Gene Cloning Of 3 Wool-Promoting Growth Factors Of Tibetan Sheep And Quantitative Analysis Of Their MRNA Expression Under Different Nutritional Conditions

Tibetan sheep, one of the three Chinese original sheep breeds, mainly live in the alpine and pastoral areas. Qinghai province is the main producing area. Tibetan sheep have long rough hair fiber and their wool is exquisite and warm to create some good raw materials, such as bed blanket, carpets, mattress and so forth. Growth of the wool is a result of interaction of various growth factors and hormones whick work together to promot the growth of hair follicles. This study focused on the three natural genes, IGF-I, VEGF and HGF. The total mRNA was extracted from the skin smaples of 1-month-old lambs whose mother is the natural grazing Tibetan sheep. We extracted total mRNA from the skin samples of 1-month-old lambs whose mother is the natural grazing Tibetan sheep. Subsequently, three sets of specific primers were designed based on the corresponding sequences of ordinary sheep available in GenBank, amplifing the growth factors genes IGF-I, VEGF and HGF respectively. Also, we cloned and analyzed these three function genes. Further more, by SYBR Green I real-time PCR assay, the mRNA expression levels of IGF-I, VEGF and HGF were quantitatively analyzed for the lambs produced by ewes with different nutrition at late gestation.The following results were obtained in this study:1. The complete CDS of VEGF(573bp), partial sequence of IGF-I(940bp) and HGF(108bp) of Tibetan sheep were cloned successfully. Sequence alignment showed that these three genes have high homology with ordinary sheep, goat, cow, horse, pig, human, chicken, duck, mouse and rat. The similarity is from 74.2% to 100%. The similarity is up to 100% with ordinary sheep, showing highest homology.2. Quantitative methods for rapid analysis of mRNA expression of IGF-I, VEGF and HGF were established. The results indicated that, by making up Nutrition Brick to the Tibetan pregnancy female sheep, the IGF-â… , VEGF and HGF expression of lamb are 1.1 times, 11.5 times and 76.1 times higher than the control group. By making up Granule Feed to the Tibetan pregnancy female sheep, the IGF-I, VEGF and the HGF expression of lamb are about 1.8 times, 0.85 times and 1.9 times higher than the control group.3. It is also showed that the real-time PCR of the IGF-I, VEGF and HGF that we established has good amplification efficiency, linear range and stability.