| The fineness of wool is a key factor in determining the price of wool and hair follicles of the skin is the foundation of wool growth, and development of research is one of the focus areas. Looking for the factors that affect wool growth, proven the regulation mechanisms of major genes, and screening of molecular markers which can be used to mark sheep wool production performance for the further development of molecular breeding foundation. The present study was to establish a real-time quantitative PCR method which is suitable for quantitative detection of mRNA expression in sheep skin tissues with the expression of candidate genes for microarray detection of fine-wool sheep fiber diameter on the basis of the group members Dijiang who detected mRNA expression of14candidate genes by using this method in the superfine and fine wool sheep skin tissues differences, from gene level to explore the intrinsic link between candidate gene expression changes with fine wool and examined TXNIP, KRT26gene SNP locus, analysis of the gene and the relevance of the fineness of wool. The main results of this study are as follows:1. Selection of the reference gene in the skin tissues of sheep. In this study, RT-PCR technique was used, six candidate reference gene was find from GeneBank and the corresponding amplified respectively fragment,18SrRNA:185bp, β-Actin:225bp, GAPDH:181bp, B2M:138bp, TBP:186bp, RPL13A:81bp. The geNorm software was used to select the most suitable reference gene of the sheep skin tissue. The results of selection is that the number of optimal reference gene for3.Reference genes expression stability of the average M value as follows:0.536>0.480>0.476>0.472>0.371, expression stability was showed from low to high. Ultimately determine the three housekeeping genes such as18SrRNA^P-Actin and GAPDH on sheep skin tissue as internal control gene.and accordingly established the quantitative detection of mRNA expression based on SYBR Green1dye technology which is suitable for sheep skin tissues with real-time fluorescent quantitative PCR.2. Real-time PCR screening fineness of wool-related candidate genes. In this study, the relative expression of14genes were detected including TXNIP, KRT26, PIK.3CA, PPARD, FZD3, TFDP1, PTPN13, RPS6KA, ABCG2, AJDAM9, GJB3, GSTA1, FAIM. LAMB1,ets. in the superfine and fine-wool sheep skin tissues by using RT-PCR methods established the former experiment.The results showed that:14candidate genes were expressed in the sheep skin tissues, the up-regulated genes in the superfine and fine-wool sheep are KRT26, TXNIP, TFDP1,FAIM; and the down-regulated genes are PIK3CA, ADAM9, FZD3. the finer fineness of wool, the higher expression levels of up-regulated genes in the skin tissue, and vice versa.3. Analysis of TXNIP and KRT26gene polymorphism associated with the fineness of wool. In this study, the PCR-SSCP technique was firstly used to detect TXNIP and the KRT26two candidate genes SNP locus in Chinese Merino (Xinjiang type), analysis of the genetic polymorphism and its correlation with the fineness of wool. The results showed that: TXNIP and KRT26genes have three genotypes:AA, AB and BB; TXNIP gene detected two mutations,52bp-/T,86bpG/T are synonymous mutations, have no differences with the fineness of wool(P>0.05); KRT26gene mutation of the five bases were83bp (G/C),86bp (T/C),112bp (C/T),140bp (G/A),247bp (C/T),5mutations were found in KRT26gene; they are showed respectively83bp (G/C)s86bp (T/C),112bp (C/T),140bp (G/A),247bp (C/T); three of which were silent mutations, remaining two mutations, Val/Ile and Asn/Lys caused amino acid changes. Three kinds of genotype have a significant difference with the fineness of wool (P<0.01)), therefore, the KRT26gene could be used a candidate gene related to the fineness of wool in sheep. |
