An Endogenous Reference Gene of Common and Durum Wheat for Detection of Genetically Modified Wheat

摘要

To develop a method for detecting GM wheat that may be marketed in the near future, we eval-uated the proline-rich protein (PRP) gene as an endogenous reference gene of common wheat (Triticum aestivum L.) and durum wheat (Triticum durum L.). Real-time PCR analysis showed that only DNA of wheat was amplified and no amplification product was observed for phylogenetically related cereals, indicating that the PRP detection system is specific to wheat. The intensities of the amplifi-cation products and Ct values among all wheat samples used in this study were very similar, with no nonspecific or additional amplification, indicating that the PRP detection system has high se-quence stability. The limit of detection was estimated at 5 haploid genome copies. The PRP region was demonstrated to be present as a single or double copy in the common wheat haploid genome. Furthermore, the PRP detection system showed a highly linear relationship between Ct values and the amount of plasmid DNA, indicating that an appropriate calibration curve could be constructed for quantitative detection of GM wheat. All these results indicate that the PRP gene is a suitable endogenous reference gene for PCR-based detection of GM wheat.%遺伝子組換えコムギ検知法への適用を目指し，コムギの内在性遺伝子候補としてプロリンリッチプロテイン（PRP）遺伝子の評価を行った．リアルタイムPCRにおいて．夕月P遺伝子は普通コムギとデュラムコムギでのみ増幅が得られ，他の10作物では増幅産物が得られなかった．コムギの銘柄?品種間の比較では，増幅産物の大きさ，強度．Ct億がほぼ同等で，非特異的な増幅産物も見られなかったことから，コムギ間に反応性の差はないと考えられた．PRP遺伝子のコピー数は，1ハブロイドあたり1もしくは2コピーであり，検出限界は5ハブロイドゲノムコピーであった．以上，PRP遺伝子はPCRを利用した遺伝子組換えコムギ検知に有用な内在性遺伝子として利用可能と判断された．