Cloning and characterization of squalene synthase gene from Fusarium fujikuroi (Saw.) Wr.

被引：16

作者：

Zhao, Rui-Yu ^{[1
,2
,3
,4
]}

Xiao, Wei ^{[1
,2
,3
,4
]}

Cheng, Hai-Li ^{[1
,2
,3
,4
]}

Zhu, Ping ^{[1
,2
,3
,4
]}

Cheng, Ke-Di ^{[1
,2
,3
,4
]}

机构：

[1] Chinese Acad Med Sci, Inst Mat Med, Dept Biosynth Nat Prod, Beijing 100050, Peoples R China

[2] Peking Union Med Coll, Beijing 100050, Peoples R China

[3] Minist Hlth PRC, Key Lab Biosynth Nat Prod, Beijing 100050, Peoples R China

[4] Minist Educ PRC, Key Lab Bioact Subst & Resources Utilizat, Beijing 100050, Peoples R China

来源：

JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY | 2010年 / 37卷 / 11期

关键词：

Fusarium fujikuroi; Squalene synthase; Recombinant protein; Conserved regions; GC-MS analysis; GC-AG INTRONS; MOLECULAR-CLONING; TRANSCRIPTIONAL REGULATION; KINETIC CHARACTERIZATION; SPLICE SITES; EXPRESSION; CDNA; BIOSYNTHESIS; DNA; PURIFICATION;

D O I：

10.1007/s10295-010-0764-z

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5'-untranslated region (UTR), 3'-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS-PAGE and western blot. GC-MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.

引用

页码：1171 / 1182

页数：12

共 46 条

[1]

ASHBY MN, 1990, J BIOL CHEM, V265, P13157

[2] Isolation and characterization of the gibberellin biosynthetic gene cluster in Sphaceloma manihoticola [J].