Baseline Survey of Root-Associated Microbes of \u3cem\u3eTaxus chinensis\u3c/em\u3e (Pilger) Rehd

Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis

The genome of Populus alba x Populus tremula var. glandulosa clone 84K

Poplar 84K (Populus alba x P. tremula var. glandulosa) is a fast-growing poplar hybrid. Originated in South Korea, this hybrid has been extensively cultivated in northern China. Due to the economic and ecological importance of this hybrid and high transformability, we now report the de novo sequencing and assembly of a male individual of poplar 84K using PacBio and Hi-C technologies. The final reference nuclear genome (747.5 Mb) has a contig N50 size of 1.99 Mb and a scaffold N50 size of 19.6 Mb. Complete chloroplast and mitochondrial genomes were also assembled from the sequencing data. Based on similarities to the genomes of P. alba var. pyramidalis and P. tremula, we were able to identify two subgenomes, representing 356 Mb from P. alba (subgenome A) and 354 Mb from P. tremula var. glandulosa (subgenome G). The phased assembly allowed us to detect the transcriptional bias between the two subgenomes, and we found that the subgenome from P. tremula displayed dominant expression in both 84K and another widely used hybrid, P. tremula x P. alba. This high-quality poplar 84K genome will be a valuable resource for poplar breeding and for molecular biology studies.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

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 9 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.Figure S1 Conserved domains of EgLBD protein family.Figure S2 The chromosomal localization of the LBD gene family in Eucalyptus grandis.Figure S3 Subcellular localization of EgLBD22, EgLBD29 and EgLBD37 proteins.Figure S4 Gel electrophoresis analysis for the presence of the transgene in EgLBD22-oe, EgLBD29-oe and EgLBD37-oe plants.Figure S5 Validation for the expression of the transgene in EgLBD22-oe, EgLBD29-oe and EgLBD37-oe plants by qRT-PCR.Table S1 All the primers used in this study.Table S2 The coding sequences of LBD genes in Eucalyptus grandis.Table S3 The information of LBD gene family in Eucalyptus grandis.Table S4 Conserved motifs predicted by MEME program in EgLBD proteins.Table S5 Protein-protein interaction prediction for possible functional protein association networks of EgLBD22.Table S6 Protein-protein interaction prediction for possible functional protein association networks of EgLBD29.Table S7 Protein-protein interaction prediction for possible functional protein association networks of EgLBD37.Table S8 The differentially expressed genes between EgLBD22-oe and WT-84k plants.Table S9 The differentially expressed genes between EgLBD29-oe and WT-84k plants.Table S10 The differentially expressed genes between EgLBD37-oe and WT-84k plants.Table S11 The information of eight key differentially expressed genes in EgLBD22-oe, EgLBD29-oe and EgLBD37-oe plants.Basic Research Fund of RIF [RIF2014-01]; Natural Science Foundation of China [31670676]; Mondi and Sappi through the Forest Molecular Genetics Programme; Technology and Human Resources for Industry Programme [UID 80118]; National Research Foundation of South Africa [UID 18312, 71255, 86936]http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1467-7652am2018Forestry and Agricultural Biotechnology Institute (FABI)Genetic

Genome sequencing and analysis of the paclitaxelproducing endophytic fungus \u3cem\u3ePenicillium aurantiogriseum\u3c/em\u3e 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

Factors influencing householder self-evacuation in two Australian bushfires

The thesis investigated householder self-evacuation decision-making during bushfires in the Perth and Adelaide Hills in 2014 and 2015. It explored the factors that influenced householders’ decisions to evacuate, identified factors that predict self-evacuation and established the characteristics of self-evacuators. The Protective Action Decision Model (PADM) provided a conceptual framework for the research. Its theoretical and analytical usefulness in an Australian context, was assessed. A mixed methods research strategy was used involving quantitative telephone surveys of 457 bushfire-affected participants and face-to-face interviews of 109 participants in 59 households. The study concluded that environmental and social cues and warnings and householders’ perceptions of the threat, of hazard adjustments and of other stakeholders, influenced self-evacuation decision-making. Protective action perceptions, particularly the effectiveness of evacuating or not evacuating in protecting personal safety or property, were most important in predicting self-evacuation. Receipt of official warnings and the perception of likely impact of the bushfire on property were also important predictors. Undertaking long-run hazard adjustments, although not predictive of self-evacuation, was pivotal in shaping perceptions of the effectiveness of evacuating and remaining in protecting personal safety and property and indirectly influenced evacuation decisions. Seven archetypes that characterised householders’ self-evacuation attitudes and behaviour were identified. These included Threat, and Responsibility Deniers, Dependent, and Considered Evacuators, Community Guided and Experienced Independents all who took different decisional ‘rules of thumb’ and routes toward evacuating or remaining . The PADM needs to be split into two separate models to incorporate the influence of long-run hazard adjustments on protective action decision-making in an Australian bushfire. The findings suggest that future research on those who wait and see during a bushfire should take account of their decisional rules of thumb and that design and targeting of Australian bushfire safety policy should better account for self-evacuator characteristics

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

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

<p>Abstract</p> <p>Background</p> <p><it>Siraitia grosvenorii </it>(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 <it>S. grosvenorii</it>, especially the late steps of the pathway.</p> <p>Results</p> <p>In this study, a cDNA library generated from of equal amount of RNA taken from <it>S. grosvenorii </it>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 <it>CYP450</it>s and five <it>UDPG</it>s were selected as the candidates most likely to be involved in mogrosides biosynthesis.</p> <p>Conclusion</p> <p>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 <it>CYP450</it>s and five <it>UDPG</it>s 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 <it>S. grosvenorii</it>.</p

Transcriptomic Analysis of <i>Betula halophila</i> 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 &#8216;Fatty acid elongation&#8217;, &#8216;Ribosome&#8217;, &#8216;Sphingolipid metabolism&#8217; and &#8216;Flavonoid biosynthesis&#8217;. 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

MULTI-TARGETS OPTIMIZATION DESIGN FOR MOVING CROSSBEAM OF MACHINE TOOL BASED ON ORTHOGONAL EXPERIMENTAL METHOD

In order to improve a machine moving beam’s comprehensive performances,which includes static and dynamic characteristics,it must have a reasonable beam ribs layout and structural properties. The crossbeam was simplified to get the mechanical vibration model,the maximum coupling deformation was solved by the theoretical calculation method. Using 3D software to establish the three-dimensional modeling of beam and ANSYS was used for static,modal analysis. Taking beam’s quality and the maximum coupling deformation,the maximum coupling stress,the first-order frequency as objective functions to judge the merits and drawbacks of crossbeam’s optimization,taking the crossbeam’s rib structure,the rib thickness,the angle of support stiffened plate as design variables for multi-objective optimization design. Orthogonal experimental method was used to design the orthogonal experiment with three levels and four factors. Using overall balance method to chose the optimal level,and then getting the best test program. The results show that: Theoretical analysis and simulation results are very similar. the beam with "井"type rib structure,rib thickness of 25 mm,the support stiffened plate tilted 55 ° design is the optimal solution.Compared with the original design,the beam deformation reduced by 7. 455% and the first-order frequency increased by 2. 956%,while the quality reduced by 473 kg. It shows that using the orthogonal experimental method to design reasonable levels and factors can achieve optimum results with fewer tests,and the orthogonal experimental method is valuable for engineering application