阿拉善双峰驼ENaCα亚基基因克隆及激素对肾皮质细胞中该基因表达的影响

doi:10.16431/j.cnki.1671-7236.2019.02.002

摘要/Abstract

摘要：

为探究阿拉善双峰驼上皮细胞钠通道的作用特点，试验采用深圳华大公司提供的ENaCα亚基基因片段序列设计PCR引物，提取阿拉善双峰驼肾皮质总RNA，反转录合成cDNA，PCR扩增获得双峰驼ENaCα亚基基因，构建克隆质粒pMD19-T-ENaCα，对重组质粒进行双酶切和测序分析；采集双峰驼肾皮质部分，细胞体外培养到P3代后，加入不同浓度的醛固酮（aldosterone）和血管紧张素Ⅱ（angiotensin Ⅱ），实时荧光定量PCR技术检测细胞中ENaCα亚基基因的表达情况，以确定ENaCα对两种激素浓度变化的敏感性。结果显示，PCR扩增得到2.2 kb的特异性条带，SmaⅠ和PstⅠ双酶切得到2.2和2.7 kb的条带，表明重组质粒pMD19-T-ENaCα构建成功。实时荧光定量PCR结果显示，1×10^-9 mol/L醛固酮和血管紧张素Ⅱ处理的双峰驼肾皮质细胞的ENaCα表达量显著高于对照组（P<0.05）；1×10^-6、1×10^-7和1×10^-8 mol/L醛固酮和血管紧张素Ⅱ表达量显著低于对照组（P<0.05）。本研究结果为揭示双峰驼水盐代谢的生理机制提供了试验依据。

关键词: 阿拉善双峰驼; 上皮细胞钠通道; 醛固酮; 血管紧张素Ⅱ

Abstract:

This study was aimed to explore the effect of sodium channel in epithelial cells of Alax League Bactrian camel.PCR primers were designed using the ENaCα subunit gene fragment which was provided by Shenzhen Huada company.Total RNA was extracted from the renal cortex of Alax League Bactrian camel,and cDNA was obtained by reverse transcription synthesis,then the renal cortex ENaCα subunit gene fragments were amplified by PCR.The cloning plasmid pMD19-T-ENaCα was constructed,the recombinant plasmid was identified by double enzyme digestion and squencing.The renal cortex of Alax League Bactrian camel was collected,after the cells were cultured in the P3 generation,different concentrations of aldosterone and angiotensin Ⅱ were added,the expression of ENaCα subunit gene was detected by Real-time quantitative PCR to determine the sensitivity of ENaCα changes in two hormone concentrations.The results showed that the ENaCα subunit gene was successfully cloned with the length of 2.2 kb,double digestion with Sma Ⅰ and Pst Ⅰ gave 2.2 and 2.7 kb bands,the recombinant plasmid pMD19-T-ENaCα was successfully constructed.Real-time quantitative PCR results confirmed that the expression levels of ENaCα treated with 1×10^-9 mol/L aldosterone and angiotensin Ⅱ were significantly higher than that of control group (P<0.05);The expression levels of ENaCα treated with 1×10^-6,1×10^-7 and 1×10^-8 mol/L aldosterone and angiotensin Ⅱ were significantly lower than that of control group (P<0.05).The results provided the experimental evidence for revealing the physiological mechanism of water and salt metabolism in Bactrian camel.

Key words: Alax League Bactrian camel; epithelial sodium channel; aldosterone; angiotensin Ⅱ

中图分类号:

S824

朱纯霄, 孙睿, 张焱如, 张东, 刘春霞, 孟凡华. 阿拉善双峰驼ENaCα亚基基因克隆及激素对肾皮质细胞中该基因表达的影响[J]. 《中国畜牧兽医》, 2019, 46(2): 338-344.

ZHU Chunxiao, SUN Rui, ZHANG Yanru, ZHANG Dong, LIU Chunxia, MENG Fanhua. Cloning of ENaCα Subunit Gene from Alax League Bactrian Camel and Effect of Hormone on the ENaCα Expression of Renal Cortex Cells[J]. , 2019, 46(2): 338-344.

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