Screening And Identification Of Endogenous Reference Gene In Maize For GMPs Detection

Endogenous reference gene (ERG) is an essential reference in Genetically Modified Plants (GMPs) identification system. In this system, PCR were applied using genomic DNA samples, through the comparison of the results for transferred gene detection and reference detection, judgments could be made on whether the sample is qualified and the PCR system works correctly. Moreover, quantitative analysis could be applied. Currently, the exploitation of ERGs for some commercialized genetically modified plants (GMPs) such as soybean and tomato has been done. However, the development of ERG in maize, is just entering its first stage.This study first took for candidate genes compared by BLAST and BLINK sequences. Then filter endogenous reference gene from intraspecific consistency and stability, species specificity, stability of copy number and other aspects. After non-corn species and different varieties of maize species-specific qualitative and quantitative PCR detectton, copy number analysis, the main results obtained are as follows:1. Using PGID, NCBI public database information, and BLINK, BLAST sequence alignment,20 specific non-intron genes as candidate genes for maize endogenous reference were identified.2. Highly conserved primers designed using each species genomic DNA as template. Screening the exon of 20 candidate genes, the minimum fragment length is restricted as 200bp. Through retrieving these genes information one by one, designed primers, PCR validation, GRMZM2G054655 was eventually chosen as the beast endogenous reference gene in maize for GMPs detection.3. Specific PCR primers were designed based on GRMZM2G054655 sequences. By ordinary PCR experiments, we verify that GRMZM2G054655 has a high stability of intraspecific consistency and species specificity.4. By optimizing the conditions for the quantitative detection, we establish a stable and efficient, reproducible quantitative detection systems for GRMZM2G054655, quantitative detection sensitivity of 0.04ng.5. Using a single-copy gene GADPH as control, quantitative PCR reaction proved GRMZM2G054655 is a constant single-copy gene in different maize varieties. GRMZM2G054655 can be used as the reference genes for detection of genetically modified maize. Establish a system of screening and detection of endogenous reference gene in maize for GMPs detection.