台灣水鹿與台灣梅花鹿類胰島素生長因子乙型片段序列探討

外文標題: 
Study on Partial Sequence of Insulin-like Growth Factor 2 in Formosan sambar deer and Formosan sika deer
校院系所: 
屏東科技大學動物科學與畜產系所
指導教授: 
張秀鑾
出版年份: 
2011年
主題類別: 
摘要: 

本研究旨在探討台灣特有亞種,台灣水鹿與台灣梅花鹿,IGF2基因部分片段序列與其多態性。哺乳類類胰島素生長因子乙型(insulin-like growth factor 2, IGF2)基因具父系銘印表現已獲證實,豬IGF2基因核苷酸變異點具影響動物脂肪沉積、瘦肉量與飼料效率;牛則具影響腰眼面積肌肉之生成。鹿茸組織中檢測出IGF1和IGF2含量,而鹿茸角頂端亦發現IGF1與IGF2接受體含量,故推測類胰島素生長因子(IGFs)於鹿茸細胞生長可能扮演重要角色。分類學研究顯示,豬科、牛科與鹿科為複系群(polyphyletic group),且比對牛與豬IGF2基因第三內含子全長序列,相似度達74%,顯示不同物種IGF2基因序列相似度高,推論IGF2基因亦可能影響鹿茸生長。試驗主要分成兩部分:試驗一應用豬隻IGF2-intron3-G3072A 引子進行台灣水鹿核內基因組(gDNA)聚合酶鏈鎖反應(PCR)擴增產物後,經膠體純化、連接、轉形、藍白菌落篩選與定序。結果發現,經PCR擴增出IGF2-intron3部分片段長度分別為78與202 bp,其中78 bp序列與豬IGF2-intron3第3051至3128個核苷酸處具相同序列。進一步比對顯示,其與豬、牛、小鼠與人等四個物種IGF2-intron3區部分片段相近,且相似度分別為100%、94%、85%與85%。因此,推論IGF2-intron3區之核苷酸序列,於不同物種間可能具高度保留性。試驗二進行台灣水鹿、台灣梅花鹿與荷士登乳牛 IGF2-intron3區部分序列擴增。台灣水鹿與台灣梅花鹿gDNA經引子分別擴增出655 bp與654 bp片段。序列比對後,具44個單核苷酸多態性位點(single nucleotide polymorphism, SNP),包括31個轉換(transition)、七個顛換(transversion)與六個插入(insertion)/缺失(deletion)。台灣水鹿與台灣梅花鹿分別有27與19個SNP位點,族群分析結果,台灣水鹿與台灣梅花鹿分別有12與13種單套型。然應用乳牛片段序列,引子組於乳牛與台灣梅花鹿gDNA產物擴增時,獲得371 bp單一泳帶。定序比對顯示,台灣梅花鹿具插入/缺失與轉換,共兩個SNPs位點與三種單套型組合;而乳牛,則有四個轉換SNPs位點與五種單套組合型。台灣梅花鹿序列相似度比對發現,與牛、豬、小鼠與人類等四個物種IGF2基因最為相近,相似度分別為95%、83%、83%與76%。綜合上述,不同物種間IGF2-intron3核苷酸序列,具高度保留性。本研究定序台灣水鹿與台灣梅花鹿之IGF2-intron3區域 655 bp 與371 bp片段,以及其核苷酸多態型。

外文摘要: 

The aims of this study were to sequence the partial fragments of insulin-like growth factor 2 (IGF2) gene, and identify the polymorphism of Formosan sambar deer (Rusa unicolor swinhoei) and and Formosan sika deer (Cervus nippon taiouanus), which is unique to Taiwan. The discovery of the paternally expressed insulin-like growth factor 2 (IGF2) gene in mammals was confirmed. Effects of SNPs in the IGF2 gene on fat deposition, lean meat content and feed efficiency in pigs, and on loin eye muscle mass in cattle were also reported. Moreover, the Suidae, Bovidae, and Cervidae are classified in polyphyletic group based on phylogeny, with 74% identity in full sequence of IGF2-intron3 between Suidae and Bovidae. Because of the existence of well conserved region among species, the effect of IGF2 gene on antler growth could be inferred. Two experiments were included in this study. Experiment 1 used the primers designed for IGF2-intron3-G3072A genotyping in pig to amplify gDNA extracted from Rusa unicolor swinhoei. The PCR product was then electrophoreses, followed by gel extraction, ligation, transformation, blue/white color screening and finally the sequencing procedure. Two PCR with 78 and 202 bp in length were obtained after amplification, in which the 78 bp nucleotide fragment was completely matched with that of the 3051th to 3128th sequence in IGF2-intron3 of pig after alignment. Furthermore, 100%, 94%, 85%, and 85% identity were obtained when alignment were made with Sus scrofa, Bos taurus, Mus musculus, and Homo sapiens, respectively. Highly conservative sequence in IGF2-intron3 region among species was then further confirmed. Experiment 2 were to design two new pairs of primers, (DF2, DR2) and (IN3F, IN3R) to amplify the IGF2-intron3 region partial sequence for Rusa unicolor swinhoei, Cervus nippon taiouanus, and Bos taurus. Both fragments with 655 and 654 bp were observed after amplification of gDNA extracted from Rusa unicolor swinhoei and Cervus nippon taiouanus via (DF2, DR2) primer pair. A total of 44 single nucleotide polymorphism (SNP) sites were obtained, which included 31 transitions, 7 transversions, and 6 insertions/deletions. However, there were 27 and 19 SNPs sites with 12 and 13 haplotypes found in Rusa unicolor swinhoei and Cervus nippon taiouanus, respectively. In addition, we obtained 371 bp fragment when (IN3F, IN3R) primer pair was used to amplify the gDNA of Cervus nippon taiouanus and Holstein cattle. Alignment results showed two and four SNPs in Cervus nippon taiouanus and Holstein cattle, respectively, which accounted for three and five haplotypes in the corresponding species. Furthermore, one insertion/deletion and one transition were found in Cervus nippon taiouanus, and four transition sites were observed in Holstein cattle. Cervus nippon taiouanus showed 95, 83, 83, and 76% identity with Bos Taurus, Sus scrofa, Mus musculus, and Homo sapiens, respectively, when alignment were conducted for the fragment described above. In conclusion, the IGF2-intron3 region showed highly conservative among species. A length of 655 bp and 371 bp fragment in IGF2-intron3 region of Rusa unicolor swinhoe and Cervus nippon taiouanus was sequenced and aligned within this study.