Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (4): 811-818.DOI: 10.1016/S2095-3119(13)60515-8

• 论文 • 上一篇    下一篇

Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

 HE Xiu-ting, LIU Cheng-cheng, LI Zhao-qun, ZHANG Zan, LI Guo-qing, LI Fei , DONG Shuang-   

  1. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education/College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, P.R.China
  • 收稿日期:2013-02-27 出版日期:2014-04-01 发布日期:2014-04-16
  • 通讯作者: DONG Shuang-lin, Tel: +86-25-84399062, E-mail: sldong@njau.edu.cn; LI Fei, Tel: +86-25-84399025, E-mail: lifei@njau.edu.cn
  • 基金资助:

    This research is supported by the National 973 Program of China (2010CB126200), the Genetically Modified Organism Breeding Project, Ministry of Agriculture, China (2009ZX08001-002B).

Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

 HE Xiu-ting, LIU Cheng-cheng, LI Zhao-qun, ZHANG Zan, LI Guo-qing, LI Fei , DONG Shuang-   

  1. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education/College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, P.R.China
  • Received:2013-02-27 Online:2014-04-01 Published:2014-04-16
  • Contact: DONG Shuang-lin, Tel: +86-25-84399062, E-mail: sldong@njau.edu.cn; LI Fei, Tel: +86-25-84399025, E-mail: lifei@njau.edu.cn
  • Supported by:

    This research is supported by the National 973 Program of China (2010CB126200), the Genetically Modified Organism Breeding Project, Ministry of Agriculture, China (2009ZX08001-002B).

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摘要： The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were further confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

关键词: cDNA cloning , housekeeping gene , qPCR , reference gene , small brown planthopper

Abstract: The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were further confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

Key words: cDNA cloning , housekeeping gene , qPCR , reference gene , small brown planthopper