Identification Of Upstream Regulatory Regions Of Two Wool Follicle-Specific Genes In Sheep:KRTAP13.1 And KRTAP3.3

Wool industry is one of the most important animal husbandry in China. In the result, genetic improvement of the capability of wool production is important for Sheep farming. Development of skin and hair follicle affects wool production and quality, therefore, cloning and identification of promoter of gene which is specially expressed in wool follicle is the foundation of improving the wool fiber quality by using the transgenic technology. Consequently, this study use wool follicle specially expressed keratin and keratin associated protein genes as candidate genes, cloning its’upstream regulatory region, to provide the gene regulating elements for improving the wool fiber quality by using the transgenic technologyWe use hair follicle, skin (contain hair follicle) heart, liver, spleen, lung, kidney, rumen, abomasum, rectum, duodenum, lymph, fat, muscle, testis and ovary of tibetan sheep, and screening for wool follicle specifically expressed gene through PCR and q-PCR, cloning its upstream regulatory region. In addition, verification of gene which was specially expressed in wool follicle was accomplished by in situ RNA hybridization.1) Screening of wool follicle and skin specifically expressed gene.According to electronic expression profile of NCBI Unigene, we chose some sheep skin (contain hair follicle) specifically expressed candidate gene. we identify their expression in tissues of Tibetan sheep by way of Semi-quantitative RT PCR and found that KRTAP6.1.KRTAP3.3, KRTAP13.1, KRT27, KRT86, ELOVL3, IRSal, KRTAP1L1 were skin(contain hair follicle) specifically expressed.2) Identification of wool follicle specifically expressed geneFurther verification of sheep skin and follicle candidate genes, we found that KRTAP13.1 and KRTAP3.3 gene are high expressed in wool follicle than skin apparently, so KRTAP13.1 and KRTAP3.3 are specifically expressed gene in wool follicle. And verification of sheep KRTAP13.1 gene was specially expressed in hair shaft of wool follicle, which was accomplished by in situ RNA hybridization.3) Cloning of sheep follicle specifically expressed gene promoter.This study use template of tibetan sheep genome, the KRTAP13.1 KRTAP3. 3 promoter primers designed according to sheep genome v2.0 (http://www.livestock genomics.csiro.au/sheep/oar2.0.php) published by Australia CSIRO(Common wealth Scientific and Industrial Research Organization), about 6900 base pairs of 5’upstre am KRTAP13.1,KRTAP3.3 sequence was amplified by PCR4) Construction of recombination plasmid KRTAP13.1P-ZsGreen1-1.This study used a promoterless ZsGreenl vector named pZsGreen 1-1,6900 base pairs of 5’upstream KRTAP13.1 sequence was amplified by PCR with sheep DNA, the activity of sheep KRTAP13.1 promoter was analyzed by Zoanthus sp. Green fluorescent protein repoter gene in transgenic mice, Which is Provided possibility and feasibility to explore exogenous gene specifically expressed in sheep follicle.