Validation of Artemisia Genus-Specific Gene, GEL4, for Use as an Endogenous Reference Gene in Qualitative PCR Detection of Putative Transgenic Artemisia annua

Artemisia annua L. is a popular medical plant used against malaria. Transgenic A. annua plants have been developed to increase the artemisinin content using genetic engineering. To develop a reliable PCR method of detecting transgenic A. annua, one Artemisia specific gene, GEL4, was selected and validated as suitable. Qualitative PCR methods were assayed with 16 different A. annua ecotypes and four Artemisia plants, and identical amplified products were obtained. No amplified products were observed when genomic DNA samples were extracted from 15 different plant species. An LOD of 0.05 ng of A. annua DNA was also determined. Furthermore, the results of a Southern blot method showed that the GEL4 gene was in three copies of the six A. annua ecotypes tested. To determine whether there was intraspecific variability in the GEL4 region between different A. annua ecotypes, the GEL4 region corresponding to the 10 A. annua ecotypes was cloned and sequenced. Alignment of the obtained sequences presented a highly conserved amplification product region in 10 A. annua ecotypes. Therefore, it is concluded that the GEL4 gene can be used as an endogenous reference gene of A. annua, and that the qualitative PCR system was reliable for the detection of transgenic A. annua.