suffolk sheep）werechosen as the object of our study. In this study, we use technology of molecularbiology to study the candidate genes (KAP6, KAP7and KAP8gene) which is relatedwith the thickness of wool. The differences of KAP6, KAP7and KPA8genebetween Chinese merino sheep and Suffolk sheep were investigated. Thepolymorphisms of the CDS (coding sequences) and flanking sequences of KAP6,KAP7and KAP8genes in four of the five sheep were acquired. Part of upstreamsequences of CDS of those three genes have been first amplified and sequenced inthis study. At last, thought to play an important role in regulation of these three geneexpression, the three newly acquired sequences were analyzed in bioinformatics. Theresults were showed following:(1) The differential expression of KAP6, KAP7and KAP8genes in hair follicletissueThe differential expression levels of KAP6, KAP7and KAP8gene in the hairfollicle tissue of Suffolk sheep and Chinese merino sheep breed were investigatedwith Real-time Quantity PCR. Results showed that mRNA expression of these threegenes positively related with diameters of the wool, and the correlation coefficientsare0.941(P=0.00),0.842(P=0.01) and0.948(P=0.00); the expression levels of KAP6, KAP7and KAP8gene in Suffolk sheep were remarkably high than that in Chinesemerino sheep, and attended4.23times,5.74times and5.87times, respectively.(2) Polymorphisms of KAP6, KAP7and KAP8genes in four Chinese sheepbreedsPCR technology was used to amplify objective fragments of KAP6, KAP7andKAP8gene. By sequencing directly and cloning sequencing, polymorphisms ofKAP6, KAP7and KAP8gene in Chinese merino sheep, Hu sheep, Romney sheepand Chinese merino×Hu sheep were investigated. The results showed that there werefive different sequences in KAP6gene locus, two different sequences in KAP7genelocus and five different sequences in KAP8gene locus. Specially, two sequences ofKAP6gene locus and one SNP of KAP7gene locus were breeds specificity.(3) Amplification of the unknown upstream sequences of KAP6, KAP7andKAP8gene and analysis of the newly acquired unknown sequencesSpecial primers were designed according to the partial sequences of KAP6,KAP7and KAP8gene provided by GeneBank. TAIL-PCR (thermal asymmetricinterlaced PCR) were adopted to amplify the5′unknown fragments of KAP6, KAP7and KAP8. The unknown fragments nucleic acid sequences of KAP6, KAP7andKAP8were acquired through cloning sequencing and their length were1877bp,3224bp and1826bp, respectively. Then, these sequences were spliced with theiralready known corresponding genes sequences via DNAman software. At last, weacquire the partial upstream sequences of CDS of KAP6, KAP7and KAP8genes andtheir length are2918bp,3619bp and2801bp, respectively. The three newlyacquired sequences were analyzed through bioinformatics analytic software(Genomatrix). According to analysis, a number of regulatory element binding sitewere found which has laid the ground work for the further analysis of tissue specialexpress and activity analysis of KAP6, KAP7and KAP8genes. |
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