[Cloning and characterization of cDNA encoding Psammosilene tunicoides squalene synthase].
The total triterpene saponins of Psammosilene tunicoides have significant pharmacologic activity. Psammosilene tunicoides squalene synthase (PSS) is a gateway enzyme to regulate the biosynthesis of total triterpene saponins extracted from the root of Psammosilene tunicoides which is an endangered species. In this paper, cDNA encoding of PSS was cloned by the degenerate primer PCR and rapid-amplification of cDNA ends (RACE). The full-length of cDNA of PSS is 1663 bp, with an open reading frame (ORF) of 1 245 bp, encoding 414 amino acid polypeptide (calculated molecular mass, 47.69 kDa), 5'UTR (untranslated region) and 3'UTR are 260 bp and 158 bp, respectively. The deduced amino acid sequence of PSS has higher homology with the known squalene synthases of several species such as Panax notoginseng (83%), Panax ginseng (82%) and Glycyrrhiza glabra (82%) than that with Schizosacharomyces pombe (35%), Candida albicans (39%) and Homo sapiens (47%). The characterization of PSS was done by a series of methods, such as prokaryotic expression, the activity of enzyme in vitro, capillary gas chromatography (GC) and capillary gas chromatography mass spectrometry (GC-MS). The results showed that the cell-free extract of E. coli transformed with the recombinant plasmid can effectively convert farnesyl diphosphate into squalene in vitro. GenBank accession number is EF585250. Our research provided important base for the study of Psammosilene tunicoides secondary metabolism and metabolic engineering.
Dai
Zhu-bo
ZB
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Qian
Zi-gang
ZG
Hu
Yun-qian
YQ
Huang
Lu-qi
LQ
D029748:Caryophyllaceae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D056727:Endangered Species; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D016366:Open Reading Frames; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010946:Plants, Medicinal / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D013186:Farnesyl-Diphosphate Farnesyltransferase