Novel gene expression system for plant cells based on induction of alpha-amylase promoter by carbohydrate starvation.
The 5' regulatory region and putative signal sequence of a rice alpha-amylase gene, alpha Amy8, was fused to a bacterial gene encoding beta-glucuronidase (GUS) and introduced into rice, tobacco, and potato via Agrobacterium-mediated transformation systems. Expression of this chimeric gene in suspension cells of transgenic plants was suppressed by the presence of sucrose in the medium and induced by its absence. Induction or suppression of GUS expression in transgenic rice could be reversed by the deprivation or replenishment, respectively, of sucrose in the medium. The expressed GUS fusion protein was translocated to the endoplasmic reticulum, modified by glycosylation, and secreted into the culture medium of transgenic cells. In the presence of a glycosylation inhibitor, tunicamycin, the enzymatically active form of GUS was assembled in the endoplasmic reticulum. The yield of GUS secreted by transgenic cells was estimated to be as high as 40% of total secreted proteins. The reversible induction of the alpha-amylase promoter in culture cells by sugar level in the medium provides an excellent inducible expression system for basic research in plant science. Combination of the alpha-amylase promoter and signal sequence also offers a novel approach for large scale production of low cost, easily purified, secreted recombinant proteins.
Chan
M T
MT
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Chao
Y C
YC
Yu
S M
SM
D000595:Amino Acid Sequence; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D012275:Oryza; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum; D014026:Nicotiana; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D004247:DNA; D000516:alpha-Amylases; D005966:Glucuronidase