Khan M R, Ceriotti A, Tabe L, Aryan A, McNabb W, Moore A, Craig S, Spencer D, Higgins T J
CSIRO, Division of Plant Industry, Australia.
Transgenic Res. 1996 May;5(3):179-85. doi: 10.1007/BF01969707.
A gene encoding a sulphur-rich, sunflower seed albumin (23% cysteine plus methionine) was modified to contain the promoter for the 35S RNA of cauliflower mosaic virus, in order to obtain leaf expression in transgenic plants. In addition, a sequence encoding an endoplasmic reticulum-retention signal was added to the 3' end of the coding region so as to stabilize the protein by diverting it away from the vacuole. The modified gene was introduced into subterranean clover (T. subterraneum L.) and its expression was detected by northern and western blots and by immunogold localization. The albumin was accumulated in the lumen of the endoplasmic reticulum, and, among six independent, transformed lines, it accumulated in the leaves of T0 transgenic plants at varying levels up to 0.3% of the total extractable protein. The level of accumulation of the sunflower albumin increased with increasing leaf age, and in the older leaves of the most highly expressing plants of the T1 generation it reached 1.3% of total extractable protein. Expression of the SSA gene was stable in the first and second generation progeny. These results indicate that there is potential for significantly improving the nutritional value of subterranean clover for ruminant animals such as sheep by expressing genes that code for sulphur-rich, rumen-stable proteins in leaves.
一个编码富含硫的向日葵种子清蛋白（半胱氨酸加蛋氨酸含量为23%）的基因经过修饰，使其包含花椰菜花叶病毒35S RNA的启动子，以便在转基因植物的叶片中表达。此外，一个编码内质网滞留信号的序列被添加到编码区的3'端，通过使蛋白质不进入液泡来稳定该蛋白质。修饰后的基因被导入地下三叶草（T. subterraneum L.），并通过Northern杂交、Western杂交和免疫金定位检测其表达。清蛋白在内质网腔中积累，在六个独立的转化株系中，它在T0转基因植物的叶片中积累，含量高达总可提取蛋白的0.3%，且积累水平各不相同。向日葵清蛋白的积累水平随着叶片年龄的增加而提高，在T1代表达量最高的植株的老叶中，其含量达到总可提取蛋白的1.3%。SSA基因在第一代和第二代后代中的表达是稳定的。这些结果表明，通过在叶片中表达编码富含硫、瘤胃稳定蛋白的基因，有潜力显著提高地下三叶草对反刍动物如绵羊的营养价值。
