Estimation of the homoplasmy degree for transplastomic tobacco using quantitative real-time PCR
Estimation of the homoplasmy of transgene in chloroplast is a necessary step in chloroplast transformation. This task is usually achieved by Southern blot analysis, which is laborious, time-consuming, requires large amounts of plant materials and needs hazardous radioisotopes in some cases. To develop a fast, sensitive, stable and effective technique for determining the homoplasmy of transgene in chloroplast, one real-time PCR system based on TaqMan probe technique was developed for evaluating the homoplasmy degree. In the real-time PCR system, one assay targets the exogenous faeG gene, and another one the tobacco chloroplast reference gene, maturase gene within the trnK intron (matK). The homoplasmy of the transgene was determined by the comparison of copy number of faeG and matK. The analyzed results of 17 transplastomic tobacco lines using the developed real-time PCR system were consistent with those from Southern blot analysis, indicating that the real-time PCR method is suitable for estimating the homoplasmy degree of transplastomic plant with the advantages of high efficiency and throughput, low cost and saving time.
KeywordsTransplastomic tobacco Real-time quantitative PCR Homoplasmy degree
- 5.Zhang ZL, Ren YG, Shen YX, Shan S, Fan GC, Wu XF, Qian KX, Shen GF (2000) Expression of Bacillus thuringiensis (Bt) crystal toxin gene in the chloroplast of tobacco. Yi Chuan Xue Bao 27:270–277Google Scholar
- 6.Zhang ZL, Chen X, Qian KX, Shen GF (1999) Studies on inset resistance of Bt transplastomic plants and the phenotype of their progenies. Acta Bot Sin 41:947–951Google Scholar
- 18.Soria-Guerra RE, Alpuche-Solis AG, Rosales-Mendoza S, Moreno-Fierros L, Bendik EM, Martinez-Gonzalez L, Korban SS (2009) Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer. Planta 229:1293–1302CrossRefGoogle Scholar
- 29.Selvaraj D, Sarma RK, Sathishkumar R (2008) Phylogenetic analysis of chloroplast matK gene from Zingiberaceae for plant DNA barcoding. Bioinformation 3:24–27Google Scholar