Cho Myeong-Je, Choi Hae-Woon, Jiang Wen, Ha Chi D, Lemaux Peggy G
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA Present address: Genenech Inc., San Francisco, CA 95616, USA Present address: Medical College of Pennsylvania, Hahnemann University, Philadelphia, PA 19129, USA.
Physiol Plant. 2002 May;115(1):144-154. doi: 10.1034/j.1399-3054.2002.1150117.x.
The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.
利用由水稻肌动蛋白启动子或大麦胚乳特异性d-醇溶蛋白启动子驱动的合成绿色荧光蛋白基因[sgfp(S65T)]，对转基因大麦（Hordeum vulgare L.）植株中绿色荧光蛋白（GFP）的表达及其遗传特性进行了研究。由玉米泛素启动子和内含子驱动的编码膦丝菌素乙酰转移酶（bar）的基因被用作选择标记，以鉴定转基因组织。在用含sgfp(S65T)构建体转化的愈伤组织细胞和T0植株的各种组织中，观察到由水稻肌动蛋白启动子驱动的强烈GFP表达。在17个独立的可再生转基因愈伤组织系中，有14个观察到由水稻肌动蛋白启动子驱动的GFP表达；然而，在二倍体系的T0及后代中，表达逐渐丧失。在14个（43%）独立的GFP表达愈伤组织系中，只有6个的T2后代观察到稳定的GFP表达。在T2后代中不表达GFP的8个系中，有4个在从愈伤组织再生T0植株的过程中丧失了GFP表达；1个在从T0到T1代的转变中丧失了GFP表达，3个系不育。同样，由玉米泛素启动子驱动的bar在T1后代中表达丧失；26个独立系中只有21个（81%）对草丁膦具有抗性。与肌动蛋白驱动的表达相反，由d-醇溶蛋白启动子驱动的GFP表达表现出胚乳特异性。所有7个用d-醇溶蛋白驱动的GFP转化的系（100%）在T1和T2代均表达GFP，无论倍性水平如何，且表达以孟德尔方式分离。我们得出结论，sgfp(S65T)基因已成功转化到大麦中，并且由d-醇溶蛋白启动子驱动的GFP表达在T1和T2后代中的遗传模式比由水稻肌动蛋白启动子驱动的或由玉米泛素启动子驱动的bar基因更稳定。
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