盤克夏豬的造骨蛋白基因啟動子區域之定序

廖仁寶 張秀鑾 吳明哲

台灣省畜產試驗所

造骨蛋白（OPN）基因啟動子區域含有GT序列重複區，本研究以PCR分別增幅四頭盤克夏豬的核內DNA，將其PCR產物接合至載體上，再轉形至大腸桿菌中。四頭豬的轉形株中，再各挑選 4、4、3和5個白色菌落，培養後萃取其重組質體，再進行插入部分定序步驟。插入部分定序結果為5’CCAATCCTATTCACGAAAAAGCTAGTTAATGACATTGTACATAAGTAATGTTTTAACTGCAGAGGGC(GT)nTGTAACCACAAAACCAGAGGGGGAAGTGTGGGAGCAAGTGGGTTG3’(首尾兩端黑粗斜體字為正反向引子)，所有選殖株的插入部分序列，除GT重複數有不同外，其餘部分皆相同。豬B9510806的重組質體插入部分序列的GT重複數皆為14。豬B9560505的重組質體插入部分序列的GT重複數有13、14和24三種。豬B100303的重組質體插入部分序列的GT重複數有24和25兩種。豬B100501的重組質體插入部分序列的GT重複數有22、23、24和25四種。本研究以四頭盤克夏豬造骨蛋白基因啟動子區域之GT序列重複數定序結果，有13、14、22、23、24和25個GT重複數的六種交替基因。

關鍵語：盤克夏、造骨蛋白、定序。

SEQUENCING OF THE PROMOTER REGION OF OSTEOPONTIN
GENE IN BERKSHIRE PIGS

R. B. Liaw, H. L. Chang and M. C. Wu

Taiwan Livestock Research Institute

There is a variable number of GT dinucleotide repeats in the promoter region of osteopontin (OPN) gene in pigs. The fragments of the promoter region of osteopontin gene from four Berkshire pigs were amplified with PCR. Then the amplicons were ligated to the cloning vector and transformed to Escherichia coli. There were 4, 4, 3, and 5 white colonies, respectively, selected from the four pigs. Recombinant plasmids were extracted from transformants and inserts were sequenced by an automated DNA sequencer. The insert sequence was 5’CCAATCCTATTCACGAAAAAGCTAGTTAATGACATTGTACATAAGTAATGTTTTAACTGCAGAGGGC(GT)nTGTAACCACAAAACCAGAGGGGGAAGTGTGGGAGCAAGTGGGTTG3’ (the forward and reverse primers were in bold and italic). All insert sequences of recombinant plasmids were the same except of the number of GT dinucleotide repeats. Number of GT repeats of the inserts from B9510806 pig was 14 only. GT repeats of the inserts from B9560505 pig were 13, 14 and 24. GT repeats of the inserts from B100303 pig were 24 and 25. GT repeats of the inserts from B100501 pig were 22, 23, 24 and 25. Results from this study indicated that there were six alleles of the 13, 14, 22, 23, 24 or 25 repeats of GT in the promoter region of osteopontin gene based upon four pigs of Berkshire breed.

Key Words: Berkshire, Osteopontin, Sequencing.