嘉磷塞(Glyphosate)為目前全球使用最普遍的除草劑，其主要作用為抑制 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS)的活性。近年台灣中部農田已出現抗嘉磷塞的美洲假蓬bonariensis L. Cron )生物型。本研究利用對嘉磷塞耐受性不同的美洲假蓬EPSPS-1及EPSPS-2基因，二者開放讀碼區（open reading frame, ORF)為1,341及1,338 bp，核酸序列的相同度(identity)為85%，編碼的EPSPS 有36個胺基酸的差異。以對藥劑較敏感之EPSPS-1為模板，運用點突變技術 (site-directed mutagenesis, SDM)置換特定胺基酸，由嘉磷塞之耐受性、酵素活性及動力學分析，探討影響美洲假蓬EPSPS與嘉磷塞鍵結的重要胺基酸與其特性。初步經由15個胺基酸突變之篩選，僅T42M、G104A、T105I及P109L 4個EPSPS-1 修飾的大腸桿菌轉殖株，可於80 mM嘉磷塞溶液中維持約80%的活性，其中以突變EPSPS-1 (G104A及P109L)的轉殖株耐受性較強。將104G置換為Pro、Leu、 Asp、Ser、Phe或Lys，含此6種突變胺基酸的大腸桿菌轉殖株，於40 mM嘉磷塞的菌株生長皆明顯受抑制；而將108P置換為Cys、Tyr、His、Asp或Gly，含此5 種突變胺基酸的大腸桿菌轉殖株，於100 mM嘉磷塞的菌株仍具有高度抗性，顯示 第104個胺基酸可能為EPSPS酵素活性表現的重要位置，不可任意更換；而第109個胺基酸為影響與嘉磷塞鍵結的重要位置。經由活性及動力學分析，G104A及 P109L單點突變的EPSPS-1對嘉磷塞的抗性，皆較原EPSPS-1模板明顯增加， G104A及P109L單點突變的EPSPS-1的ki值分別為EPSPS-1模板的33.3及14.7 倍。後續可利用點突變技術，於美洲假蓬第104及109個胺基酸的相關位置，修飾作物自身的EPSPS為抗性基因，進而發展為抗嘉磷塞作物。

Glyphosate is a non-selective broad spectrum herbicide that inhibits 5-enolpyruvyl-shikimate- 3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. Glyphosate-resistant and glyphosate-susceptible hairy fleabane (Conyza bonariensis L. Cron ) were obtained from orchards in Taiwan in 2008. There are two EPSPS cDNAs of both resistant and susceptible hairy fleabane with open reading frame for EPSPS-1 and EPSPS-2 is 1,341 and 1,338 bp, respectively. Sequence data revealed 85% identity and 36 amino acids differences between EPSPS-1 and EPSPS-2. In this study, we used the site directed mutagenesis (SDM) to induce point mutations in the hairy fleabane EPSPS-1 gene and to analyze the glyphosate tolerance in 15 transformed Escherichia coli EPSPS-1 mutants. Only T42M、G104A、T105I and P109L mutants with high tolerance could survive in 80 mM glyphosate. Replace the 104Gly with Pro、Leu、Asp、Ser、Phe or Lys, and the 109Pro with Cys、Tyr、His、Asp or Gly and analysis of these mutants indicated that all the 104Gly mutations resulted in glyphosate-susceptible E. coli in 40 mM, suggested that the 104Gly residue is probably involved in EPSPS-1 catalysis. While all the 109Pro replacements are tolerant in 100 mM glyphosate, revealed that 109Pro residue is one of the critical binding site of glyphosate. Kinetic analysis of EPSPS activity of G104A and P109L mutants indicated that the tolerance were 353-fold and 15-fold increased Ki for glyphosate, compared to wild-type EPSPS-1, respectively. The G104A and P109L mutants of EPSPS will be instrumental for generation of transgenic crops.