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六溴环十二烷胁迫下红树蚬荧光定量PCR内参基因稳定性分析
车志群1,2, 孙仁杰1, 刘文爱1, 廖思明3, 阎冰1
0
(1.广西科学院广西红树林研究中心, 广西红树林保护与利用重点实验室, 广西北海 536000;2.广西大学, 广西南宁 530000;3.广西科学院, 广西南宁 530007)
摘要:
[目的]筛选出不同浓度六溴环十二烷(HBCD)胁迫下红树蚬(Polymesoda erosa)实时荧光定量PCR (qRT-PCR)的最适内参基因,用以准确校准目的基因的表达,为进一步开展分子毒理研究和开发分子生物标志物奠定基础。[方法]通过荧光定量PCR技术监测候选内参18S核糖体RNA (18S rRNA)、β-肌动蛋白(β-actin)、甘油醛-3-磷酸脱氢酶(GAPDH)、α-微管蛋白(α-tubulin)等管家基因,在红树蚬各组织受不同浓度(0 μg/L、0.86 μg/L、8.6 μg/L)六溴环十二烷(HBCD)胁迫后的表达量,利用qRT-PCR及geNorm、NormFinder、BestKeeper等分析软件对不同条件下的表达数据进行处理,从而筛选出适合荧光定量PCR的最佳内参基因。[结果]受不同浓度HBCD胁迫后,鳃及肝胰中各管家基因的表达稳定性排序整体为β-actin=α-tubulin > GAPDH > 18S rRNA[结论]可单独或共同使用两个内参基因(β-actinα-tubulin)校准荧光定量结果。
关键词:  红树蚬  六溴环十二烷  内参基因  稳定性分析
DOI:10.13657/j.cnki.gxkxyxb.20180807.001
投稿时间:2018-03-20
基金项目:广西红树林保护与利用重点实验室开放基金(GKLMC-201306),广西红树林保护与利用重点实验室基金课题(GMLMC-201310)和科技部林业公益项目(201504413)资助。
The Expression Stability Analysis of Reference Genes for qRT-PCR in Polymesoda erosa under Hexabromocyclododecane Stress
CHE Zhiqun1,2, SUN Renjie1, LIU Wen'ai1, LIAO Siming3, YAN Bing1
(1.Guangxi Key Lab of Mangrove Conservation and Utilization, Guangxi Mangrove Research Center, Guangxi Academy of Sciences, Beihai, Guangxi, 536000, China;2.Guangxi University, Nanning, Guangxi, 530000, China;3.Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China)
Abstract:
[Objective] This experiment was to select the best reference gene for quantitative PCR under different concentrations of hexabromocyclododecane treatments, which will accurately calibrate the expression of objective genes and lay a foundation for further study on molecular toxicology research and development of molecular biomarkers. [Methods] The expression level of four candidate genes(18S rRNAβ-actinGAPDHα-tubulin)on different tissues of Polymesoda erosa under different concentrations of hexabromocyclododecane treatments were monitored by using qRT-PCR technology, and the expression data obtained from qRT-PCR was analyzed with different software, such as BestKeeper, geNorm, NormFinder to select the best reference gene suitable for real-time PCR. [Results] The results showed that after being stressed by different concentrations of HBCD, the order of candidate genes expression in gill and hepatopancreas was:β-actin=α-tubulin > GAPDH > 18S rRNA. [Conclusion] The qRT-PCR results can be calibrated using two reference genes (β-actin, α-tubulin) alone or in combination.
Key words:  Polymesoda erosa  hexabromocyclododecane  reference genes  stability analysis

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