Screening And Identifying Regulatory Genes Associated With Taihang Black Goat Hair Follicle Anagen-catagen Transition

Cashmere is an excellent textile material, a derivative of skin tissue, and enjoys a "softgold" reputation in the international community. Its development and differentiation occurs inthe secondary follicles in goat skin. Studies have shown that goat secondary follicle presentscyclical changes with seasons, turns into the anagen, catagen and telogen. Recent researchesfind that these properties have associated with a unique pattern of genes expression, involvingcomplex regulatory mechanisms. Particular attention has been paid to anagen-catagentransition regulatory molecules. Because the lengths of cashmere were determined by theduration of hair follicle anagen, it is very meaningfull to find the regulators involving insecondary hair follicle anagen-catagen transition. In mammals, the mouse has been used asthe model for the study of these regulatory genes. Over the past decade, many of theanagen-catagen transition regulatory molecules were identified. However, little is knownregarding the characteristics of these genes and their expression pattern in goat. In this study,we generated a library enriched in up-regulated ESTs at anagen-catagen transition of Taihangblack goat secondary hair follicle using suppressive and subtractive hybridization (SSH), anddifferentially expressed ESTs from forward SSH library was confirmed and analyzed. Themain results were as follows:1. Both the forward (tester: the cDNA from skin during early catagen, driver: thecDNA from skin during late anagen) and reverse (tester: the cDNA from skin during lateanagen, driver: the cDNA from skin during early catagen) SSH analyses were conducted.The subtraction efficiency was estimated by a housekeeping gene GAPDH, and the resultshowed that GAPDH was subtracted efficiently by211and28folds for forward and reversesubtracted cDNA library respectively, which indicated that the reduction of GAPDH cDNAwas observed in the subtracted library.2. The products from forward SHH were cloned. The clones were were selected forfurther PCR amplifications with the nest primers and screened using dot blotting assay. Ourstringent judgment led to the selection of72positive clones from3000clones. Aftersequencing and analysis, forty-five high-quality sequences were aligned to the GenBankdatabase. forty-two clones were unknown in goats but have high homology with Ovis aries, Bos taurus or other species with identity over84%, Two clones were known in goats, andone clone was found without any significant matches with the GenBank database..3. Six genes that occured more frequently in library and the potential novel gene wereselected for quantitative real-time PCR validation assay to confirm the expression profilesgenerated by SSH. The result showed that their expression quantity varied from one to threefolds (2.79(TA2212),2.53(EDA),1.64(BMPR1A),1.59(MSTN),1.56(Catenin),1.42(TP53INP1) and1.41(IGFBP3)), indicating that those genes increasingly expressed in theanagen-catagen transition.4. The potential novel gene, TA2212, was successfully amplified by RT-PCR and thesequences were analyzed. The expression of TA2212in different stages of hair folliclescycle was analyzed by real-time quantitative PCR. An open reading frame of171bp wasfound at16-171nucleotides. Analysis by SMART suggested that a26amino acid residue ofterminal signal peptide was found. The real-time quantitative expression analyses showedthat the TA2212was expressed at a lower level during telogen (0.093296±0.018871) and itsexpression decreased by12%during anagen and it had no significant difference (P>0.05)compared to telogen (0.082684±0.007827). Its expression increased by3.3folds duringearly catagen (0.310684±0.021578) and it was significantly higher than the anagen andtelogen (P <0.01).5. The BMPR1A gene was cloned and the sequences were analyzed. The expression ofBMPR1A mRNA in different stages of hair follicles cycle was analyzed by real-timequantitative PCR. We found the size of BMPR1A CDS was1599bp encoding532aminoacid residues. The amino acid sequences shares more than92%identity with other speciesBMPR1A. Analysis by SMART suggested that the encoded protein contained signal peptide,Activin_recp motif, GS, transmembrane segment and S_TKc domain. The BMPR1A mRNAlevels in skin were abundant at telogen (0.112077±0.01942). The expression levelsdecreased by41%at anagen (0.066293±0.002288) and it had no siginificant difference(P>0.05) compared to the telogen. The expression levels increased by14%at categen and ithad no siginificant difference (P>0.05) compared to the telogen and the anagen.6. The Eda gene was cloned and the sequences were analyzed. The expression of EdamRNA in different stages of hair follicles cycle was analyzed by real-time quantitative PCR.We found the size of Eda-A1CDS was1176bp encoding391amino acid residues; TheEda-A2was only6bases (nt1161-1166) shorter than Eda-A1encoding389amino acidresidues. The amino acid sequences shares more than90%identity with other species Eda.Analysis by SMART suggested that the encoded protein contained TNF motif, collagen andtransmembrane segment. Analysis with SWISS-MODEL suggested that the surface shape of the Eda-A2protein showed distinct difference compared to Eda-A1. The expressionlevels were lowest at telogen (0.015793±0.001407). The expression levels increased by1.9folds at anagen (0.030191±0.002114) and were significantly higher than that at telogen(P<0.05). The Eda mRNA levels in skin increased by7folds at categen(0.206635±0.026615) and were significantly higher than that at anagen and telogen...