SEFA-PCR法克隆灵芝鲨烯合酶基因启动子及其序列分析

鲨烯合酶是灵芝三萜生物合成的关键酶,灵芝鲨烯合酶基因的表达和活性的高低决定了灵芝中三萜含量的高低。根据已经获得的灵芝鲨烯合酶全长cDNA序列设计一对专一引物,通过PCR扩增得到了灵芝鲨烯合酶基因的基因组全长,序列长1984bp,含有3个内含子。根据其基因组序列设计引物,采用SEFA-PCR的方法,以总DNA为模板,克隆了灵芝鲨烯合酶基因的启动子序列,长1042bp。序列分析发现灵芝鲨烯合酶基因启动子中没有明显的TATA和CAAT框,但是含有CCAAT-bindingfactor、GATA-1、GC-box、TFⅡD等重要的转录因子的结合位点,以及在人和酿酒酵母鲨烯合酶基因启动子中发现的甾醇调节相关的顺式调控元件。     

Molecular cloning and expression analysis of a squalene synthase gene from a medicinal plant, Euphorbia pekinensis Rupr.

Euphorbia pekinensis Rupr., which is also known as a medicinal plant, produces a large amount of alkaloids, phytosterols and triterpenes. In this study, we reported on the cDNA cloning and characterization of a novel squalene synthase (SQS) from E. pekinensis. Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol and triterpene biosynthesis. The full length cDNA named EpSQS (Genbank Accession Number JX509735) contained 1,614 bp with an open reading frame of 1,236 bp encoding a polypeptide of 411 amino acids. The deduced amino acid sequence of the EpSQS named EpSQS exhibited a high homology with other plant SQSs, and contained a single domain surrounded by helices. Phylogenetic analysis showed that EpSQS belonged to the plant SQS kingdom. Tissue expression analysis revealed that EpSQS expressed strongly in roots, weakly in stems and leaves, implying that EpSQS was a constitutive expression gene. The recombinant protein was expressed in Escherichia coli and detected by SDS-PAGE and western blot. The high performance liquid chromatography (HPLC) analysis showed that EpSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene.     

Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei

The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E. coli and completely sequenced. The DNA encodes five closely packed, very large open reading frames reading in one direction. As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each. The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and β-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis.     

Isolation and characterization of squalene synthase cDNA fromcentella asiatica (L) urban

We have cloned and characterized a gene for squalene synthase (SQS) fromCentella asiatica (L) Urban, a species that produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Its full-length cDNA clone was isolated by RACE PCR. The sequence ofpSQS contains an open reading frame of 1248 nucleotides, which code for 416 amino acids with a molecular mass of 47.3 kDa. Southern analysis revealed that one copy might exist in the C.asiatica genome. We also determined that 0.1 mM methyl jasmonate was sufficient to up-regulate those levels ofCaSQS mRNA.     

Biosynthesis of squalene and other triterpenes in Mentha piperita from mevalonate-2-14C

Unlike mono- and sesqui-terpenes, squalene and other triterpenes in peppermint readily incorporate mevalonate-2-¹⁴C label (greater than 30% incorporation of R-mevalonate in 4 hr). The labelled squalene produced turns over rapidly. Squalene derived from mevalonate-2-¹⁴C in incorporation times of 1, 4 and 7 hr was degraded chemically and shown to be equivalently labelled, according to theory, in the isopentenyl pyrophosphate-derived and dimethylallyl pyrophosphate-derived portions of the molecule. This contrasts with earlier studies on the biosynthesis of mono- and sesqui-terpenes in peppermint from ¹⁴C-precursors, in which the isopentenyl pyrophosphate-derived portions of the terpene molecules were found to be preferentially labelled, suggesting the presence of endogenous dimethylallyl pyrophosphate pools. The kinetics of squalene biosynthesis, and the labelling pattern of squalene, suggest that sites of triterpene biosynthesis are readily accessible to exogenous mevalonate and that endogenous dimethylallyl pyrophosphate pools do not participate in triterpene biosynthesis to any appreciable extent. The triterpene biosynthetic sites in peppermint thus appear to differ significantly from the monoterpene and sesquiterpene biosynthetic sites.     

Cloning and Functional Characterization of a Squalene Synthase from Paris polyphylla var. yunnanensis

Paris polyphylla Smith var. yunnanensis (Franch.) Hand. – Mazz. is a precious traditional Chinese medicine, and steroidal saponins are its major bioactive constituents possessing extensive biological activities. Squalene synthase (SQS) catalyzes the first dedicated step converting two molecular of farnesyl diphosphate (FDP) into squalene, a key intermediate in the biosynthetic pathway of steroidal saponins. In this study, a squalene synthase gene (PpSQS1) was cloned and functionally characterized from P. polyphylla var. yunnanensis, representing the first identified SQS from the genus Paris. The open reading frame of PpSQS1 is 1239 bp, which encodes a protein of 412 amino acids showing high similarity to those of other plant SQSs. Expression of PpSQS1 in Escherichia coli resulted in production of soluble recombinant proteins. Gas chromatography-mass spectrometry analysis showed that the purified recombinant PpSQS1 protein could produce squalene using FDP as a substrate in the in vitro enzymatic assay. qRT-PCR analysis indicated that PpSQS1 was highly expressed in rhizomes, consistent with the dominant accumulation of steroidal saponins there, suggesting that PpSQS1 is likely involved in the biosynthesis of steroidal saponins in the plant. The findings lay a foundation for further investigation on the biosynthesis and regulation of steroidal saponins, and also provide an alternative gene for manipulation of steroid production using synthetic biology.     

麻疯树鲨烯合酶基因SQS的克隆及生物信息学分析

以麻疯树(Jatropha curcas L.)总RNA为模板,根据已报道的鲨烯合酶基因序列设计简并引物,用RACE方法克隆得到麻疯树鲨烯合酶基因全长cDNA,命名为JcSQS。JcSQS全长1609 bp,包含1个1242 bp的开放阅读框,预测麻疯树鲨烯合酶基因编码的蛋白含有413个氨基酸。JcSQS具有鲨烯合酶类的保守结构域,JcSQS 蛋白与蓖麻、柿、木榄等植物中SQS基因编码的氨基酸序列具有高度同源性。这为研究麻疯树萜烯类物质的生物合成和调控机制奠定了基础。     

Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum

This report provides the complete nucleotide sequences of the full-length cDNA encoding squalene synthase (SQS) and its genomic DNA sequence from a triterpene-producing fungus, Ganoderma lucidum. The cDNA of the squalene synthase (SQS) (GenBank Accession Number: DQ494674) was found to contain an open reading frame (ORF) of 1,404 bp encoding a 468-amino-acid polypeptide, whereas the SQS genomic DNA sequence (GenBank Accession Number: DQ494675) consisted of 1,984 bp and contained four exons and three introns. Only one gene copy was present in the G lucidum genome. The deduced amino acid sequence of Ganoderma lucidum squalene synthase (Gl-SQS) exhibited a high homology with other fungal squalene synthase genes and contained six conserved domains. A phylogenetic analysis revealed that G. lucidum SQS belonged to the fungi SQS group, and was more closely related to the SQS of U. maydis than to those of other fungi. A gene expression analysis showed that the expression level was relatively low in mycelia incubated for 12 days, increased after 14 to 20 days of incubation, and reached a relatively high level in the mushroom primordia. Functional complementation of Gl-SQS in a SQS-deficient strain of Saccharomyces cerevisiae confirmed that the cloned cDNA encoded a squalene synthase.     

Cloning and functional identification of farnesyl diphosphate synthase from <Emphasis Type="Italic">Pinus massoniana</Emphasis> Lamb

Farnesyl diphosphate synthase (FPPS) is a key isoprenyl diphosphate synthase (IDS), which provides synthetic precursors to the terpenoid metabolic pathway. We isolated and characterized a Pinus massoniana FPPS (PmFPPS) gene which encodes a putative farnesyl diphosphate synthase from P. massoniana Lamb. In silico domain analysis revealed that PmFPPS contained all five conserved IDS domains and was homologous to FPPSs from other plant species. An in vitro enzymatic activity assay resulted in an optimum pH, temperature, and Mg²⁺ concentration of 7.0–7.5, 25 °C, and 1.2 mM, respectively. To identify the function of PmFPPS in vivo, sense and antisense expression vectors were constructed and transformed into tobacco using a constitutive cauliflower mosaic virus-35S promoter. The overexpression of PmFPPS in transgenic plants had higher squalene contents than the control, and the downregulated transgenic plants had lower squalene contents than the control. These results indicate that PmFPPS performs a regulatory role in triterpene biosynthesis.     

Molecular characterization and differential expression studies of an oxidosqualene cyclase (OSC) gene of Brahmi (Bacopa monniera)

Triterpenoid saponins are the class of secondary metabolites, synthesized via isoprenoid pathway. Oxidosqualene cyclases (OSCs) catalyzes the cyclization of 2, 3-oxidosqualene to various triterpene skeletons, the first committed step in triterpenoid biosynthesis. A full-length oxidosqualene cyclase cDNA from Bacopa monniera (BmOSC) was isolated and characterized. The open reading frame (ORF) of BmOSC consists of 2,292 bp, encoding 764 amino acid residues with an apparent molecular mass of 87.62 kDa and theoretical pI 6.21. It contained four QxxxxxW motifs, one Asp-Cys-Thr-Ala-Glu (DCTAE) motif which is highly conserved among the triterpene synthases and another MWCYCR motif involved in the formation of triterpenoid skeletons. The deduced amino acid sequence of BmOSC shares 80.5 % & 71.8 % identity and 89.7 % & 83.5 % similarity with Olea europaea mixed amyrin synthase and Panax notoginseng dammarenediol synthase respectively. Phylogenetic analysis revealed that BmOSC is closely related with other plant OSCs. Quantitative real-time PCR (qRT-PCR) data showed that BmOSC is expressed in all tissues examined with higher expression in stem and leaves as compared to roots and floral parts.     

Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in <Emphasis Type="Italic">Bupleurum falcatum</Emphasis>

The root of Bupleurum falcatum L. (Apiaceae) has long been one of the most important traditional herbal medicines in Asian countries. A group of triterpene saponins (saikosaponins) are the major constituents of this plant. Squalene synthase (SS) may play a regulatory role in directing triterpene intermediates and sterol pathways. Here, we investigated the regulatory role of the squalene synthase (BfSS1) gene in the biosynthesis of phytosterol and triterpene in B. falcatum. BfSS1 mRNA accumulated ubiquitously in plant organs and markedly increased in roots after treatment with methyl jasmonate (MeJA), ABA and ethephon. Transgenic B. falcatum constructs overexpressing BfSS1 in the sense and antisense orientations were assembled using the Agrobacterium-mediated method. Transgenic roots overexpressing BfSS1 in the sense orientation resulted in enhanced production of both phytosterol and saikosaponins. Overexpression of the BfSS1 gene in the sense orientation increased the mRNA accumulation of downstream genes such as squalene epoxidase and cycloartenol synthase but unexpectedly decreased the mRNA levels of β-amyrin synthase (β-AS), a triterpene synthase mRNA. MeJA treatment of wild-type roots strongly stimulated β-AS mRNA accumulation and saikosaponin production but suppressed phytosterol production. MeJA treatment of transgenic roots overexpressing BfSS1 in the sense orientation failed to stimulate β-AS mRNA accumulation but still enhanced saikosaponin and phytosterol production. These results indicate that overexpression of BfSS1 in B. falcatum regulates more powerfully the downstream genes than elicitor (MeJA) treatment in triterpene and phytosterol biosynthesis.     

Squalene-hopene cyclase (Spterp25) from Streptomyces peucetius: sequence analysis, expression and functional characterization

Squalene-hopene cyclase, which catalyzes the complex cyclization of squalene to the pentacyclic triterpene, hopene, is a key enzyme in the biosynthesis of hopanoids. The deduced amino acid sequence of the Streptomyces peucetius gene (spterp25) had significant similarity to other prokaryotic squalene-hopene cyclases. Like other triterpene cyclases, the S. peucetius squalene-hopene cyclase contains eight so-called QW-motifs with an aspartate-rich domain. The 2,025-bp squalene-hopene cyclase-encoding gene was expressed in Escherichia coli BL21(DE3)pLySs, and the in vitro activity of the recombinant cyclase was demonstrated using purified membrane protein. The cyclization product hopene was identified by gas chromatography/mass spectrometry (GC/MS).