台灣市售飼料玉米抗固殺草基因特性及檢測利用之探討

Characterization and detection of the glufosinate-resistant gene in corn products in Taiwan

收集一般雜糧店販售之飼料玉米，測定其種子發芽及幼株對固殺草之反應，並由抗性植株葉片中分離核酸，探討抗藥基因之特性。本研究以市售玉米於溫室內播種，於植株幼苗期噴施固殺草0.9 kg/ha，施藥後3-4日，多數玉米植株呈現傷害徵狀，並於2週內枯死，12組測定樣品中均有抗藥性之植株，其所佔比率介於1-14.7%之間，應為抗固殺草之轉基因玉米。再以固殺草次致死劑量0.4 kg/ha處理植株，區別感抗植株後，進一步抽取玉米幼葉之genomic DNA，由CaMV 35S promoter、terminator及NOS terminator DNA序列設計引子，進行聚合酵素鏈鎖反應，結果具抗藥之疑似轉基因玉米可分別擴增約1.0、1.2及1.35 kb的DNA片段，非抗藥植株則無此等DNA片段。PCR產物經接合、轉型反應及定序，比對核酸及胺基酸序列，顯示此3組PCR產物由35S promoter、IVS2、pat基因、bar基因、35S terminator或NOS terminator DNA等片段組成，顯示此3種玉米為似抗固殺草之T25、Event176及Bt11品系。經由專一性引子之設計及multiplex PCR分析結果，可分別檢出6、5及14株抗固殺草玉米為似T25、Event 176及Bt11品系。市售之玉米粒、玉米仁、玉米片及玉米餅乾等食品以multiplex PCR分析，可檢出含抗固殺草基因之玉米物料。

"Five million tons of corn are imported annually into Taiwan. Most seeds sampled from local shops were found to be viable. We established seedlings in a greenhouse and applied 0.9 kg/ha of glufosinate to the foliage of these plants. Sensitive seedlings showed symptoms of injury in 3~4 days and died within 2 weeks. Glufosinate-resistant seedlings accounted for 1 %~14.7 % of the original seed samples. We used plants that survived glufosinate treatment for PCR detection and genomic characterization. PCR assay of genomic DNA extracted from corn leaves, using the CaMV 35S promoter, terminator, and NOS terminator as primers, produced a 1.0-, 1.2- and 1.35-kb fragments. DNA sequencing using PCR products showed that the fragments consisted of the 35S promoter, IVS2, bar, pat, the 35S terminator and/or the NOS terminator. We designed specific primer sets for the DNA sequence of glufosinate-resistant corn; PCR products of 159, 211, 225, 253, 285, and 329 bp, respectively, were produced using these specific primers. A primer producing low molecular DNA fragments were suitable for detecting of implanted genes from processed products, such as field corn, corn kernels, corn fries, and flakes."