Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L.
Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5'- and 3'-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.
Uchida
Hidenobu
H
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa, 921-8836, Japan.
Yamashita
Hirofumi
H
Kajikawa
Masataka
M
Ohyama
Kiyoshi
K
Nakayachi
Osamu
O
Sugiyama
Ryuji
R
Yamato
Katsuyuki T
KT
Muranaka
Toshiya
T
Fukuzawa
Hideya
H
Takemura
Miho
M
Ohyama
Kanji
K
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D028481:Euphorbia / Q000201:enzymology / Q000235:genetics*; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics*; D008401:Gas Chromatography-Mass Spectrometry; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010840:Phytosterols / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes / Q000378:metabolism; D013185:Squalene / Q000032:analysis / Q000378:metabolism
D018076:DNA, Complementary; D010840:Phytosterols; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D009249:NADP; C004808:farnesyl pyrophosphate; D013185:Squalene; D013186:Farnesyl-Diphosphate Farnesyltransferase