Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants. Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants. To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A. rhizogenes strain 1724. By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids. As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes. In addition, a virulence-related gene, tzs was located independently of the vir region.
Background
To provide the consumer with choices of GMO or non-GMO, official food labeling systems were established in many countries. Because the threshold GMO content values were set to distinguish between “non-GMO” and “GMO” designations, GMO content quantification method are required for ensuring the appropriateness of labeling.
Objective
As the number of GMOs is continuously increasing around the world, we set out to develop a low-cost, simple and less-biased analytical strategy to cover all necessary detection targets.
Methods
Digital PCR methods are advantageous compared to the conventional quantitative real-time PCR methods. We developed a digital PCR-based GMO quantification method to evaluate the GMO content in maize grains. To minimize the analytical workload, we adopted multiplex digital PCR targeting 35S promoter and NOS terminator, which are genetic elements commonly introduced in many GMOs.
Results
Our method is significantly simpler and more precise than the conventional real-time PCR-based methods. Additionally, we found that this method enables to quantify the copy number of GM DNA without double counting multiple elements (P35S and TNOS) tandemly placed in a recombinant DNA construct.
Conclusion
This is the first report on the development of a GM maize quantification method using the multiplexed genetic element-specific digital PCR method. The tandem effect we report here is quite useful for reducing the bias in the analytical results.
Highlights
Multiplexed genetic element-specific digital PCR can simplify weight-based GMO quantification and thus should prove useful in light of the continuous increase in the numbers of GM events.
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