Cloning and expression of squalene synthase cDNA from hot pepper (Capsicum annuum L.).

We isolated and artificially expressed a cDNA clone of the Capsicum annuum squalene synthase (CASS) gene to elucidate the pattern of alternatively regulated two-branch point enzymes. The 1,674-bp CASS cDNA contained an open reading frame of 411 amino acids, yielding a predicted molecular mass of about 45 kDa. A deduced amino acid sequence comparison to other squalene syntheses showed identities with Nicotiana tabacum (91%), Nicotiana benthamiana (90%), Arabidopsis thaliana (79%), and rats (40%). The artificially expressed soluble form of the CASS enzyme was identified by the enzyme activity that converted FPP to squalene and by SDS-PAGE. A Southern blot analysis indicated that at least two copies of the squalene synthase gene exist in the hot pepper genome. In hot pepper, the regulation of the branch point enzymes, squalene synthase and sesquiterpene cyclase was investigated in the UV-challenged leaves of Capsicum annuum. The transcript level and enzyme activity of the CASS were slightly reduced by UV. However, those of the CASC were rapidly induced within 24 h and slowly decreased thereafter.

Lee JH ，Yoon YH ，Kim HY ，Shin DH ，Kim DU ，Lee IJ ，Kim KU ... -  《MOLECULES AND CELLS》

Cloning and bacterial expression of sesquiterpene cyclase, a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of Capsicum annuum.

Sesquiterpene cyclase, a branch point enzyme in the general isoprenoid pathway for the synthesis of phytoalexin capsidiol, was induced in detached leaves of Capsicum annuum (pepper) by UV treatment. The inducibility of cyclase enzyme activities paralleled the absolute amount of cyclase protein(s) of pepper immunodetected by monoclonal antibodies raised against tobacco sesquiterpene cyclase. A cDNA library was constructed with poly(A)+ RNA isolated from 24 h UV-challenged leaves of pepper. A cDNA clone for sesquiterpene cyclase in pepper was isolated by using a tobacco 5-epi aristolochene synthase gene as a heterologous probe. The predicted protein encoded by this cDNA was comprised of 559 amino acids and had a relative molecular mass of 65,095. The primary structural information from the cDNA clone revealed that it shared 77%, 72% and 49% identity with 5-epi aristolochene, vetispiradiene, and cadinene synthase, respectively. The enzymatic product catalyzed by the cDNA clone in bacteria was identified as 5-epi aristolochene, as judged by argentation TLC. RNA blot hybridization demonstrated the induction of an mRNA consistent with the induction of cyclase enzyme activity in UV-treated pepper.

Back K ，He S ，Kim KU ，Shin DH ... -  《PLANT AND CELL PHYSIOLOGY》

Functional characterization and differential expression studies of squalene synthase from Withania somnifera.

Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His-Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni-NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph-mass spectrometer (GC-MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.

Gupta N ，Sharma P ，Santosh Kumar RJ ，Vishwakarma RK ，Khan BM ... -  《-》

Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor.

The enzyme squalene synthase (SS) represents the first commitment of carbon from the general isoprenoid pathway toward sterol biosynthesis and is a potential point for regulation of sterol biosynthesis. The isolation and characterization of tobacco (Nicotiana tabacum) squalene synthase (TSS) cDNA and genomic DNA clones, as well as determination of the steady state level of TSS mRNA in response to elicitor treatment, were investigated. cDNA clones for TSS were isolated from poly (A)+ RNA using a reverse transcription/polymerase chain reaction (RT/PCR) method. A 1233-bp cDNA clone was generated that contained an open reading frame of 411 amino acids giving a predicted molecular mass of 46.9 kDa. Comparison of the TSS deduced amino acid sequence with currently described SS from different species showed the highest identify with Nicotiana benthamiana (97%), followed by Glycyrrhiza glabra (81%), Arabidopsis thaliana (74%), rat (40%), and yeast (37%). Expression of a soluble form of the TSS enzyme with enzymatic activity in Escherichia coli was achieved by truncating 24 hydrophobic amino acids at the carboxy terminus. Characterization of genomic TSS (gTSS) revealed a gene of 7.086 kb with a complex organization of small exons and large introns not typical of plant genes. Southern blot hybridization indicated only two copies of the SS gene in the tobacco genome. Treatment of tobacco cell suspension cultures with a fungal elicitor dramatically reduced TSS enzymatic activity, lowering it to zero within 24 h. Analysis of TSS mRNA levels, by RNA blot hybridization and primer extension assays, in elicitor-treated cells indicated that the transcript level remained largely unchanged over this 24-h period. These results suggest that the suppression of TSS enzyme activity in elicitor-treated cells may result from a posttranscriptional modification of TSS.

Devarenne TP ，Shin DH ，Back K ，Yin S ，Chappell J ... -  《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》

Molecular cloning, in vitro expression and characterization of a plant squalene synthetase cDNA.

Squalene synthetase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the first committed step for sterol biosynthesis and is thought to play an important role in the regulation of isoprenoid biosynthesis in eukaryotes. Using degenerate oligonucleotides based on a conserved region found in yeast and human squalene synthetase genes, a cDNA was cloned from the plant Nicotiana benthamiana. The cloned cDNA contained an open reading frame of 1234 bp encoding a polypeptide of 411 amino acids (M(r) 47002). Northern blot analysis of poly(A)+ mRNA from N. benthamiana and N. tabacum cv. MD609 revealed a single band of ca. 1.6 kb in both Nicotiana species. The identity and functionality of the cloned plant squalene synthetase cDNA was further confirmed by expression of the cDNA in Escherichia coli and in a squalene synthetase-deficient erg9 mutant of Saccharomyces cerevisiae. Antibodies raised against a truncated form of the protein recognized an endogenous plant protein of appropriate size as well as the full-length bacterially expressed protein as detected by western analysis. Comparison of the deduced primary amino acid sequences of plant, yeast, rat and human squalene synthetase revealed regions of conservation that may indicate similar functions within each polypeptide.

Hanley KM ，Nicolas O ，Donaldson TB ，Smith-Monroy C ，Robinson GW ，Hellmann GM ... -  《PLANT MOLECULAR BIOLOGY》