Duwenig E, Steup M, Willmitzer L, Kossmann J
Institut für Genbiologische Forschung Berlin GmbH, Berlin, Germany.
Plant J. 1997 Aug;12(2):323-33. doi: 10.1046/j.1365-313x.1997.12020323.x.
To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.
为了确定胞质磷酸化酶(Pho2;EC 2.4.1.1)的功能,构建了转基因马铃薯植株,通过导入一个嵌合基因来抑制该酶的表达,该嵌合基因包含胞质磷酸化酶编码区的一部分,以反义方向与35S CaMV启动子相连。Northern印迹分析和天然聚丙烯酰胺凝胶电泳结果显示,转基因植株的叶片和块茎中胞质磷酸化酶的表达均受到强烈抑制。通过茎切段繁殖的转基因植株在形态上与野生型无法区分。然而,转基因马铃薯块茎的发芽情况发生了显著变化:与野生型相比,转基因块茎产生的芽多2.4至8.1倍。在温室中栽培时,转基因种薯产生的茎比野生型多两到三倍。所得植株的花序出现得更早。许多转基因植株连续开花两到三次。由种薯衍生的转基因植株单株形成的块茎数量是未转化对照的1.6至2.4倍。单个块茎的大小和干物质含量没有明显改变。转基因植株的块茎产量显著更高。对新鲜收获和储存的块茎进行碳水化合物测定结果表明,胞质磷酸化酶的反义抑制对淀粉和蔗糖库没有明显影响;然而,长时间储存后葡萄糖和果糖水平明显降低。这些结果支持胞质磷酸化酶不参与淀粉降解的观点。文中还讨论了胞质磷酸化酶水平降低与观察到的发芽和开花变化之间可能的联系。