| 17β-hydroxysteroid dehydrogenase type2(17β-HSD2)enzyme is an isozyme of17β-hydroxysteroid dehydrogenase superfamily (17β-HSDs). To date, a total of14mammalian17p-HSDs have been identified of which at least11exist within the human genome, encoded by different genes.14different mammalian17β-HSDs are all members of the short-chain dehydrogenase/reductase(SDR)family, with the exception of17β-HSD type5, which is an aldo-keto reductase(AKR).17β-HSD type2can catalyze estradiol (E2) into estrone (E1), besides it also can catalyze testosterone (T) into4-dione and5-dione into dehydroepiandrosterone (DHEA). In this experiment, we choose17β-HSD type2as a candidate gene, in order to have further recognization of its physiological function in Hu-Sheep, we cloned the full-length cDNA sequence. There are two groups in our study, the low energy (0.5×energy requirements) diet group(n=6), the high energy(1.5×energy requirements) diet group (n=5),28tissues were collected from each sheep. RT-PCR and real-timePCR were used to examining the expression of17β-HSD type2mRNA in tissues we collected, besides, we also do research in the expression of17β-HSD type2mRNA in tissues between the two groups we mentioned above. At the same time, softwares of bioinformatics were used to analysis of17β-HSD type2gene,include amino acid composition, protein secondary and tertiary structure. The results are as follows:1. Cloning the full-length cDNA of17β-HSD type2Bos Taurus shares a high homology of most gene with Ovis, based on the information of that,2primers were designed according to the sequences of17β-HSD type2of Bos Taurus.835bp of that gene were successfully cloned from Hu-Sheep rumen tissue, two pairs of specific primers were used to clone the5’end and the3’ end, the products were 540bp and330bp respectively. After splicing, the full-length of17β-HSD type2(1,317bp) of Hu-Sheep were obtained. Two primers were designed to amplify the completed CDS.2. Expression analysis of Hu-Sheep17β-HSD type228tissues of each Hu-Sheep were collected in our study, including rumen, reticulum, omasum, abomasums, duodenum, jejunum, ileum, cecum, rectum, colon, heart, liver, spleen, lung, kidney, uterine horn, oviduct, uterus, medulla oblongata, hypothalamus, pons, hypophysis, cerebrum, pineal gland, cerebellum, adrenal gland, olfactory bulb, and ovary. Expression analysis with real-time PCR showed that the Hu-Sheep17β-HSD type2was expressed in all the28tissues we collected with the exception of lung tissue.17β-HSD type2was high expression both in liver and GI tract, so we predict live and GI tract maybe the improtant palce where sex steroids were etabolized. The widely expression of17β-HSD type2demonstrate that it plays an important role in the regulation of intracellular levels of biologically active sex steroid hormones in various Hu-Sheep tissues.3. Expression of17β-HSD type2of Hu-Sheep under different energy level11adult ewes(3-4years) were selected, all the sheep were similar in weight and body condition. Two groups were assigned, the low energy (0.5×energy requirements) diet group(n=6), and the high energy(1.5x energy requirements) diet group (n=5). All ewes received a total mixed ration (TMR) diet. After pre-fed a maintenance diet7days, a pessary was inserted. At the12day after inserted the pessary, the sheep were killed, and28tissues each sheep were collected. Real-time PCR was used to obtain the data of expression of17(3-HSD type2mRNA in tissues between the two groups. In our study, Statistics indicate that expression of17β-HSD type2in rumen, omasum, duodenum, and hypophysis tissues between the two energy diet groups were significant difference(p<0.05), besides it was close to the remarkable level in cecum tissue(p=0.059).17β-HSD type2gene was low expressed in hypophysis tissues though the expression was significant difference, it indicated that short-term feed restriction during the luteal phase may affect the expression of17β-HSD type2, then affect the development of follicle by the effection of genital rachis. High expressed in four tissues of GI tract indicated that GI tract may be the important places where sex steroids degradation and excretion. Besides,17β-HSD type2which high expression both in liver and GI tract play key roles in regulating exposure to orally sex steroids via inactivation of these compounds. It is reported that,17β-HSD Type2express in liver and GI tract also have a critical function in protecting the body against exposure to environmental and bacterially synthesized sex steroids. And more research of the function of17β-HSD Type2expressed in GI tract is needed.4. Bioinformatics analysis of the sheep17β-HSD type2geneBased on the full-lengh cDNA sequence of17β-HSD type2, we predicted its amino acid composition, analysis of signal peptides, subcellular location and the secondary structure of17β-HSD type2protein by bioinformatics softwares. Statistics indicate that it encodes a17β-HSD type2precursor of389AA. Alignment of Hu-Sheep17β-HSD type2with other species shows that it shares high AA sequence identity with that of bos taurus(96.13%), sus scrofa(77.06%), canis lupus familiaris(70.44%), callithrix jacchus(65.72%), nomascus leucogenys(65.46%), pan troglodytes(65.21%), human(64.69%), mus musculus (58.35%), and a comparatively lower identity to danio rerio(37.85%). |
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