Showing papers by "Hongmei Luo published in 2013"

Transcriptome analysis reveals ginsenosides biosynthetic genes, microRNAs and simple sequence repeats in Panax ginseng C. A. Meyer

Abstract: Panax ginseng C A Meyer is one of the most widely used medicinal plants Complete genome information for this species remains unavailable due to its large genome size At present, analysis of expressed sequence tags is still the most powerful tool for large-scale gene discovery The global expressed sequence tags from P ginseng tissues, especially those isolated from stems, leaves and flowers, are still limited, hindering in-depth study of P ginseng Two 454 pyrosequencing runs generated a total of 2,423,076 reads from P ginseng roots, stems, leaves and flowers The high-quality reads from each of the tissues were independently assembled into separate and shared contigs In the separately assembled database, 45,849, 6,172, 4,041 and 3,273 unigenes were only found in the roots, stems, leaves and flowers database, respectively In the jointly assembled database, 178,145 unigenes were observed, including 86,609 contigs and 91,536 singletons Among the 178,145 unigenes, 105,522 were identified for the first time, of which 656% were identified in the stem, leaf or flower cDNA libraries of P ginseng After annotation, we discovered 223 unigenes involved in ginsenoside backbone biosynthesis Additionally, a total of 326 potential cytochrome P450 and 129 potential UDP-glycosyltransferase sequences were predicted based on the annotation results, some of which may encode enzymes responsible for ginsenoside backbone modification A BLAST search of the obtained high-quality reads identified 14 potential microRNAs in P ginseng, which were estimated to target 100 protein-coding genes, including transcription factors, transporters and DNA binding proteins, among others In addition, a total of 13,044 simple sequence repeats were identified from the 178,145 unigenes This study provides global expressed sequence tags for P ginseng, which will contribute significantly to further genome-wide research and analyses in this species The novel unigenes identified here enlarge the available P ginseng gene pool and will facilitate gene discovery In addition, the identification of microRNAs and the prediction of targets from this study will provide information on gene transcriptional regulation in P ginseng Finally, the analysis of simple sequence repeats will provide genetic makers for molecular breeding and genetic applications in this species

Identification of genes related to agarwood formation: transcriptome analysis of healthy and wounded tissues of Aquilaria sinensis.

Abstract: Agarwood is an expensive resinous heartwood derived from Aquilaria plants that is widely used in traditional medicines, incense and perfume. Only wounded trees can produce agarwood, and the huge demand for the agarwood products has led all Aquilaria spp. being endangered and listed in the Appendix II of the CITES ( http://www.cites.org ). The major components of agarwood are sesquiterpenes and phenylethyl chromones. Owing to a lack of genomic information, the molecular basis of wound-induced sesquiterpenes biosynthesis and agarwood formation remains unknown. To identify the primary genes that maybe related to agarwood formation, we sequenced 2 cDNA libraries generated from healthy and wounded A. sinensis (Lour.) Gilg. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at bioinformatics levels. Of those associated with agarwood formation, 30 putatively encoded enzymes in the sesquiterpene biosynthesis pathway, and a handful of transcription factors and protein kinases were related to wound signal transduction. Three full-length cDNAs of sesquiterpene synthases (ASS1-3) were cloned and expressed in Escherichia coli, and enzyme assays revealed that they are active enzymes, with the major products being δ-guaiene. A methyl jasmonate (MJ) induction experiment revealed that the expression of ASS was significantly induced by MJ, and the production of sesquiterpenes was elevated accordingly. The expression of some transcription factors and protein kinases, especially MYB4, WRKY4, MPKK2 and MAPK2, was also induced by MJ and coordinated with ASS expression, suggesting they maybe positive regulators of ASS. This study provides extensive transcriptome information for Aquilaria spp. and valuable clues for elucidating the mechanism of wound-induced agarwood sesquiterpenes biosynthesis and their regulation.

Discovery of WRKY transcription factors through transcriptome analysis and characterization of a novel methyl jasmonate-inducible PqWRKY1 gene from Panax quinquefolius

Abstract: Transcription factors (TFs) are important regulating factors that can mediate many life processes. However, no TF genes have previously been reported in Panax quinquefolius (American ginseng), with the exception of a few expressed sequence tags. In this study, 753 unigenes (unique sequences) have been annotated in the plant transcription factor database PlnTFDB (version 3.0) by mining a 454 transcriptome dataset of P. quinquefolius. After classification of the unigenes, 45 unigenes were discovered to be annotated as WRKY transcripts in the public databases. Furthermore, PqWRKY1, one of the WRKY family TF genes that respond to methyl jasmonate, was isolated according to the sequences in the 454 transcriptome dataset. The cDNA of PqWRKY1 (P. quinquefolius WRKY1) encodes a putative protein of 358 amino acids, including a WRKY domain and a zinc finger motif. A subcellular localization assay demonstrated that the protein localizes to the nucleus and has strong transcriptional activation activity in transgenic yeast. In comparison to control lines, the PqWRKY1 transgenic Arabidopsis line exhibited insensitive phenotypes when exposed to high salt or mannitol. Correspondingly, in transgenic Arabidopsis lines, the expression levels of some genes involved in the anti-stress process were relatively higher than those in the control lines. Additionally, genes involved in triterpene biosynthesis were expressed onefold to fivefold higher in the transgenic line compared to the control line. These results suggest that the transcription factor PqWRKY1 is a positive regulator related to osmotic stress and triterpene ginsenoside biosynthesis in P. quinquefolius.

Complete chloroplast genome sequence of Magnolia grandiflora and comparative analysis with related species

Abstract: Magnolia grandiflora is an important medicinal, ornamental and horticultural plant species. The chloroplast (cp) genome of M. grandiflora was sequenced using a 454 sequencing platform and the genome structure was compared with other related species. The complete cp genome of M. grandiflora was 159623 bp in length and contained a pair of inverted repeats (IR) of 26563 bp separated by large and small single copy (LSC, SSC) regions of 87757 and 18740 bp, respectively. A total of 129 genes were successfully annotated, 18 of which included introns. The identity, number and GC content of M. grandiflora cp genes were similar to those of other Magnoliaceae species genomes. Analysis revealed 218 simple sequence repeat (SSR) loci, most composed of A or T, contributing to a bias in base composition. The types and abundances of repeat units in Magnoliaceae species were relatively conserved and these loci will be useful for developing M. grandiflora cp genome vectors. In addition, results indicated that the cp genome size in Magnoliaceae species and the position of the IR border were closely related to the length of the ycf1 gene. Phylogenetic analyses based on 66 shared genes from 30 species using maximum parsimony (MP) and maximum likelihood (ML) methods provided strong support for the phylogenetic position of Magnolia. The availability of the complete cp genome sequence of M. grandiflora provides valuable information for breeding of desirable varieties, cp genetic engineering, developing useful molecular markers and phylogenetic analyses in Magnoliaceae.

Salvia miltiorrhiza as medicinal model plant

Abstract: Research on medicinal model organism is one of the core technologies to promote the modernization of traditional Chinese medicine (TCM). The research progress of Salvia miltiorrhiza as medicinal model plant is summarized in this paper. The genome of S. miltiorrhiza is small and its life cycle is short, as well as this plant can be stably genetically transformed. Because S. miltiorrhiza possesses the important medicinal and economic values, recently the transcriptome and genome of S. miltiorrhiza have been significantly recovered. The research prospect of S. miltiorrhiza as medicinal model plant in TCM was discussed, including biosynthesis of active components and their genetic regulation, relationship between quality of TCM and ecological environments, and selective breeding of good quality lines. Furthermore, as medicinal model plant, the construction of mutant library for S. miltiorrhiza, the genome map with high quality, and the functional genome should be investigated. Accompanying modern investigation of life sciences, the platform for medicinal model plant, S. miltiorrhiza, will be promoted to be established. It is important to develop the ethnopharmacology and new drugs around the world.

Investigation and integrated molecular diagnosis of root-knot nematodes in Panax notoginseng root in the field

Abstract: Root-knot nematodes (RKNs, Meloidogyne spp.) are damaging pests that can infect thousands of plant species and cause enormous crop losses worldwide. Panax notoginseng is a common host of root-knot nematodes. In this study, we surveyed notoginseng gardens and determined the incidence of RKNs. Among the gardens surveyed, 71 % were infected with RKNs, and the RKN incidence index ranged from 8 % to 47 % in three randomly infected gardens. Meloidogyne hapla was identified as the pathogenic nematode based on 18S ribosomal RNA analysis by DNA barcoding. The results were qualitatively and quantitatively confirmed using a real-time PCR assay according to variations in the ITS1 and ITS2 regions. These results indicated that the combination of DNA barcoding and real-time PCR is a reliable and precise method for identifying parasitic nematodes from mixed-infected plant roots in the field. In addition, the abundance of ITS1 and ITS2 displayed a similar trend to the numbers of RKNs in the three gardens, which suggests that the results of real-time PCR can be used to determine the damage caused by M. hapla in the field. Our studies show that RKNs are common and can cause serious damage to notoginseng. We present an integrated method of detecting mixed nematode species in the field and confirm M. hapla as the target for parasitic nematode control in notoginseng gardens. Our results contribute to the improvement of RKN control in notoginseng and further promote the sustainable development of medicinal plants.

Cloning and expression analysis of a key device of HMGR gene involved in ginsenoside biosynthesis of Panax ginseng via synthetic biology approach

Abstract: 3-Hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), the first enzyme of mavalonic acid pathway, is one of the key devices involved in ginsenoside biosynthesis based on synthetic biology approach. The open reading frame of a novel HMGR gene from Panax ginseng (PgHMGR2) was cloned and analyzed in this study. PgHMGR2-encoding protein showed 71.6% sequence similarity to a P. ginseng HMGR in GenBank. The full-length cDNA sequence of PgHMGR2 containing 1 770 bp, which encodes 589 amino acids, was cloned by RT-PCR strategy from P. ginseng. The bioinformatic analysis showed that PgHMGR2-encoding protein contained two transmembrane regions and the HMG_CoA_reductase domain, without signal peptide. The protein sequence of PgHMGR2 had the highest sequence similarity (99%) with Panax quinquefolius HMGR (GenBank accession No. ACV65036). The expression level of PgHMGR2 was the highest in flower based on a real-time PCR analysis, followed by leaf and root, and the lowest was in stem. The result will provide a foundation for exploring the molecular function of PgHMGR2 involved in ginsenoside biosynthesis based on synthetic biology approach in P. ginseng plants.

[Development of the devices for synthetic biology of triterpene saponins at an early stage: cloning and expression profiling of squalene epoxidase genes in panax notoginseng].

Abstract: Synthetic biology of traditional Chinese medicine (TCM) is a new and developing subject based on the research of secondary metabolite biosynthesis for nature products. The early development of synthetic biology focused on the screening and modification of parts or devices, and establishment of standardized device libraries. Panax notoginseng (Burk.) F.H.Chen is one of the most famous medicinal plants in Panax species. Triterpene saponins have important pharmacological activities in P. notoginseng. Squalene epoxidase (SE) has been considered as a key rate-limiting enzyme in biosynthetic pathways of triterpene saponins and phytosterols. SE acts as one of necessary devices for biosynthesis of triterpene saponins and phytosterols in vitro via synthetic biology approach. Here we cloned two genes encoding squalene epoxidase (PnSE1 and PnSE2) and analyzed the predict amino acid sequences by bioinformatic analysis. Further, we detected the gene expression profiling in different organs and the expression level of SEs in leaves elicited by methyl jasmonate (MeJA) treatment in 4-year-old P notoginseng using real-time quantitative PCR (real-time PCR). The study will provide a foundation for discovery and modification of devices in previous research by TCM synthetic biology. PnSE1 and PnSE2 encoded predicted proteins of 537 and 545 amino acids, respectively. Two amino acid sequences predicted from PnSEs shared strong similarity (79%), but were highly divergent in N-terminal regions (the first 70 amino acids). The genes expression profiling detected by real-time PCR, PnSE1 mRNA abundantly accumulated in all organs, especially in flower. PnSE2 was only weakly expressed and preferentially in flower. MeJA treatment enhanced the accumulation of PnSEI mRNA expression level in leaves, while there is no obvious enhancement of PnSE2 in same condition. Results indicated that the gene expressions of PnSE1 and PnSE2 were differently transcribed in four organs, and two PnSEs differently responded to MeJA stimuli. It was strongly suggested that PnSEs play different roles in secondary metabolite biosynthesis in P. notoginseng. PnSE1 might be involved in triterpenoid biosynthesis and PnSE2 might be involved in phytosterol biosynthesis.

Ganoderma lucidum : an Emerging Medicinal Model Fungus for Study of the Biosynthesis of Natural Medicines

Abstract: Model organisms play critical roles in life science At present, lack of mature model systems is one of the major bottlenecks that hinder the study of the biosynthesis of natural medicines Ganoderma lucidum is one of the most widely studied medicinal organisms and has the general features of model organisms: short life cycle, ability to produce large number of progenies, small genome size, easy to culture and genetically transform, and no harm for human being and the environment In addition, G lucidum involves multiple biosynthetic pathways of secondary metabolites, making it an ideal model to study the biosynthesis of secondary metabolites and their regulations Recently, the elucidation of the chromosome-level genome has laid the solid foundation for further application of G lucidum as a medicinal model organism G lucidum is supposed to play important roles in the study on the diversity of secondary metabolites, development of medicinal fungi and synthetic biology of natural medicines In addition, the proposal of G lucidum as a medicinal model fungus will greatly contribute to introducing state-of-the-art technologies and strategies of modern life sciences into the study of medicinal organisms and uncovering the common mechanisms and principles in the biosynthesis of secondary metabolites, thus paving the way for the construction of effective and controllable biosynthetic platform of natural medicines

Construction of the coexpression vector containing key element GLCYP450 involved in Ganoderma triterpene biosynthesis and its reductase gene GLNADPH

Abstract: Cytochrome P450 (CYP450) is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5' and 3' ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.