The Development Of The Matrix Reference Material Of MON89788GM Soybean And Evaluao N The Applicability Of Four Wheat Endogenous Reference Genes

To ensure the implement of genetically modified organisms (GMO) labeling regulation in the world and perfect the detection methods system for GMOs, we developed the matrix reference materias for the detection of the MON89788transgenic soybean. In addtion, we analyzed the applicability of four wheat reference genes for GM wheat detection.The development of matrix reference materials of MON89788GM soybean. The matrix reference materials (MRMs) for the detection of transgenic soybean MON89788were developed by mixing seed powder of non-transgenic soybean A3244and homozygous MON89788. The MON89788and A3244seeds were finely ground under the optimized conditions. Water content, particle size distribution, and DNA extraction efficiency of MON89788powder were proved to be close to that of the control line A3244. The MRMs with5%,1%, and0.5%mass fractions of MON89788were prepared by mixing the seed powders of MON89788andA3244, respectively. Particle size distribution of the MON89788MRMs was ranging from180μm to200μm. The relative humidity of the MON89788MRMs was2.96%～3.03%. The homogeneity of MON89788MRMs was tested using quantitaivc PCR. and the result showed that the MON89788MRMs were homogeneous.The applicability of four wheat endogenous reference genes. The quantitative assay for genetically modified (GM) food/feed quantification depends on the reliable endogenous reference genes. Four previously reported Triticum aestivum.L endogenous reference genes, including acetyl-CoA carboxlyase (acc1)、ABA kinase(PKABA)、ALMT、 waxy-D1has been analyzed for their species specificity in Triticum aestivum.L and other species. In this study, we analyzed nucleotide sequence variation in the target genomic regions in43conventional wheat varieties from different geographic and phylogenic origins, also quantitative real-time PCR and GeNorm software evaluated their quantification performance via calculate a series of Ct value to get "Q value ". The sequencing analysis results showed that the reported ALMT and PKABA taqman probe regions had detectable single nucleotide polymorphisms (SNPs) among the tested wheat cultivars, and the forward primer of ALMT and accl had detectable single nucleotide polymorphisms (SNPs), while no SNPs were observed for waxy-D1amplicons. Using the new primers and probe which were designed according the sequencing result, the amplification efficiency, R2value and slope are perfect good in these cultivars with SNPs using ALMT、PKABA and aceluantitative PCR systems. We think waxy-Dl gene is more suitable for GM wheat quantitative detection than other wheat reference genes.