qRT-PCR-生物分析检测技术.pptVIP

Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR.Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D.School of life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, Peoples Republic of China. Plant Cell Rep. 2005 Mar;23(10-11):759-63. Epub 2004 Oct 1. 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. Report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. Use 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 beta-glucuronidase (GUS) and hygromycin phosphotransferase (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, achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. 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. An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana.Jang JY, Kim DG, Kim YO, Kim JS, Kang H.Division of Applied Plant Science and Agricultural Plant Stress Research C