Wheat-Specific Gene, Ribosomal Protein L21, Used as the Endogenous Reference Gene for Qualitative and Real-Time Quantitative Polymerase Chain Reaction Detection of Transgenes

• Published in: Journal of agricultural and food chemistry Vol. 62; no. 43; pp. 10405 - 10413
• Main Authors: Liu, Yi-Ke, Li, He-Ping, Huang, Tao, Cheng, Wei, Gao, Chun-Sheng, Zuo, Dong-Yun, Zhao, Zheng-Xi, Liao, Yu-Cai
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 29.10.2014; American Chemical Society, Books and Journals Division
• Subjects: Aegilops speltoides; Aegilops tauschii; Arabidopsis; detection limit; diploidy; Gene Dosage; haploidy; Plant Proteins - genetics; Plants, Genetically Modified - genetics; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Reference Standards; Ribosomal Proteins; sequence analysis; Species Specificity; Transgenes; transgenic plants; Triticum - genetics; Triticum urartu; wheat; ribosomal protein L21; genetically modified organisms; endogenous reference gene; real-time PCR; Triticum aestivum
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf503559b

Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5–4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.