《植物生理学报》 2015, 51(5): 642-648

小麦与叶锈菌互作过程中实时定量PCR内参基因的选择及TaSGR基因的表达分析

李晖^1,*, 牛宏伟^2,*, 刘娜¹, 陈琰¹, 侯春燕^1,**, 王冬梅^1,**

¹河北农业大学生命科学学院植物逆境实验室, 河北保定071001; ²河北省环保产品质量监督检验院, 石家庄050000

通信作者：李晖；E-mail: dongmeiwang63@126.com, houchunyan@126.com；Tel: 0312-7528276; 0312-7528249

摘　要：

实时定量PCR (RT-qPCR)是确定基因功能的重要手段之一。为了量化基因表达水平, 选择合适的内参基因是实验准确的先决条件。本文以小麦ACTIN1、ACTIN2、GAPDH、GAPDH2、ATUB、BTUB、EF1A1、EF1A2和TA共9个基因作为候选内参基因, 在小麦(Triticum aesetrum)与叶锈菌(Puccinia triticina)互作过程中, 利用RT-qPCR技术, 检测这9个基因的表达情况。经geNorm和NormFinder软件分析可知, 在小麦接种叶锈菌后不同时间点, GAPDH和TA基因的表达稳定性较高, 可以作为RT-qPCR的内参基因。在此基础上, 利用筛选得到的最适内参GAPDH对小麦与叶锈菌互作过程中, TaSGR基因的相对表达量做了定量分析。结果显示, 在亲和组合接种后中后期, TaSGR基因的相对表达量迅速增加, 达到了对照的近7倍水平, 而在不亲和组合接种后不同时间点, TaSGR基因的表达量与对照0 h相比差异均不明显。

关键词：小麦; 叶锈菌; RT-qPCR; 内参基因; geNorm; NormFinder

收稿：2015-02-02   修定：2015-03-24

资助：国家自然科学基金(31171472)、高等学校博士学科点专项科研基金(优先发展领域) (20111302130001)、河北省科技厅河北省应用基础研究计划重点基础研究项目(12967149D)和河北省人事厅留学人员科技活动项目(20120316) * 共同第一作者。

Reference Gene Selection for RT-qPCR and Expression Analysis of TaSGR Gene in Defense Response of Wheat to Puccinia triticina

LI Hui^1,*, NIU Hong-Wei^2,*, LIU Na¹, CHEN Yan¹, HOU Chun-Yan^1,∗∗, WANG Dong-Mei^1,∗∗

¹Plant Stress Laboratory, College of Life Sciences, Agricultural University of Hebei, Baoding, Hebei 071001, China; ²Hebei Provincial Institute of Environmental Protection Products Quality Supervision and Inspection, Shijiazhuang 050000, China

Corresponding author: LI Hui; E-mail: dongmeiwang63@126.com, houchunyan@126.com; Tel: 0312-7528276; 0312-7528249

Abstract:

Real time quantitative PCR (RT-qPCR) has many potential applications in the determination of gene function. The quality of reference gene plays an essential role in analyzing gene expression. In this study, we took nine genes widely used in wheat (Triticum aesetrum) as candidate reference genes and evaluated the expression stability of the nine candidate genes, in the interaction between wheat and Puccinia triticina, by using RT-qPCR. With the help of geNorm and NormFinder, we analyzed the data of RT-qPCR and got the expression stability of the nine candidate genes. Our results showed that GAPDH and TA were most stable two genes which can be reliably used in the stress conditions assessed. Based on this, we took GAPDH as the reference gene and analyzed gene expression profiles of TaSGR in the interaction between wheat and Puccinia triticina. Our data showed that the relative expression of TaSGR had a rapid increase in the compatible combinations at middle and later time after inoculation, nearly seven times than the level of control. While in the incompatible combinations, the relative expression of TaSGR did not change obviously.

Key words: wheat; Puccinia triticina; RT-qPCR; reference gene; geNorm; NormFinder