Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in Lesquerella fendleri | Science Publications

American Journal of Agricultural and Biological Sciences

Grace Q. Chen¹  Jiann-Tsyh Lin¹ 

关键词: Lesquerella fendleri;    quantitative PCR;    polymerase chain reaction;    transgenes;   

DOI  :  10.3844/ajabssp.2010.415.421

学科分类：农业科学（综合）

来源: Science Publications

【 摘 要 】

Problem statement: In transgenic plants, the number of transgene copies could greatlyinfluence the level of expression and genetic stability of the target gene, thus it is important to developan efficient method for accurate estimation of transgene copies. The quantitative Polymerase ChainReaction (qPCR) technique is becoming more efficient nowadays to determine copy numbers oftransgenes in transgenic plants, being used here, for the first time in quantifying copy numbers oftransgenes in Lesquerella fendleri. Approach: The system utilized a known one copy gene, LfKCS4/5,from L. fendleri as an endogenous calibrator and the threshold Crossing point (Ct) measured byApplied Biosystem 7500 system to calculate the copy numbers of transgenes in primary transgeniclines (T0 generation). Results: The qPCR condition was optimized and each primer set had a PCRefficiency of 0.99 or 1.01. Our data demonstrated unambiguous 2-fold discrimination of the copynumber of β-glucuronidase gene (gusA) and hygromycine phosphotransferase II (hptII) genes in 12 T0lines. Most of the lines contained one or two copies of each gene. Eight out of 12 samples (66.7%)showed more copies of gusA gene than that of hptII gene, suggesting rearrangements of theTransferred (T)-DNA. Possible modifications of the T-DNA cassette in L. fendleri are discussed basedon main models of T-DNA integration in the plant genome. Conclusion: The qPCR described in thisstudy is an efficient method and it is particularly useful in identification and selection of transgenicplants with desirable copy numbers at early stage.

【 授权许可】

Unknown