摘要
【目的】克隆徐淮山羊硬脂酰辅酶A脱氢酶1(SCD1)基因的cDNA并分析该基因生物信息学功能。通过EGFP融合蛋白在亚细胞水平定位该基因表达产物,研究该基因在异种动物体内表达情况,探索制备异种转基因动物的可能性及外源基因能否稳定遗传。【方法】采用RT-PCR方法从徐淮山羊脂肪组织中克隆SCD1基因cDNA,进行生物信息学分析,并构建含有增强型绿色荧光蛋白(EGFP)报告基因的融合表达载体pEGFP-C1-SCD1,脂质体(LTX)介导基因转染NHI-3T3细胞,48 h后荧光倒置显微镜下观察基因表达产物的分布,RT-PCR检测mRNA在体外水平的表达。利用睾丸注射法制备转基因小鼠,在F1代和F2代个体DNA水平和蛋白水平上检测目的基因的表达情况。【结果】成功克隆出徐淮山羊SCD1基因的全序列cDNA,大小为1 074 bp,编码357个氨基酸,GenBank登录号为JX854036,徐淮山羊SCD1序列与人、褐家鼠、小家鼠、绵羊、牛和野猪相应编码区的同源性分别为86%、83%、82%、98%、95%、90%,氨基酸序列同源性分别为84%、79%、79%、96%、92%、95%;成功构建了融合表达载体pEGFP-C1-SCD1;RT-PCR检测mRNA表达明显;EGFP-SCD1融合蛋白定位在NIT-3T3细胞质中,与在线软件预测一致;通过尾静脉注射和睾丸注射,该基因可以在小鼠体内暂态表达和持续性表达,且可以稳定遗传。【结论】体外克隆的徐淮山羊SCD1基因cDNA在单细胞水平上表达于细胞质中,在小鼠体内也可以表达。
[ Objective ] The aim of this paper was to clone the stearoyl-CoA desaturase 1 （SCD 1） gene from Xuhuai goat, and analyze it＇s sub-cellular localization through enhanced green fluorescent （EGFP） fusion protein. Study the gene expression in heterologous animals, explore the possibility of preparing dissimilar transgenic animals and verify whether the foreign gene can stably inherited. [Method] cDNA was cloned by reverse transcription polymerase chain reaction （RT-PCR）, it＇s biological information characteristics was analyzed by online software, and fusion expression vector named pEGFP-C1-SCD1 was constructed successfully. Then NIH-3T3 cells were transfected with pEGFP-C1-SCD1 through lipofectamine TMLTX＆PLUS（LTX）. At 48 h post-transfection, the transfected cells were observed under fluorescence inverted microscope. Then the mRNA expression level of SCD1 was detected by RT-PCR. The transgenic mice was produced by testicular injection, and the target gene expression in the F1 and F2 generations of individual was detected at DNA and protein levels. [ Result] The results showed that the coding sequence （CDS） length of Xuhuai goat SCD1 gene was 1 074 bp, encoding 357 amino acids with GenBank accession number （JX854036）. The SCD1 eDNA coding sequence was compared with the corresponding region of Human, Rattus norvegicus, Mus, Ovis aries, Bostaurus, Sus scrofa, respectively, the similarity was 86%, 83%, 82%, 98%, 95%, and 90%, respectively, and amino acid sequence homology was 84%, 79%, 79%, 96%, 92%, and 95%, respectively, pEGFP-SCD1 was successfully constructed. The RT-PCR results showed the SCD1 mRNA expressed successfully in vitro. The SCD1 protein was localized in the cytoplasm which was in line with the result of online prediction. The gene can also be expressed in mice transiently and persistencely after intravenous and testicular injection and the gene can be genetically stable. [ Conclusion ] The SCD 1 gene eDNA of Xuhuai goat was cloned successfully and in the single-cell level can express in the cytoplasm, also can express in mice.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第17期3695-3703,共9页
Scientia Agricultura Sinica
基金
国家转基因重大专项(2011ZX08008-003
2009ZX08008-003B)
