Novel Reference Gene, High-mobility-group protein I/Y, Used in Qualitative and Real-Time Quantitative Polymerase Chain Reaction Detection of Transgenic Rapeseed Cultivars

Abstract

With the development of transgenic crops, regulations to label the genetically modified organisms (GMOs) and their derived products have been issued in many countries. Polymerase chain reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods are generally needed to amplify the transgene and compare the amplified results with that of a corresponding reference gene to get the reliable results. Specific primers were developed for the rapeseed (Brassica napus), High-mobility-group protein I/Y (HMG-I/Y) single-copy gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 15 different rapeseed varieties, and identical amplified products were obtained with all of them. No amplification was observed when templates were the DNA samples from the other species of Brassica genus or other species, such as broccoli, stem mustard, cauliflower, Chinese cabbage, cabbage, sprouts, Arabidopsis thaliana, carrot, tobacco, soybean, mung bean, tomato, pepper, eggplant, plum, wheat, maize, barley, rice, lupine, and sunflower. This system was specific for rapeseed. Limits of detection and quantitation in qualitative and quantitative PCR systems were about 13 pg DNA (about 10 haploid genomes) and about 1.3 pg DNA (about 1 haploid genome), respectively. To further test the feasibility of this HMG-I/Y gene as an endogenous reference gene, samples containing transgenic rapeseed GT73 with the inserted glyphosate oxidoreductase (GOX) gene were quantitated. These demonstrated that the endogenous PCR detection systems were applicable to the qualitative and quantitative detection of transgenic rapeseed.

The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis

“…Primers ( BnGRF2a RTf, BnGRF2a RTr, BnGRF2b RTf, and BnGRF2b RTr) were designed to detect expression of BnGRF2a and BnGRF2b in rapeseed. Rapeseed β-actin2 and β-actin3 ( Gao et al , 2004 ; Weng et al , 2005 ) served as endogenous reference genes and were amplified using the primers Bnactin2-F, Bnactin2-R, and Bnactin3F, Bnactin3R. Primers for BnGRF2a ( BnGRF2a RTf and BnGRF2a RTr) and AtGRF2 ( AtGRF2 RTf and AtGRF2 RTr) were used to detect BnGRF2a expression levels in transgenic Arabidopsis .…”

The Development and Standardization of Testing Methods for Genetically Modified Organisms and their Derived ProductsF

“…The Standardization Committee for Agricultural GMOs is engaged in large numbers of research projects and the development of GM molecular characterization, analytical methods, and CRM etc. Several endogenous reference genes including rice SPS , tamato Lat52 , (Ding et al 2004; Weng et al 2005; Yang et al 2005a, 2005b; Guo et al 2009a) suitable for GMO detection and event‐specific qualitative and quantitative PCR detection methods were validated (http://gmdd.shgmo.org/), and about 40 technical standards were developed (http://www.stee.agri.gov.cn/biosafety/). Several international collaborative validations for GM detection methods were also organized for the international harmonization of the detection.…”

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events

“…On the other hand, the number of copies for CruA and Ccf varied from 11,800 to 13,500 for 10 ng DNA. HMG-I/Y and CruA have been reported to be single and two copy genes, respectively [ 5 ]. A drawback reported for HMG-I/Y was that it may not be stable to be used as a reference gene for canola [ 4 ].…”

“…Two different sequences were reported for the cloned fragments of HMG-I/Y, PEP and CruA , indicating the presence of genes in two copies [ 4 ]. On the other hand, HMG-I/Y was reported to be a single copy reference gene that can be used for quantification of GE canola events by real-time PCR [ 5 ]. It was also reported that the five endogenous reference genes mentioned above were not suitable for real-time PCR quantification of GE canola events as they were not specific between different species and also not stable among cultivars [ 4 ].…”