Molecular cloning and characterization of the yew gene encoding squalene synthase from Taxus cuspidata.
The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semiquantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.
Huang
Zhuosh
Z
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai 200433, People's Republic of China.
Jiang
Keji
K
Pi
Yan
Y
Hou
Rong
R
Liao
Zhihua
Z
Cao
Ying
Y
Han
Xu
X
Wang
Qian
Q
Sun
Xiaofen
X
Tang
Kexuan
K
D000085:Acetates / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005875:Gibberellins / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D054883:Oxylipins / Q000494:pharmacology; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020946:Taxus / Q000201:enzymology / Q000235:genetics*
D000085:Acetates; D003517:Cyclopentanes; D018076:DNA, Complementary; D005875:Gibberellins; D054883:Oxylipins; D010937:Plant Growth Regulators; D010940:Plant Proteins; C072239:methyl jasmonate; C007842:gibberellic acid; D013186:Farnesyl-Diphosphate Farnesyltransferase