Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous β-glucuronidase (GUS) and hygromycin phosphortransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.

[1]  Paolo Provero,et al.  Estimating the number of integrations in transformed plants by quantitative real-time PCR , 2002, BMC biotechnology.

[2]  P. Mourrain,et al.  Transgene-induced gene silencing in plants. , 1998, The Plant journal : for cell and molecular biology.

[3]  D J Ingham,et al.  Quantitative real-time PCR assay for determining transgene copy number in transformed plants. , 2001, BioTechniques.

[4]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[5]  Karhu,et al.  Gene Copy Number Analysis by Fluorescence in Situ Hybridization and Comparative Genomic Hybridization , 1996, Methods.

[6]  P. Song,et al.  Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS™-derived transgenic maize , 2002, Plant Cell Reports.

[7]  Clive James,et al.  Global status of commercialized transgenic crops : 1999 , 1999 .

[8]  F. Speleman,et al.  Quantification of MYCN, DDX1, and NAG Gene Copy Number in Neuroblastoma Using a Real-Time Quantitative PCR Assay , 2002, Modern Pathology.

[9]  A. Protopopov,et al.  NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Esther J Kok,et al.  Comparative safety assessment for biotech crops. , 2003, Trends in biotechnology.

[11]  Dabing Zhang,et al.  Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. , 2004, Journal of agricultural and food chemistry.

[12]  M. Wigler,et al.  Detecting gene copy number fluctuations in tumor cells by microarray analysis of genomic representations. , 2000, Genome research.

[13]  Jianqun Chen,et al.  A novel real-time quantitative PCR method using attached universal template probe. , 2003, Nucleic acids research.

[14]  Farid E Ahmed,et al.  Detection of genetically modified organisms in foods. , 2002, Trends in biotechnology.

[15]  E. Hollox,et al.  DNA copy number analysis by MAPH: molecular diagnostic applications , 2002, Expert review of molecular diagnostics.

[16]  S. Knuutila,et al.  Comparative genomic hybridization reveals differences in DNA copy number changes between sporadic gastric carcinomas and gastric carcinomas from patients with hereditary nonpolyposis colorectal cancer. , 1998, Cancer genetics and cytogenetics.

[17]  R. Flavell.  Inactivation of gene expression in plants as a consequence of specific sequence duplication. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[18]  C. Sismani,et al.  Measurement of locus copy number by hybridisation with amplifiable probes. , 2000, Nucleic acids research.