CAJ | 학술논문

[目的]为研究转生长相关基因对猪的作用。[方法]采用RT-PCR方法,从13/17罗伯逊易位杂合子猪小肠组织中提取总RNA,将其纯化后作为PCR扩增模板,参考GenBank公布的猪Ghrelin的mRNA序列设计合成具有Nhe I和Hind III双酶切位点引物,扩增获得Ghrelin基因cD-NA全长序列。将准确的Ghrelin基因片段克隆于 pMD19-T simple Vector后进行序列分析,获得猪Ghrelin基因cDNA全长基因片段。经Nhe I和Hind III双酶切,将 Ghrelin基因cDNA片段连接到真核表达载体pEGFP-N1,获得真核表达载体的重组质粒pEGFP-Ghrelin。重组质粒转染猪成纤维细胞,观察标记基因荧光蛋白的表达。[结果]13/17罗伯逊易位杂合子猪的Ghrelin基因与已发表的序列相同,并成功获得具备猪Ghrelin基因全长cDNA序列的真核表达载体pEGFP-Ghrelin。[结论]构建的真核表达载体可以用于转基因猪的进一步试验,同时也为研究Ghrelin的调节机制奠定基础。
[목적]위연구전생장상관기인대저적작용。[방법]채용RT-PCR방법,종13/17라백손역위잡합자저소장조직중제취총RNA,장기순화후작위PCR확증모판,삼고GenBank공포적저Ghrelin적mRNA서렬설계합성구유Nhe I화Hind III쌍매절위점인물,확증획득Ghrelin기인cD-NA전장서렬。장준학적Ghrelin기인편단극륭우 pMD19-T simple Vector후진행서렬분석,획득저Ghrelin기인cDNA전장기인편단。경Nhe I화Hind III쌍매절,장 Ghrelin기인cDNA편단련접도진핵표체재체pEGFP-N1,획득진핵표체재체적중조질립pEGFP-Ghrelin。중조질립전염저성섬유세포,관찰표기기인형광단백적표체。[결과]13/17라백손역위잡합자저적Ghrelin기인여이발표적서렬상동,병성공획득구비저Ghrelin기인전장cDNA서렬적진핵표체재체pEGFP-Ghrelin。[결론]구건적진핵표체재체가이용우전기인저적진일보시험,동시야위연구Ghrelin적조절궤제전정기출。
[Objective] This study aimed to investigate the functions of transgenic growth related gene in pig growth. [Method] A pair of primers containing Nhe I and Hind Ⅲ restriction sites were designed by referring to the pig Ghrelin mRNA sequence published in Genbank. Total RNA was extracted from the small intestine tissue of 13/17 Robertson translocation heterozygous pig, and then was purified and used as the template in later RT-PCR reaction to amplify the full-length pig Ghrelin gene. The correct pig Ghrelin gene fragment was cloned into the pMD19-T simple vector for sequencing analysis. The obtained full-length cDNA of pig Ghrelin gene fragment was digested with both Nhe I and Hind Ⅲ, and then was linked into the eukaryotic expression vector pEGFP-N1 to obtain the recombinant plasmid pEGFPGhrelin. The recombinant plasmid was transected into the fibroblast cells to detect the fluorescence labeled gene expression. [Result] The nucleotide sequence extracted from 13/17 Robertson translocation heterozygous pig was the same as expected; and the eukaryotic expression vector pEGFP-Ghrelin was successfully constructed. [Conclusion] The eukaryotic expression vector constructed in this study can be further used in research on transgenic pigs, but also lays foundation for research on the regulatory mechanism of Ghrelin gene.