臺灣市售飼料玉米抗嘉磷塞基因特性及檢測利用之探討

Characterization and Detection of the Glyphosate-resistant Gene in Corn Products in Taiwan

袁秋英(Chiou-Ing Yuan) ； 謝玉貞(Yu-Chen Hsieh) ； 林李昌(Li-Chang Lin) ； 蔣慕琰(Mou-Yen Chiang)

《作物、環境與生物資訊》 3卷1期 (2006/03) Pp. 20-32

https://doi.org/10.30061/CEB.200603.0003

摘要

於2005年1月至2月期間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嘉磷塞之反應，並由抗性植株葉片中分離核酸，探討抗藥基因之特性。將市售玉米於溫室內播種，於4葉齡幼苗期噴施嘉磷塞（41%溶液）1.8 kg ha^(-1)，施藥後5~7日，多數玉米植株呈現傷害徵狀，並於2週內枯死，14組測定樣品中均有抗藥性之植株，其所佔比率介於0.9~3.7%之間，應為抗嘉磷塞之轉基因玉米。再以嘉磷塞次致死劑量0.5kg ha^(-1)處理植株，區別感抗植株後，進一步抽取玉米幼葉之genomic DNA，由花椰菜嵌紋病毒啟動子(CaMV 35S promoter)、水稻actin 基因啟動子及nopaline synthase基因之終結子(NOS terminator)核酸序列設計兩組引子，進行聚合酶鏈鎖反應，結果具抗藥之疑似轉基因玉米可分別擴增約2.0及2.6kb片段或2.1kb之單一片段，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2.0 PCR產物含水稻actin promoter、阿拉伯芥ribulose bisphosphate carboxylase/oxygenase small subunit (RuBisCo SSU)之chloroplast transit peptide (CTP)、CP4 EPSPS及NOS terminator片段。2.6 kb之基因構築為35S promoter、玉米熱休克蛋白(SHP70)基因、CP4 EPSPS及NOS terminator片段。2.1 kb片段則由水稻actin promoter、向日葵RuBisCo SSU之CTP、玉米RuBisCo small subunit之CTP、修飾之玉米抗嘉磷塞EPSPS及NOS terminator等片段組成。顯示此2種玉米為似抗嘉磷塞之NK603及GA21品系。經由專一性引子之設計及multiplex PCR分析結果，可分別檢出4及11株抗嘉磷塞玉米為似NK603及GA21品系。市售之玉米粉、玉米仁、爆米花及玉米罐頭等食品以multiplex PCR分析，可檢出含抗嘉磷塞基因之玉米物料。

關鍵字

基因改造生物體 ； 玉米 ； 嘉磷塞抗藥性 ； CP4 EPSPS基因 ； 修飾之玉米抗嘉磷塞EPSPS基因 ； 複合－聚合酶鏈鎖反應

並列摘要

We germinated corn seeds collected from retail shops in central Taiwan and tested seedling tolerance against glyphosate. Of all 14 samples tested, seedlings survived herbicide treatment (1.8kg ha^(-1) foliar spray) ranged from 0.9-3.7%. These indicated that a small portion of marked feed corn was genetically modified for glyphosate tolerance. We extracted genomic DNA from leaves of seedlings survived low rate of glyphosate spray (0.5kg ha^(-1)), and conducted PCR with two sets of primers (CaMV 35S promoter/NOS terminator, rice actin promoter/NOS terminator). PCR amplified fragments were 2.0 and 2.1 kb for promoter using rice actin and 2.6 kb using 35S. Nucleic acid sequences of these fragments were analyzed after conjugation and transformation. Comparison on nucleic and amino acid (NCBI, blast analysis) suggested that components of PCR fragments were: 1) actin promoter, chloroplast transit peptide (CTP) of RuBisCo small subunit from Arabidopsis thaliana, CP4 EPSPS and NOS terminator for 2.0 kb fragment; 2) 35S promoter, heat shock protein from maize (SHP70), CP4 EPSPS and NOS terminator for 2.6 kb fragment; 3) Rice actin promoter, CTP of RuBisCo small subunit from Helianthus annuus, CTP of RuBisCo small subunit from corn, modified EPSPS from corn and NOS terminator for 2.1 kb product. These results also suggested that genetically modified corn lines NK603 and GA21 were in our samples. Multiplex PCR using sequence based primers were conducted on seedlings and corn products. Analysis of 15 glyphosate tolerant seedlings showed that 4 and 11 were NK603 and GA21, respectively. Gene fragments, glyphosateresistant related, were detected in 11 of 17 corn products that sampled in local market. Positive identified products included flour, kernel (fresh, canned), popcorn and corn flake.

並列關鍵字

GMO ； Corn ； Glyphosate resistance ； CP4 EPSPS gene ； Modified EPSPS of maize ； Multiplex-PCR