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| 从江香猪ApoA1基因的克隆及亚细胞定位研究 | |
| 引用本文： | 赵佳福,段志强,杨远青,嵇辛勤,王圆圆,孙成娟.从江香猪ApoA1基因的克隆及亚细胞定位研究[J].中国畜牧兽医,2017,44(7):1954-1960. |
| 作者姓名： | 赵佳福 段志强 杨远青 嵇辛勤 王圆圆 孙成娟 |
| 作者单位： | 1. 贵州大学, 高原山地动物遗传育种与繁殖省部共建教育部重点实验室, 贵阳 550025; 2. 贵州大学动物科学学院, 贵阳 550025; 3. 贵州省毕节市畜禽遗传资源管理站, 毕节 551700 |
| 基金项目： | 省校联合基金（黔科合LH字[2014]7668）；贵州省科技重大专项（黔科合重大专项字[2013]6008号）；贵州大学青年基金（贵大自青基合字[2012]010号） |
| 摘 要： | 试验旨在克隆从江香猪载脂蛋白A1（apolipoprotein A1，ApoA1）基因，研究ApoA1基因在真核细胞中的亚细胞定位情况。通过提取从江香猪总RNA，采用RT-PCR、目的基因的连接、转化等方法构建携带有绿色荧光蛋白的pEGFP-C1-ApoA1重组质粒，并经菌落PCR、双酶切及测序鉴定正确后，转染HEK-293T细胞，36 h后观察荧光，分析ApoA1蛋白在真核细胞中的亚细胞定位情况。结果表明，从江香猪ApoA1基因与GenBank上公布的野猪序列相比，有6处发生了碱基突变，其中5处为有义突变，分别导致180位氨基酸由丙氨酸变为谷氨酸、185位氨基酸由组氨酸变为谷氨酰胺、186位氨基酸由缬氨酸变为亮氨酰胺、209位氨基酸由天冬氨酸变为甘氨酸；PSORT Ⅱ Prediction和荧光共定位试验结果均表明，ApoA1蛋白的表达主要集中在细胞外基质，约占总表达量的77.8%。本试验成功克隆了从江香猪ApoA1基因CDS区，且ApoA1蛋白的表达主要集中在细胞外基质中，为进一步构建ApoA1基因转基因动物模型、开展ApoA1基因与人类因肥胖引起的相关疾病关系的研究奠定基础。 |
| 关 键 词： | 丛江香猪 ApoA1基因 pEGFP-C1载体 HEK-293T细胞 生物信息学分析 亚细胞定位 |
| 收稿时间： | 2017-02-23 |
Cloning and Subcellular Localization of ApoA1 Gene in Congjiang Xiang Pig |
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| ZHAO Jia-fu,DUAN Zhi-qiang,YANG Yuan-qing,JI Xin-qin,WANG Yuan-yuan,SUN Cheng-juan.Cloning and Subcellular Localization of ApoA1 Gene in Congjiang Xiang Pig[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):1954-1960. | |
| Authors: | ZHAO Jia-fu DUAN Zhi-qiang YANG Yuan-qing JI Xin-qin WANG Yuan-yuan SUN Cheng-juan |
| Institution: | 1. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China; 2. College of Animal Science, Guizhou University, Guiyang 550025, China; 3. Animal Genetic Resources Management of Bijie City of Guizhou Province, Bijie 551700, China |
| Abstract: | This experiment was aimed to clone apolipoprotein A1(ApoA1) gene of Congjiang Xiang pig, and study the subcelluar localiztion of ApoA1 gene in eukaryocyte. The recombination plasmid pEGFP-C1-ApoA1 was constructed with RT-PCR and other methods, and detected by colony PCR,double digestion and sequencing, after successful construction of the recombination plasmid pEGFP-C1-ApoA1,the subcellular localization of ApoA1 protein were analyzed by fluorescence co-localization technique in the 36 h-transfected HEK-293T cells. Compared with ApoA1 gene of Sus scrofa submission in GenBank, the results showed that six base mutations were found in ApoA1 gene of Congjiang Xiang pig, five of above mentioned mutations were sense mutations, causing alanine to glutamic acid, histidine to glutamine, valine to leucine and aspartic acid to glycine in 180,185,186 and 209 amino acid residues, respectively. Using PSOR Ⅱ Prediction and fluorescence co-localization, it was found that the expression of ApoA1 protein was observed mainly in the extracellular matrix (77.8%). In conclusion, ApoA1 gene of Congjiang Xiang pig was cloned successfully, and the expression of ApoA1 protein was mainly concentrated in the extracellular matrix. These results would provide a knowledge for further constructing the ApoA1 gene transgenic animal models, and contribute to understanding the relation between ApoA1 gene and the human obesity-induced diseases. |
| Keywords: | Congjiang Xiang pig ApoA1 gene pEGFP-C1 vector HEK-293T cell bioinformatics analysis subcellular localization |
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