Genome-wide Screening Of Endogenous Reference Gene In Bos Taurus And Its Applications In National Standard And Identification Of Meat Products

Endogenous reference gene (ERG) is an essential reference in Genetically-Modified Organisms (GMOs) identification system. It is a kind of species-specified gene with stable Copy Number (CN), as well as non-allelic variation and conservative DNA sequences. The trait of species-specify contain two parts:interspecies specificity and intraspecies consistency. Interspecies specificity refers to the low homolog at DNA levels which could be detected using Polymerase Chain Reaction (PCR) distinctively, while intraspecies consistency refers to the high conservation of gene structures and sequences within target species which could be detect in different breeds entirely. Stable CN provides the ability of quantitative analysis when used against target genes. In this identification system, PCR were applied using genomic DNA samples, through the comparison of the results for transferred gene detection and reference detection (ERG, Positive, Negative and Blank), 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 all commercialized genetically-modified Plants (GMPs) has been done and multi-ERGs exist in some crops such as maize and soybean. However, the development of ERG in animals, cows in this study, is just entering its first stage due to the lacking of applied genetically modified animals. In this study genome wide selection of ERGs for cows (Bos taurus) and its system construction, together with its application in beef products identification, were done.After the comparison of methods in ERG selection within crops, genomic data of sequencing and annotation material and Basic local alignment search tool (BLAST) were used as materials and methods respectively. Then validation of candidate ERGs was employed using several breeds of cows and buffalo, goat, sheep, pig, mouse, etc. as target and references. Finally we successfully constructed the national standardized detection system of ERG.Our research results were as follows:1. Protein/nucleotide sequence query information were gathered from Ensembl gene annotation and UMD3.1genomic sequencing data, BLASTp/BLASTn of the whole gene set in cows were run using NCBI nr/nt as subject.138/69cow protein-specified/nucleotide-specified gene were detected, among those are48protein&nucleotide specified genes. Information gathered via this BLAST could provide foundations for further functional study.2. An Exon targeted BLASTn was carried out in order to create a better list for candidate ERGs of length larger than200bp. After removal of multi-CN and sex-chromosome genes, a list of9candidate ERGs was generated.3. Validation of the9candidate ERGs were completed by design specific primers after characterizations of gene sequence/structure. Tumor Necrosis Factor Receptor Superfamily, member10a gene, TNFRSF10A was selected to be the ERG in this study. Sequence of TNFRSF10A was obtained via sequencing.4. Optimizations for primer set and reaction condition were done for ERG TNFRSF10A. Specification and sensitivity tests were also finished, ultimately the National standard of "Detection of genetically modified animals and derived products: Target-taxon-specific qualitative PCR method for Bos taurus" was established.5. In the understanding of the TNFRSF10A sequence specification, patent of identification methods in beef products was applied.