绵羊ApoER2 基因克隆及其干扰质粒对睾丸共培养细胞硒蛋白表达的影响

doi:10.11843/j.issn.0366-6964.2014.08.005

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (8): 1237-1245.doi: 10.11843/j.issn.0366-6964.2014.08.005

绵羊ApoER2 基因克隆及其干扰质粒对睾丸共培养细胞硒蛋白表达的影响

张春香，赵辉，张国林，郭丽娜，任有蛇^*

（山西农业大学动物科技学院，太谷 030801）

收稿日期:2014-02-18 出版日期:2014-08-23 发布日期:2014-08-23
通讯作者: 任有蛇，Tel：0354-6285990，E-mail：rys925@126.com
作者简介:张春香(1972-)，女，山西侯马人，博士，副教授，主要从事动物遗传育种与繁殖研究，Tel：0354-6285990，E-mail：zhchx66@126.com
基金资助:
高等学校博士学科点专项科研基金（20091403120001）

The Clone of ApoER2 Gene and the Inhibitory Effect of Interference Plasmid ApoER2-siRNA on Selenoprotein Expression in Co-culture Sertoli Cells of Sheep

ZHANG Chun-xiang，ZHAO Hui，ZHANG Guo-lin，GUO Li-na，REN You-she^*

(College of Animal Science and Veterinary Medicine，Shanxi Agricultural University，Taigu 030801,China)

Received:2014-02-18 Online:2014-08-23 Published:2014-08-23

摘要/Abstract

摘要：

本研究旨在克隆绵羊ApoER2基因，构建ApoER2-siRNA质粒载体，研究ApoER2基因沉默对睾丸共培养细胞Sel P和PHGPx表达的影响。利用RT-PCR技术，克隆ApoER2序列，在此基础上构建4种pGPU6/GFP/Neo-ApoER2-siRNA质粒表达载体，利用脂质体将其转染到体外共培养绵羊睾丸细胞中，采用Real-time PCR和Western blotting方法，检测其抑制效应，并研究ApoER2基因沉默对Sel P和PHGPx mRNA表达的影响。本研究成功获得了长度为2 490 bp的绵羊睾丸ApoER2完整编码区序列，共编码892个氨基酸，绵羊ApoER2核苷酸序列与牛的同源性最高，相似性为97.7%。ApoER2-siRNA-1565和ApoER2-siRNA-2567位点重组质粒的干扰效果较好（P<0.01），选取ApoER2-siRNA-2567重组质粒，结果显示转染组Sel P和PHGPx表达量显著降低（P<0.01）。获得了绵羊ApoER2编码区序列和有效抑制该基因表达的2个质粒载体，ApoER2沉默可能影响了睾丸硒蛋白Sel P 和 PHGPx的表达，为进一步研究ApoER2转运硒机理提供了理论依据。

Abstract:

The aim of this study was to clone ovine ApoER2 (apolipoprotein E receptor-2)，construct the plasmid vector ApoER2-siRNA，and investigate the inhibitory effect of ApoER2 gene silencing on expression of Sel P and PHGPx genes in co-culture Sertoli cells of sheep.The ovine ApoER2 was cloned by RT-PCR.Four pGPU6/GFP/Neo-ApoER2 plasmid expression vectors were constructed，and transfected into the co-culture Sertoli cells of sheep by Lipofectamine 2000^TM.The inhibitory effect of ApoER2-shRNA was detected by RT-PCR and Western blotting.The effect of ApoER2-shRNA inhibitory on expression of Sel P and PHGPx genes was detected by RT-PCR.The complete CDS of ApoER2 was 2 490 bp encoding a 892-amino-acid protein.The BLAST analysis showed that the entire nucleotide sequence of ApoER2 gene had 97.7% homology with bovine.ApoER2 mRNA levels in co-culture Sertoli cells were decreased significantly after transfection by the recombinant plasmids located in ApoER2-siRNA-1565 (P<0.01)and ApoER2-siRNA-2567 (P<0.01).Expression of Sel P and PHGPx mRNA were significantly decreased in the ApoER2-siRNA-2567 recombinant plasmid transfection group (P<0.01).The complete CDS of ApoER2 was obtained.Recombinant plasmid pGPU6/GFP/Neo-ApoER2-1565 and pGPU6/GFP/Neo-ApoER2-2567 were successfully constructed with effective inhibitory.Interference of ApoER2 gene decreased the expression of of Sel P and PHGPx mRNA，which could provide the scientific basis for further study of ApoER2 roles in pathway of selenium transport.

中图分类号:

S826
Q786

张春香，赵辉，张国林，郭丽娜，任有蛇. 绵羊ApoER2 基因克隆及其干扰质粒对睾丸共培养细胞硒蛋白表达的影响[J]. 畜牧兽医学报, 2014, 45(8): 1237-1245.

ZHANG Chun-xiang，ZHAO Hui，ZHANG Guo-lin，GUO Li-na，REN You-she. The Clone of ApoER2 Gene and the Inhibitory Effect of Interference Plasmid ApoER2-siRNA on Selenoprotein Expression in Co-culture Sertoli Cells of Sheep[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1237-1245.

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绵羊ApoER2 基因克隆及其干扰质粒对睾丸共培养细胞硒蛋白表达的影响

The Clone of ApoER2 Gene and the Inhibitory Effect of Interference Plasmid ApoER2-siRNA on Selenoprotein Expression in Co-culture Sertoli Cells of Sheep

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