Cloning of a trans-spliced glyceraldehyde-3-phosphate-dehydrogenase gene from the potato cyst nematode Globodera rostochiensis and expression of its putative promoter region in Caenorhabditis elegans.
Reverse genetics to determine the relative importance of individual pathogenicity factors of the potato cyst nematode Globodera rostochiensis depends, apart from an efficient transformation protocol for this obligatory plant parasite, on the availability of an efficient promoter. PCR-based cloning was used to isolate a cDNA encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, a crucial enzyme in glycolysis and gluconeogenesis; this gene was designated gpd) and its 5'-flanking region. The cDNA includes 1047 nucleotides encoding an open reading frame that shows high homology with GAPDHs from Caenorhabditis elegans and other species. Analysis of the 745 bp 5'-flanking region of the gpd gene showed no homology with a similar region in C. elegans. In this region several eukaryotic promoter elements are present. 5' Rapid amplification of cDNA ends revealed this gene was trans-spliced with a SL1 spliced leader. The 5'-flanking region of the gpd gene was fused to green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. Green fluorescent protein expression, under the transcriptional control of the 5'-flanking region of gpd, was mainly observed in body wall muscles of transgenic animals. This putative promoter region of GAPDH could be a valuable tool to drive gene expression in transgenic G. rostochiensis and other related plant-parasitic nematode species.
Qin
L
L
Wageningen Agricultural University, Department of Nematology, The Netherlands.
Smant
G
G
Stokkermans
J
J
Bakker
J
J
Schots
A
A
Helder
J
J
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics; D003001:Cloning, Molecular; D018899:CpG Islands; D018076:DNA, Complementary; D015870:Gene Expression; D017238:Genes, Helminth; D017930:Genes, Reporter; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D016385:TATA Box; D020040:Trans-Splicing; D014170:Transformation, Genetic; D014431:Tylenchoidea / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases