- 期刊
畜試黑豬肌肉生長抑制素互補DNA選殖及其mRNA表現量與新生仔豬出生體重相關性分析
Cloning of myostatin cDNA and analysis of the relation between birth weight and myostatin mRNA expression levels of neonatal TLRI No.1 black pigs
摘要
本試驗目的探討畜試黑豬仔豬之出生體重與肌肉生長抑制素mRNA含量之相關性，同時進行畜試黑豬肌肉生長抑制素互補DNA（cDNA）核酸序列選殖、以及畜試黑豬與不同豬種間肌肉生長抑制素cDNA核酸序列分析。試驗結果顯示，在採集6胎出生24小時內新生畜試黑豬仔豬平均體重最輕和最重仔豬各6頭之後腿部位肌肉樣品，利用半定量RT-PCR方法分析肌肉生長抑制素之mRNA含量，出生體重較輕仔豬組（1.0 ± 0.1 kg）之肌肉生長抑制素 mRNA表現量顯著高於體重較重仔豬組（1.5 ± 0.2 kg）約1.97倍，顯示肌肉生長抑制素基因表現量高可能具有抑制畜試黑豬仔豬出生體重之作用。試驗並利用逆轉錄作用聚合鏈反應產生cDNA產物，再用nested PCR方法，選殖畜試黑豬肌肉生長抑制素cDNA之編碼區域1128 bp，並將其接合於pGEM-T Easy載體，轉形至大腸桿菌中，經大量培養後，萃取質體DNA進行序列分析，並將定序完成之核酸資料，利用BLAST（Basic Local Alignment Search Tool）與 DNAMAN套裝軟體比對序列，確認畜試黑豬其肌肉生長抑制素之cDNA序列與梅山豬、杜洛克與約克夏等豬種相似度為100%，將可供後續進行基因功能分析與調控之研究。
並列摘要
The objective of this study was to survey the relation between myostatin mRNA amounts and birthweight of neonatal TLRI No.1 black pigs, and to clone the myostatin cDNA. Skeletal muscle of hind legs from neonatal piglets of lower (1.0 ± 0.1 kg, n = 6) and heavier birthweight (1.5 ± 0.2 kg, n = 6) were sampled, and the amounts of myostatin mRNA were determined by using semi-quantitative RT-PCR. The result showed the myostatin mRNA amounts of the piglets with lower birthweight was 1.97 times higher than those with heavier birthweight. The results suggested that myostatin gene might possess unique impact on neonatal TLRI No.1 black pig. In myostatin cDNA cloning, methodologies adopted the reverse transcription- polymerase chain reaction (RT-PCR) to yield first strand cDNA and performed the nest-PCR to generate 1128 bp myostatin cDNA. Afterward the amplified cDNA was ligated into pGEN-T vector, and then transformed the ligated vectors into competent cells and inoculated the transformed competent cells into LB broth and culture at 37℃ for overnight. The plasmid DNA was extracted from cultivated competent cells which was applied to perform nucleotides sequencing using the dideoxy chain termination method and aligned its sequencing identity using the BLAST (Basic Local Alignment Search Tool). The results showed that sequencing identity of myostatin cDNA sequences extracted from TLRI No. 1 black pig was consistent with other porcine species such as Meishan, Duroc and Yorkshire. Therefore, the preliminary results suggested that we might further apply the cloned myostatin cDNA to investigate its gene function or regulative mechanism.
延伸閱讀
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