《植物生理学报》 2014, 50(12): 1809-1815

紫花苜蓿18S rRNA基因的克隆及内参基因表达稳定性评价

付媛媛^1,2, 穆春生¹, 高洪文², 李俊², 王学敏^2,*

¹东北师范大学草地科学研究所, 植被生态科学教育部重点实验室, 长春130024; ²中国农业科学院北京畜牧兽医研究所, 北京100193

通信作者：王学敏；E-mail: wangxuemin@caas.cn；Tel: 010-62816008

摘　要：

本文克隆紫花苜蓿常用内参基因18S rRNA, 并筛选出稳定的内参基因, 以确保紫花苜蓿基因表达分析结果的精确性和可靠性。从紫花苜蓿中克隆常用内参基因18S rRNA的cDNA全长, 在此基础上结合β-actin、EF-1α、UBC2、TUB 4个常用的内参基因, 应用实时定量PCR技术对5个候选内参基因在紫花苜蓿不同组织的表达情况进行分析。经BestKeeper和geNorm软件综合分析, 5个候选基因在紫花苜蓿不同组织中的表达稳定性不同, 其中18S rRNA和EF-1α最稳定。

关键词：18S rRNA; 内参基因; 实时荧光定量PCR; 紫花苜蓿

收稿：2014-11-27  

资助：国家自然科学基金(31101755)和中国农业科学院科技创新工程(ASTIP-IAS10)。 致谢 感谢中国农业科学院北京畜牧兽医研究所杨青川研究员惠赠紫花苜蓿‘中苜3号’种子。

Cloning of 18S rRNA Gene and Stability Evaluation of Reference Genes in Medicago sativa

FU Yuan-Yuan^1,2, MU Chun-Sheng¹, GAO Hong-Wen², LI Jun², WANG Xue-Min^2,*

¹Key Laboratory of Vegetation Ecology of Ministry of Education, Institute of Grassland Science, Northeast Normal University, Changchun 130024, China; ²Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

Corresponding author: WANG Xue-Min; E-mail: wangxuemin@caas.cn; Tel: 010-62816008

Abstract:

The objective of this research was to clone reference gene of 18S rRNA from Medicago sativa, and select stable reference genes to ensure the reliability and accuracy of gene expression. The full length cDNA sequence of 18S rRNA which was frequently used as reference gene was obtained from M. sativa. Furthermore, we analyzed the stability of five candidate reference genes (18S rRNA, β-actin, EF-1α, UBC2, TUB) in different tissues by using the real-time quantitative PCR. The expression stabilities were assessed using two statistical algorithms BestKeeper and geNorm, respectively. The analysis results showed that the expression stability of five candidate genes varied in different tissues of M. sativa were different, and 18S rRNA and EF-1α were the most stably expressed genes.

Key words: 18S rRNA; reference genes; real-time fluorescence quantitative PCR; Medicago sativa