Metabolic engineering of cultured tobacco cells.
Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.
Shinmyo
A
A
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-01, Japan. shinmyo@bs.aist-nara.ac.jp
Shoji
T
T
Bando
E
E
Nagaya
S
S
Nakai
Y
Y
Kato
K
K
Sekine
M
M
Yoshida
K
K
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis; D002478:Cells, Cultured; D005285:Fermentation; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*
C489905:HSP18 protein, plant; D006360:Heat-Shock Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D010940:Plant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000426:Alcohol Dehydrogenase; D005966:Glucuronidase; D006180:Proton-Translocating ATPases