Grausem B, Chaubet N, Gigot C, Loper J C, Benveniste P
Département d'Enzymologie Moléculaire et Cellulaire, UPR 406 du CNRS, Strasbourg, France.
Plant J. 1995 May;7(5):761-70. doi: 10.1046/j.1365-313x.1995.07050761.x.
Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to gamma-ketotriazole (gamma-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of gamma-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of gamma-kt showed that their delta 5-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substate obtusifoliol. In contrast, control calli when treated with gamma-kt, displayed a sterol content strongly enriched in 14 alpha-methyl sterols and depressed in physiological delta 5-sterols. When the transgenic calli were cultured in mixtures of gamma-kt and LAN14DM inhibitors sterol compositions enriched in 14 alpha-methyl sterols were obtained, reflecting a strong inhibition of both 'endogenous' OBT14DM and 'exogenous' LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.
含有T-DNA的根癌农杆菌已转化烟草原生质体，该T-DNA中来自酿酒酵母的编码羊毛甾醇-14-脱甲基酶（LAN14DM）的CYP51A1基因受花椰菜花叶病毒（CaMV）35S启动子控制。Northern和Western实验表明，两个转化体强烈表达LAN14DM。这些转基因愈伤组织被LAB 170250F（LAB，一种抑制植物 obtusifoliol-14-脱甲基酶（OBT14DM）和LAN14DM的植物毒性杀菌剂）杀死，但对γ-酮三唑（γ-kt）具有抗性，γ-kt是一种除草剂，已证明在对对照愈伤组织致死的浓度下可抑制OBT14DM但不抑制LAN14DM。然而，这些转基因愈伤组织被γ-kt与LAN14DM的杀菌剂抑制剂（如酮康唑、伊曲康唑或氟硅唑）的混合物杀死，这些抑制剂单独使用时无效。对在γ-kt存在下生长的转基因愈伤组织的进一步分析表明，它们的δ5-甾醇含量与用对照质粒转化原生质体获得的未处理对照愈伤组织的δ5-甾醇含量接近；这与以下证据一致，即转基因表达的LAN14DM可以通过使用植物底物 obtusifoliol绕过被阻断的OBT14DM。相比之下，对照愈伤组织用γ-kt处理时，甾醇含量在14α-甲基甾醇中强烈富集，而在生理δ5-甾醇中降低。当转基因愈伤组织在γ-kt和LAN14DM抑制剂的混合物中培养时，获得了富含14α-甲基甾醇的甾醇组成，这反映了“内源性”OBT14DM和“外源性”LAN14DM均受到强烈抑制。综上所述，这些结果表明，在用CYP51A1转化的烟草愈伤组织中，对三唑类除草剂的抗性源于功能性LAN14DM酶的表达；当甾醇生物合成途径的14-脱甲基酶水平被OBT14DM除草剂抑制剂阻断时，其在转基因组织中的活性在该水平上产生了甾醇生物合成途径的旁路。
