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| Using REAL-TIME PCR to Determine Transgene Copy Number in Wheat |
| Research Institute of Biotechnology, Gansu Academy of Agricultural Science, Lanzhou 730070, China |
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Abstract SYBR GreenⅠ real-time PCR was developed to determine the copy number of exogenous pta gene in transgenic wheat. A conserved wheat housekeeping gene, wx012, was used as an internal control and 5 times diluted non-transgenic wheat genome DNA is used as an initial template copy of wx012.So the y=-0.2667x+6.98, the standard curves of the cycle threshold (CT ) relative to the log of each initial template copy of wx012 was obtained. In the same way, the standard curves of pta y=-0.2118x+4.53 is obtained using 5 times diluted plasmid DNA which contains pta gene. By SYBR GreenⅠ real-time PCR the CT values of wx012 and pta gene in each transgenic wheat was got, the transgene copy number is calculated by comparing the initial template copy of pta to wx012 gene in the same transgenic wheat. The conclusion is, among the seven PCR putative transgenic plants: one was non-transgenic plant，one had one copy number，one had two copies，and the other had three，four copies，respectively.
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Received: 01 December 2009
Published: 25 March 2010
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