雞Equarin長鏈基因之選殖及該基因與產蛋性能之關係

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Title:	雞Equarin長鏈基因之選殖及該基因與產蛋性能之關係
Other Titles:	Molecular Cloning of Chicken Equarin-L Gene and the Relationship between Equarin-L and Egg Production
Authors:	陳明軒 Chen, Ming-Syuan
Contributors:	歐柏榮 Ou, Bor-Rung 東海大學畜產與生物科技學系
Keywords:	台灣土雞;生殖力;產蛋性能;equarin Taiwan Country Chicken;reproductive ability;egg production;equarin
Date:	2009
Issue Date:	2011-03-07T06:14:03Z (UTC)
Abstract:	臺灣土雞為國人較偏好之禽肉，但其繁殖效率偏低，而產蛋性能與其相關基因之關係仍有許多是未知之處。前人針對高產蛋 (L2品系) 及低產蛋 (B品系) 性能之台灣土雞卵巢進行抑制性雜合扣除法 (suppression subtractive hybridization, SSH)，而獲得一些與生殖力相關的基因，如myostatin、ferritin heavy chain、glutathione S-transferase A1與equarin。Equarin為一種分泌型基因。Equarin蛋白質有短鏈 (short form; equarin-S) 及長鏈 (long form; equarin-L) 兩種型式，短鏈含有601個胺基酸，而長鏈於C端多了366個胺基酸。本研究從臺灣土雞選殖equarin基因，並表現重組蛋白及產製其專一性抗體，以探討其基因表現與不同品系雞間產蛋性能之關係。利用反轉錄聚合?連鎖反應 (reverse transcription-polymerase chain reaction; RT-PCR) 方法，從土雞脂肪組織RNA擴增equarin-L部分cDNA，將之構築於pQE80L表現載體，並轉殖於大腸桿菌以表現重組蛋白，再利用Ni-NTA大量純化重組蛋白，最後以紐西蘭白兔產製多株抗體。西方吸漬法顯示所產製之抗體可專一性辨認equarin-L重組蛋白。以RT-PCR分析第9、16、21天的雞胚各組織的equarin-L基因表現情形，結果顯示equarin-L基因廣泛表現於心臟、肝臟、脾臟、肺臟、腎臟、肌肉、脂肪、腦、胃、小腸、眼等。RT-PCR分析結果顯示，於脂肪及卵巢組織中，來亨雞之equarin-L基因表現量皆顯著高於B品系土雞，但在肝臟及肌肉組織中，皆無品系間差異。以西方吸漬法分析各品系之血清樣本，結果顯示B品系土雞之equarin-L顯著高於L2品系土雞及來亨雞者 (P<0.05)。另外，在雞肝臟之初代細胞培養中，添加睪固酮 (testosterone) 顯著增加 (P<0.05) equarin-L基因表現量，但添加雌素二醇 (estradiol) 及助孕固酮 (progesterone) 則無顯著影響。以上結果顯示，血清中equarin-L之含量可能與產蛋性能有關，且在生殖及胚發育中，可能扮演重要角色。 Taiwan Country chicken is a popular poultry in Taiwan. The reproductive ability of Taiwan Country chicken is low and the relationship between egg production and its related genes is not known. In previous study, some egg production related genes, such as myostatin, ferritin heavy chain, glutathione S-transferase A1, Equarin were identified by using suppression subtractive hybridization(SSH) method. Equarin is a secretary protein. Equarin protein has two forms, equarin-S (short form) and equarin-L (long form). Equarin-S consists of 601 amino acids and equarin-L has additional 366 amino acids at its C-terminal. The objectives of this study were to clone equarin gene from Taiwan Country chicken and generate the recombinant protein to produce specific antibody to investigate the relationship between equarin gene expression and egg production in different lines of chickens. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify partial equarin-L cDNA from total RNA of adipose tissue in Taiwan Country chicken. The cDNA was cloned into pQE80L expression vector and E. coli was used as the host system to overexpress the recombinant protein. The recombinant protein was purified by Ni-NTA affinity chromatography and polyclonal antibody was generated in New Zealand rabbit. The result of Western blot analysis showed that the polyclonal antibody recognized the recombinant protein specifically. The expression of equarin-L gene in different tissue of chicken embryos at days 9, 16 or 21 were analyzed by RT-PCR and the results showed that equarin-L gene expressed ubiquitously in heart, liver, spleen, lung, kidney, muscle, fat, brain, stomach, intestine and eye. The expression levels of equarin-L gene in fat and ovary tissue in Leghorn chicken were higher than that in B line, but there was no significant difference between strains in liver or muscle tissue. The equarin-L protein in serum samples of each lines were analyzed by Western blotting, and the results indicated that the equarin-L levels in B line was significantly (P<0.05) higher than that in L2 line and Leghorn chicken. The result of primary culture of isolated chicken liver cells indicated that the expression of equarin-L gene increased significantly (P<0.05) by testosterone treatment, but didn’t change by estradiol or progesterone treatment. In conclusion, the equarin-L level in serum appears to be related to egg production, and it may play an important role in reproduction and among embryonic development.
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