Cloning, prokaryotic expression and functional analysis of squalene synthase (SQS) in Magnolia officinalis.

Magnolia officinalis Rehder et Wilson is a traditional Chinese herbal medicine that is used to treat various diseases such as neurosis, anxiety, and stroke. The main secondary metabolites in magnolia bark are phenolic compounds and terpenoids. Squalene synthase plays a significant role in catalyzing two farnesyl diphosphate molecules to form squalene, the first precursor of triterpenoid, phytosterol, and cholesterol biosynthesis. In this study, a full-length cDNA of squalene synthase was cloned from M. officinalis and designated MoSQS (GenBank accession no. KT223496). The gene contains a 1240-bp open reading frame and it encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that MoSQS shared high similarity with squalene synthases among other plants. Prokaryotic expression showed that a transmembrane domain-deleted (385-409 aa) MoSQS mutant (MoSQSΔTM) could be expressed in its soluble form in Escherichia coli Transetta (DE3). GC-MS analysis showed that squalene was detected in an in vitro reaction mixture. These results indicated that MoSQSΔTM was functional, thereby establishing an important foundation for the study of triterpenoid biosynthesis in M. officinalis.

[Screening target genes for bimolecular marking methods of Magnolia quality].

The study used use bimolecular marking methods to evaluate the lignans of Magnolia officinalis and M. officinalis var. biloba. First, we compare the chemical constituents between M. officinalis and M. officinalis var.biloba. There were significant differences in concentration of magnolignan I between leaves of these two varieties. Then we further select the p-hydroxyphenyl lignin to mining the key enzyme genes of biosynthesis from Magnolia transcriptome, and screened an encoding cinnamyl alcohol dehydrogease gene as the candidate marker of bimolecular marking methods of Magnolia quality by comparing of the expression level and structure variation in homologous gene between M. officinalis and M. officinalis var.biloba. The established method provides the technical support for bimolecular marking methods of Magnolia quality evaluation.

Molecular characterization and expression analyses of an anthocyanin synthase gene from Magnolia sprengeri Pamp.

Anthocyanin synthase (ANS), which catalyzes the conversion of colorless leucoanthocyanins into colored anthocyanins, is a key enzyme in the anthocyanin biosynthetic pathway. It plays important roles in plant development and defense. An ANS gene designated as MsANS was cloned from Magnolia sprengeri using rapid amplification of complementary DNA (cDNA) ends technology. The full-length MsANS is 1171-bp long and contains a 1080-bp open reading frame encoding a 360 amino acid polypeptide. In a sequence alignment analysis, the deduced MsANS protein showed high identity to ANS proteins from other plants: Prunus salicina var. cordata (74 % identity), Ampelopsis grossedentata (74 % identity), Pyrus communis (73 % identity), and Prunus avium (73 % identity). A structural analysis showed that MsANS belongs to 2-oxoglutarate (2OG)- and ferrous iron-dependent oxygenase family because it contains three binding sites for 2OG. Real-time quantitative polymerase chain reaction analyses showed that the transcript level of MsANS was 26-fold higher in red petals than in white petals. The accumulation of anthocyanins in petals of white, pink, and red M. sprengeri flowers was analyzed by HPLC. The main anthocyanin was cyanidin-3-o-glucoside chloride, and the red petals contained the highest concentration of this pigment.

Loss of two introns from the Magnolia tripetala mitochondrial cox2 gene implicates horizontal gene transfer and gene conversion as a novel mechanism of intron loss.

Intron loss is often thought to occur through retroprocessing, which is the reverse transcription and genomic integration of a spliced transcript. In plant mitochondria, several unambiguous examples of retroprocessing are supported by the parallel loss of an intron and numerous adjacent RNA edit sites, but in most cases, the evidence for intron loss via retroprocessing is weak or lacking entirely. To evaluate mechanisms of intron loss, we designed a polymerase chain reaction (PCR)-based assay to detect recent intron losses from the mitochondrial cox2 gene within genus Magnolia, which was previously suggested to have variability in cox2 intron content. Our assay showed that all 22 examined species have a cox2 gene with two introns. However, one species, Magnolia tripetala, contains an additional cox2 gene that lacks both introns. Quantitative PCR showed that both M. tripetala cox2 genes are present in the mitochondrial genome. Although the intronless gene has lost several ancestral RNA edit sites, their distribution is inconsistent with retroprocessing models. Instead, phylogenetic and gene conversion analyses indicate that the intronless gene was horizontally acquired from a eudicot and then underwent gene conversion with the native intron-containing gene. The models are presented to summarize the roles of horizontal gene transfer and gene conversion as a novel mechanism of intron loss.

Biochemical and genomic characterization of terpene synthases in Magnolia grandiflora.

Magnolia grandiflora (Southern Magnolia) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young leaves were isolated, and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted farnesyl diphosphate (C(15)) predominantly to beta-cubebene, while Mg17 converted geranyl diphosphate (C(5)) to alpha-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all three genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 messenger RNAs in stamens. A putative amino-terminal signal peptide of Mg17 targeted the reporter green fluorescent protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, the intron-exon organizations for the three Magnolia TPS genes were different from one another and from other well-characterized TPS gene sets. The Mg17 gene consists of six introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only four introns, and Mg25 has only a single intron located near the 5' terminus of the gene. Our results suggest that the structural diversity observed in the Magnolia TPS genes could have occurred either by a rapid loss of introns from a common ancestor TPS gene or by a gain of introns into an intron-deficient progenote TPS gene.