Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla.

Uncaria rhynchophylla is an evergreen vine plant, belonging to the Rubiaceae family, that is rich in terpenoid indole alkaloids (TIAs) that have therapeutic effects on hypertension and Alzheimer's disease. GATA transcription factors (TF) are a class of transcription regulators that participate in the light response regulation, chlorophyll synthesis, and metabolism, with the capability to bind to GATA cis-acting elements in the promoter region of target genes. Currently the charactertics of GATA TFs in U. rhynchophylla and how different light qualities affect the expression of GATA and key enzyme genes, thereby affecting the changes in U. rhynchophylla alkaloids have not been investigated. In this study, 25 UrGATA genes belonging to four subgroups were identified based on genome-wide analysis. Intraspecific collinearity analysis revealed that only segmental duplications were identified among the UrGATA gene family. Collinearity analysis of GATA genes between U. rhynchophylla and four representative plant species, Arabidopsis thaliana, Oryza sativa, Coffea Canephora, and Catharanthus roseus was also performed. U. rhynchophylla seedlings grown in either red lights or under reduced light intensity had altered TIAs content after 21 days. Gene expression analysis reveal a complex pattern of expression from the 25 UrGATA genes as well as a number of key TIA enzyme genes. UrGATA7 and UrGATA8 were found to have similar expression profiles to key enzyme TIA genes in response to altered light treatments, implying that they may be involved in the regulation TIA content. In this research, we comprehensively analyzed the UrGATA TFs, and offered insight into the involvement of UrGATA TFs from U. rhynchophylla in TIAs biosynthesis.

Identification and characterization of two O-methyltransferases involved in biosynthesis of methylated 2-(2-phenethyl)chromones in agarwood.

The 2-(2-phenethyl)chromones (PECs) are the signature constituents responsible for the fragrance and pharmacological properties of agarwood. O-Methyltransferases (OMTs) are necessary for the biosynthesis of methylated PECs, but there is little known about OMTs in Aquilaria sinensis. In this study, we identified 29 OMT genes from the A. sinensis genome. Expression analysis showed they were differentially expressed in different tissues and responded to drill wounding. Comprehensive analysis of the gene expression and methylated PEC content revealed that AsOMT2, AsOMT8, AsOMT11, AsOMT16, and AsOMT28 could potentially be involved in methylated PECs biosynthesis. In vitro enzyme assays and functional analysis in Nicotiana benthamiana demonstrated that AsOMT11 and AsOMT16 could methylate 6-hydroxy-2-(2-phenylethyl)chromone to form 6-methoxy-2-(2-phenylethyl)chromone. A transient overexpression experiment in the variety 'Qi-Nan' revealed that AsOMT11 and AsOMT16 could significantly promote the accumulation of three major methylated PECs. Our results provide candidate genes for the mass production of methylated PECs using synthetic biology.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis.

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

Genomewide analysis of the lateral organ boundaries domain gene family in Eucalyptus grandis reveals members that differentially impact secondary growth.

Lateral Organ Boundaries Domain (LBD) proteins are plant-specific transcription factors playing crucial roles in growth and development. However, the function of LBD proteins in Eucalyptus grandis remains largely unexplored. In this study, LBD genes in E. grandis were identified and characterized using bioinformatics approaches. Gene expression patterns in various tissues and the transcriptional responses of EgLBDs to exogenous hormones were determined by qRT-PCR. Functions of the selected EgLBDs were studied by ectopically overexpressing in a hybrid poplar (Populus alba × Populus glandulosa). Expression levels of genes in the transgenic plants were investigated by RNA-seq. Our results showed that there were forty-six EgLBD members in the E. grandis genome and three EgLBDs displayed xylem- (EgLBD29) or phloem-preferential expression (EgLBD22 and EgLBD37). Confocal microscopy indicated that EgLBD22, EgLBD29 and EgLBD37 were localized to the nucleus. Furthermore, we found that EgLBD22, EgLBD29 and EgLBD37 were responsive to the treatments of indol-3-acetic acid and gibberellic acid. More importantly, we demonstrated EgLBDs exerted different influences on secondary growth. Namely, 35S::EgLBD37 led to significantly increased secondary xylem, 35S::EgLBD29 led to greatly increased phloem fibre production, and 35S::EgLBD22 showed no obvious effects. We revealed that key genes related to gibberellin, ethylene and auxin signalling pathway as well as cell expansion were significantly up- or down-regulated in transgenic plants. Our new findings suggest that LBD genes in E. grandis play important roles in secondary growth. This provides new mechanisms to increase wood or fibre production.

Transcriptomic Analysis of Betula halophila in Response to Salt Stress.

Soil salinization is a matter of concern worldwide. It can eventually lead to the desertification of land and severely damage local agricultural production and the ecological environment. Betula halophila is a tree with high salt tolerance, so it is of importance to understand and discover the salt responsive genes of B. halophila for breeding salinity resistant varieties of trees. However, there is no report on the transcriptome in response to salt stress in B. halophila. Using Illumina sequencing platform, approximately 460 M raw reads were generated and assembled into 117,091 unigenes. Among these unigenes, 64,551 unigenes (55.12%) were annotated with gene descriptions, while the other 44.88% were unknown. 168 up-regulated genes and 351 down-regulated genes were identified, respectively. These Differentially Expressed Genes (DEGs) involved in multiple pathways including the Salt Overly Sensitive (SOS) pathway, ion transport and uptake, antioxidant enzyme, ABA signal pathway and so on. The gene ontology (GO) enrichments suggested that the DEGs were mainly involved in a plant-type cell wall organization biological process, cell wall cellular component, and structural constituent of cell wall molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment showed that the top-four enriched pathways were 'Fatty acid elongation', 'Ribosome', 'Sphingolipid metabolism' and 'Flavonoid biosynthesis'. The expression patterns of sixteen DEGs were analyzed by qRT-PCR to verify the RNA-seq data. Among them, the transcription factor AT-Hook Motif Nuclear Localized gene and dehydrins might play an important role in response to salt stress in B. halophila. Our results provide an important gene resource to breed salt tolerant plants and useful information for further elucidation of the molecular mechanism of salt tolerance in B. halophila.

Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that are important regulators of gene expression, and play major roles in plant development and their response to the environment. Root extracts from Panax notoginseng contain triterpene saponins as their principal bioactive constituent, and demonstrate medicinal properties. To investigate the novel and conserved miRNAs in P. notoginseng, three small RNA libraries constructed from 1-, 2-, and 3-year-old roots in which root saponin levels vary underwent high-throughput sequencing. METHODS: P. notoginseng roots, purified from 1-, 2-, and 3-year-old roots, were extracted for RNA, respectively. Three small libraries were constructed and subjected to next generation sequencing. RESULTS: Sequencing of the three libraries generated 67,217,124 clean reads from P. notoginseng roots. A total of 316 conserved miRNAs (belonging to 67 miRNA families and one unclassified family) and 52 novel miRNAs were identified. MIR156 and MIR166 were the largest miRNA families, while miR156i and miR156g showed the highest abundance of miRNA species. Potential miRNA target genes were predicted and annotated using Cluster of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Comparing these miRNAs between root samples revealed 33 that were differentially expressed between 2- and 1-year-old roots (8 increased, 25 decreased), 27 differentially expressed between 3- and 1-year-old roots (7 increased, 20 decreased), and 29 differentially expressed between 3- and 2-year-old roots (8 increased, 21 decreased). Two significantly differentially expressed miRNAs and four miRNAs predicted to target genes involved in the terpenoid backbone biosynthesis pathway were selected and validated by quantitative reverse transcription PCR. Furthermore, the expression patterns of these six miRNAs were analyzed in P. notoginseng roots, stems, and leaves at different developmental stages. CONCLUSIONS: This study identified a large number of P. notoginseng miRNAs and their target genes, functional annotations, and gene expression patterns. It provides the first known miRNA profiles of the P. notoginseng root development cycle.

De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-alzheimer's properties.

BACKGROUND: The major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid ß protein (a pathological marker of Alzheimer's disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown. RESULTS: In this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis. CONCLUSION: A combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria, and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.

Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431.

BACKGROUND: Paclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer. RESULTS: The ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely. CONCLUSIONS: Our findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells.

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.

Genome-Wide Mining of CULLIN E3 Ubiquitin Ligase Genes from Uncaria rhynchophylla.

CULLIN (CUL) protein is a subtype of E3 ubiquitin ligase that is involved in a variety of biological processes and responses to stress in plants. In Uncaria rhynchophylla, the CUL gene family has not been identified and its role in plant development, stress response and secondary metabolite synthesis has not been studied. In this study, 12 UrCUL gene members all contained the typical N-terminal domain and C-terminal domain identified from the U. rhynchophylla genome and were classified into four subfamilies based on the phylogenetic relationship with CULs in Arabidopsis thaliana. They were unevenly distributed on eight chromosomes but had a similar structural composition in the same subfamily, indicating that they were relatively conserved and potentially had similar gene functions. An interspecific and intraspecific collinearity analysis showed that fragment duplication played an important role in the evolution of the CUL gene family. The analysis of the cis-acting elements suggests that the UrCULs may play an important role in various biological processes, including the abscisic acid (ABA) response. To investigate this hypothesis, we treated the roots of U. rhynchophylla tissue-cultured seedlings with ABA. The expression pattern analysis showed that all the UrCUL genes were widely expressed in roots with various expression patterns. The co-expression association analysis of the UrCULs and key enzyme genes in the terpenoid indole alkaloid (TIA) synthesis pathway revealed the complex expression patterns of 12 UrCUL genes and some key TIA enzyme genes, especially UrCUL1, UrCUL1-likeA, UrCUL2-likeA and UrCUL2-likeB, which might be involved in the biosynthesis of TIAs. The results showed that the UrCULs were involved in the response to ABA hormones, providing important information for elucidating the function of UrCULs in U. rhynchophylla. The mining of UrCULs in the whole genome of U. rhynchophylla provided new information for understanding the CUL gene and its function in plant secondary metabolites, growth and development.

Genome-Wide Identification and Expression Analysis of WRKY Transcription Factors in Siraitia siamensis.

WRKY transcription factors, as the largest gene family in higher plants, play an important role in various biological processes including growth and development, regulation of secondary metabolites, and stress response. In this study, we performed genome-wide identification and analysis of WRKY transcription factors in S. siamensis. A total of 59 SsWRKY genes were identified that were distributed on all 14 chromosomes, and these were classified into three major groups based on phylogenetic relationships. Each of these groups had similar conserved motifs and gene structures. We compared all the S. siamensis SsWRKY genes with WRKY genes identified from three diverse plant species, and the results implied that segmental duplication and tandem duplication play an important roles in the evolution processes of the WRKY gene family. Promoter region analysis revealed that SsWRKY genes included many cis-acting elements related to plant growth and development, phytohormone response, and both abiotic and biotic stress. Expression profiles originating from the transcriptome database showed expression patterns of these SsWRKY genes in four different tissues and revealed that most genes are expressed in plant roots. Fifteen SsWRKY genes with low-temperature response motifs were surveyed for their gene expression under cold stress, showing that most genes displayed continuous up-regulation during cold treatment. Our study provides a foundation for further study on the function and regulatory mechanism of the SsWRKY gene family.

De novo sequencing and a comprehensive analysis of purple sweet potato (Impomoea batatas L.) transcriptome.

High-throughput RNA sequencing was performed for comprehensively analyzing the transcriptome of the purple sweet potato. A total of 58,800 unigenes were obtained and ranged from 200 nt to 10,380 nt with an average length of 476 nt. The average expression of one unigene was 34 reads per kb per million reads (RPKM) with a maximum expression of 1,935 RPKM. At least 40,280 (68.5%) unigenes were identified to be protein-coding genes, in which 11,978 and 5,184 genes were homologous to Arabidopsis and rice proteins, respectively. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that 19,707 (33.5%) unigenes were classified to 1,807 terms of GO including molecular functions, biological processes, and cellular components and 9,970 (17.0%) unigenes were enriched to 11,119 KEGG pathways. We found that at least 3,553 genes may be involved in the biosynthesis pathways of starch, alkaloids, anthocyanin pigments, and vitamins. Additionally, 851 potential simple sequence repeats (SSRs) were identified in all unigenes. Transcriptome sequencing on tuberous roots of the sweet potato yielded substantial transcriptional sequences and potentially useful SSR markers which provide an important data source for sweet potato research. Comparison of two RNA-sequence datasets from the purple and the yellow sweet potato showed that UDP-glucose-flavonoid 3-O-glucosyltransferase was one of the key enzymes in the pathway of anthocyanin biosynthesis and that anthocyanin-3-glucoside might be one of the major components for anthocyanin pigments in the purple sweet potato. This study contributes to the molecular mechanisms of sweet potato development and metabolism and therefore that increases the potential utilization of the sweet potato in food nutrition and pharmacy.

Genome-Wide Identification of m6A Writers, Erasers and Readers in Poplar 84K.

N6-methyladenosine (m6A) RNA modification is a conserved mechanism to regulate gene expression that plays vital roles in the development of plants. However, the m6A RNA modification in forest trees remains limited. Here, we performed a complete analysis of m6A writers, erasers and readers in Poplar 84K, including gene location, gene structures, conserved motifs, phylogenetic relationships, promoter analysis, expression profiles and the homology modeling. We have identified 61 m6A pathway genes in Poplar 84K (Populus alba × Populus glandulosa), including 14 m6A writers, 14 m6A erasers and 33 m6A readers. Phylogenetic analysis indicated that the m6A writers and erasers were clustered into four groups and m6A readers were clustered into two groups. Promoter analysis showed that m6A pathway genes were mainly responsive to low oxygen followed by ABA and ethylene. The expression of the identified m6A pathway genes showed tissue-specific expression patterns in leaves, xylem, phloem and roots. Moreover, 17 genes were significantly up-regulated and 13 genes were significantly down-regulated in poplar overexpressing the transcription factor LBD15. Homology modeling and molecular docking results suggested that PagFIP37b was most likely to be regulated by LBD15, and the qPCRshowed that PagFIP37b were up-regulated in the LBD15-oe plants. The results provide insights that aid in the future elucidation of the functions of these m6A pathway genes and the epigenetic regulation mechanism of these genes in Poplar 84K.

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis.

BACKGROUND: Siraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway. RESULTS: In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis. CONCLUSION: A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven CYP450s and five UDPGs were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from S. grosvenorii.

De novo assembly of Euphorbia fischeriana root transcriptome identifies prostratin pathway related genes.

BACKGROUND: Euphorbia fischeriana is an important medicinal plant found in Northeast China. The plant roots contain many medicinal compounds including 12-deoxyphorbol-13-acetate, commonly known as prostratin that is a phorbol ester from the tigliane diterpene series. Prostratin is a protein kinase C activator and is effective in the treatment of Human Immunodeficiency Virus (HIV) by acting as a latent HIV activator. Latent HIV is currently the biggest limitation for viral eradication. The aim of this study was to sequence, assemble and annotate the E. fischeriana transcriptome to better understand the potential biochemical pathways leading to the synthesis of prostratin and other related diterpene compounds. RESULTS: In this study we conducted a high throughput RNA-seq approach to sequence the root transcriptome of E. fischeriana. We assembled 18,180 transcripts, of these the majority encoded protein-coding genes and only 17 transcripts corresponded to known RNA genes. Interestingly, we identified 5,956 protein-coding transcripts with high similarity (> = 75%) to Ricinus communis, a close relative to E. fischeriana. We also evaluated the conservation of E. fischeriana genes against EST datasets from the Euphorbeacea family, which included R. communis, Hevea brasiliensis and Euphorbia esula. We identified a core set of 1,145 gene clusters conserved in all four species and 1,487 E. fischeriana paralogous genes. Furthermore, we screened E. fischeriana transcripts against an in-house reference database for genes implicated in the biosynthesis of upstream precursors to prostratin. This identified 24 and 9 candidate transcripts involved in the terpenoid and diterpenoid biosyntehsis pathways, respectively. The majority of the candidate genes in these pathways presented relatively low expression levels except for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) and isopentenyl diphosphate/dimethylallyl diphosphate synthase (IDS), which are required for multiple downstream pathways including synthesis of casbene, a proposed precursor to prostratin. CONCLUSION: The resources generated in this study provide new insights into the upstream pathways to the synthesis of prostratin and will likely facilitate functional studies aiming to produce larger quantities of this compound for HIV research and/or treatment of patients.

Synthesis of water soluble quantum dots for monitoring carrier-DNA nanoparticles in plant cells.

Fluorescent quantum dots (QDs) have shown great promise for use as biolabels in cell and animal biology and more recently in plant sciences. An important use of QDs is for monitoring the dynamics, intracellular trafficking, and fate of carrier-DNA nanocomplexes in cell transfection and potentially in plant transformation. In this study, a low cost aqueous procedure has been developed to efficiently prepare biocompatible QDs for monitoring nanoparticle-mediated gene transfer in conjunction with molecular breeding of Jatropha curcas. Water-soluble CdSe nanoparticles were synthesized by self-assembly using L-Cysteine as stabilizer and optimal synthesis scheme established by fluorescence spectroscopy. The QDs were used to label chitosan-DNA nanoparticles via electrostatic interaction and the resultant QD-labeled chitosan-DNA complexes were shown to have superior fluorescence properties with red shift of emission and absorption spectra relative to the CdSe QDs alone. This system is being explored as a superior alternative to Agrobacterium-mediated genetic transformation of Jatropha curcas cells. PCR amplification of the full length of the carried reporter gene (GFP) suggests that the DNA was not digested in Jatropha curcas cells transfected with CdSe/CS-DNA complexes. Furthermore, GFP gene expression in the transfected callus cells, as evidenced by fluorescence detection, suggests that the target DNA was integrated into the plant genome.

The Research Progress of Taxol in Taxus.

BACKGROUND: Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production. OBJECTIVE: In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed. RESULTS: The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol. CONCLUSION: Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants.

The R2R3-MYB transcription factor family in Taxus chinensis: identification, characterization, expression profiling and posttranscriptional regulation analysis.

The MYB transcription factor family is one of the largest gene families playing regulatory roles in plant growth and development. The MYB family has been studied in a variety of plant species but has not been reported in Taxus chinensis. Here we identified 72 putative R2R3-MYB genes in T. chinensis using a comprehensive analysis. Sequence features, conversed domains and motifs were characterized. The phylogenetic analysis showed TcMYBs and AtMYBs were clustered into 36 subgroups, of which 24 subgroups included members from T. chinensis and Arabidopsis thaliana, while 12 subgroups were specific to one species. This suggests the conservation and specificity in structure and function of plant R2R3-MYBs. The expression of TcMYBs in various tissues and different ages of xylem were investigated. Additionally, miRNA-mediated posttranscriptional regulation analysis revealed that TcMYBs were the targets of miR858, miR159 and miR828, suggesting the posttranscriptional regulation of MYBs is highly conserved in plants. The results provide a basis for further study the role of TcMYBs in the regulation of secondary metabolites of T. chinensis.