育成期及產蛋期鵝隻基因表現之差異

Abstract

鵝為季節性繁殖者，在自然日照條件下，每年在特定的時段進行繁殖。在台灣地區，鵝的繁殖季節始於十月至隔年五月，其產蛋於一到三月間到達產蛋高峰，隨後產蛋量逐漸下降，並於六月休產，平均的年產蛋數約在20到70顆之間。為維持產蛋，與脂質生成以及蛋組成分形成之相關基因應正常表現。本研究於試驗一建構產蛋鵝肝臟全長cDNA基因庫並完成2,400條基因序列之定序，經過NCBI/GGGI資料庫比對後共獲得729種基因序列，其中包含34%為未知的新穎基因(Novel gene)。在已知的基因中10個大量表現的基因被挑選進行後續的研究。組織基因表現比較之結果顯示ApoVLDL II、L-FABP、vitellogenin I 以及 vitellogenin II為產蛋鵝肝臟特異表現之基因。Ovoinhibitor, preproalbumin, α-2-hs-glycoprotein 及 vitamin D binding protein 之mRNA於肝臟中的表現量明顯大於其他組織。Ovotransferrin則在肝臟、卵巢、輸卵管、殼腺、大腦及脂肪組織中表現。產蛋鵝肝臟中ovotransferrin, ovoinhibitor, preproalbumin, ApoVLDL II, vitellogenin I, vitellogenin II 以及 vitamin D binding protein mRNA的表現量明顯大於非產蛋鵝，顯示此等基因可能參與產蛋功能或與蛋形成相關之脂質代謝作用。試驗二與試驗三則利用抑制性雜合扣除法(suppression subtractive hybridization) 進行產蛋鵝與非產蛋鵝肝臟及腦垂腺中基因表現差異之研究，發現apoVLDL II, Vitellgenin I, ethanolamine kinase, G-protein gamma-5 subunit, leuayl –tRNA synthase, 以及 8個新穎的基因在產蛋鵝肝臟中特異表現，且部分基因的表現受到雌性激素的調控，證明此等基因與鵝的產蛋功能相關。而本研究亦發現prolactin, visinin-like protein 以及 4 個新穎基因於產蛋鵝腦垂腺中特異表現。 本研究中亦探討多不飽和脂肪酸(polyunsaturated fatty acid; PUFA)及硒(selenium; Se) 對產蛋鵝肝臟中脂質新生合成相關基因之影響，發現飼糧中添加3.5%魚油及0.3 mg/kg Se並不能影響包含sterol regulatory element binding proteins 1, malic enzyme, Searoyl CoA desturase 1 and fatty acid synthease等脂質生成相關基因的表現。 綜言之，本研究中取得大量產蛋鵝表現序列標籤 (expressed sequence tag ; EST)資料，且其中包含許多新穎的基因，此等基因在未來種鵝產蛋機制相關研究中具有重要的價值，有助於種鵝生殖生理之相關研究。另外於營養分對基因表現之影響方面，亦證明多不飽和脂肪酸的添加並不能對產蛋鵝肝臟中脂質生成相關基因的表現造成影響，此結果與其他產蛋家禽類似。

Geese have a short egg laying period and a low egg production rate. In order to induce and maintain egg laying, genes related to generating hepatic lipid for yolk deposition should be adequately expressed. Liver mRNA from 6 geese was extracted and used for construction of full length enriched cDNA library. About 2400 gene sequences were determined and NCBI/GGGI data bases were used to analyze these sequences, 10 highly expressed genes were selected for further research. The tissue distribution data showed that ApoVLDL II, liver type fatty acid binding protein (L-FABP), vitellogenin I and vitellogenin II transcripts were specifically expressed in the liver of laying geese. Ovoinhibitor, preproalbumin, -2-hs-glycoprotein and vitamin D binding protein mRNA were highly expressed in the liver and to a lesser extend in other tissues. Ovotransferrin mRNA was expressed in the liver, ovary, oviduct, shell gland, brain and adipose tissue. The concentrations of hepatic ovotransferrin, ovoinhibitor, preproalbumin, ApoVLDL II, vitellogenin I, vitellogenin II and vitamin D binding protein mRNA were higher in the liver of laying geese than in pre-lay geese, suggesting that these genes may involved in laying function or lipid metabolism related to egg formation. We also used a suppression subtractive hybridization technique to explore the genes which were specifically expressed in the liver and pituitary gland of laying geese. We found that there were 13 genes, including apoVLDL II, vitellgenin I, ethanolamine kinase, G-protein gamma-5 subunit, leuayl –tRNA synthase, and 8 novel genes specifically expressed in the liver of laying geese. Several of these differentially expressed genes were found to be responsive to estrogen stimulation, confirming the involvement of these genes in egg laying function of the goose. We also found that there were 6 genes, including prolactin, visinin-like protein and 4 novel genes highly expressed in the pituitary gland of laying geese. The effect of dietary DHA and selenium (Se) supplementation on the mRNA expression of lipogenic genes in livers of lying geese were also examined. Laying geese were fed with 3.5% fish oil diets containig different Se concentration (0, 0.15, 0.3 mg/kg) for 3 month. The mRNA concentration of sterol regulatory element binding proteins 1, malic enzyme, stearoyl CoA desturase 1 and fatty acid synthease were determined. Dietary DHA and Se supplement had no effect on the lipogenic genes expression in the liver of laying geese. Taken together, the data show that there are laying specific genes that may involve in supporting the laying physiology for the geese, and hepatic lipogenic genes of laying geese were not affected by DHA and Se treatment.