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

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• 作者：Yi-Ke Liu ; He-Ping Li ; Tao Huang ; Wei Cheng ; Chun-Sheng Gao ; Dong-Yun Zuo ; Zheng-Xi Zhao ; Yu-Cai Liao
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2014
• 出版时间：October 29, 2014
• 年：2014
• 卷：62
• 期：43
• 页码：10405-10413
• 全文大小：516K
• ISSN：1520-5118

文摘

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鈥?-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.

Keywords:

Triticum aestivum; ribosomal protein L21; endogenous reference gene; genetically modified organisms; real-time PCR