Screening and cloning of differentially expressed genes in Dendrobium nobile induced by orchid mycorrhizal fungus promoting the growth

Appropriate mycorrhizal fungi could effectively promote plant growth and development. Our previous research results showed that the growth of Dendrobium nobile was obviously promoted under inoculating one orchid mycorrhizal fungi, Epulorhiza sp. AR-18. To understand the growth-promoting molecular mechanisms, differential displayed real time polymerase chain reaction (DDRT-PCR), reverse Northern blot and Southern blot were used to isolate and identify genes whose transcription were altered in cultured D. nobile plants that were treated with Epulorhiza sp. AR-18. Amplified by 8 primer combinations from one anchor primers and 8 random primers, a total of 14 complementary DNA (cDNA) fragments including 12 differentially expressed cDNA bands were isolated. Reverse northern blot analysis showed that only 2 genes were differentially displayed cDNA bands. One band was an especially expressed fragment, expressed in the treated group but not in the control; while another was a differentially expressed fragment, weak in the treated and strength in the control. Southern blot analysis demonstrated that the two gene fragments were from the plant and not from the fungus. Sequence analysis and database searches revealed no significant homology to any known sequences. The results suggested that the usefulness of messenger RAN (mRNA) differential display technique for the detection of differentially expressed genes in D. nobile whose growth could be promoted by mycorrhizal fungi.Keywords: Dendrobium nobile, differential displayed real time polymerase chain reaction (DDRT-PCR), orchid mycorrhizal fungus, Epulorhiza sp., reverse northern blo

Untargeted metabolomics in Anectocillus roxburghii with habitat heterogeneity and the key abiotic factors affecting its active ingredients

IntroductionAnoectochilus roxburghii is a rare, endangered herb with diverse pharmacological properties. Understanding the main metabolite types and characteristics of wild A. roxburghii is important for efficiently utilizing resources and examining quality according to origin.MethodsSamples were collected from the main production areas across five regions in Fujian Province, China. An untargeted metabolomics analysis was performed on the entire plants to explore their metabolic profiles. We utilized UPLC-MS/MS to specifically quantify eight targeted flavonoids in these samples. Subsequently, correlation analysis was conducted to investigate the relationships between the flavonoids content and both the biological characteristics and geographical features.ResultsA comprehensive analysis identified a total of 3,170 differential metabolites, with terpenoids and flavonoids being the most prevalent classes. A region-specific metabolite analysis revealed that the Yongchun (YC) region showed the highest diversity of unique metabolites, including tangeretin and oleanolic acid. Conversely, the Youxi (YX) region was found to have the smallest number of unique metabolites, with only one distinct compound identified. Further investigation through KEGG pathway enrichment analysis highlighted a significant enrichment in pathways related to flavonoid biosynthesis. Further examination of the flavonoid category showed that flavonols were the most differentially abundant. We quantified eight specific flavonoids, finding that, on average, the YX region exhibited higher levels of these compounds. Correlation analysis highlighted a significant association between flavonoids and habitat, especially temperature and humidity.DiscussionUntargeted metabolomics via LC-MS was suitable for identifying region-specific metabolites and their influence via habitat heterogeneity. The results of this study serve as a new theoretical reference for unique markers exclusively present in a specific sample group

Stability improvement of multi-beam picosecond–petawatt laser system for ultrahigh peak-power applications

Pointing fluctuations of beams reduce the possibility of incoherent or coherent addition for ultrahigh peak power in a multi-beam picosecond–petawatt laser system. Pointing fluctuations on the target were observed on Shenguang Upgrade Petawatt (SGII-UP-Petawatt) beam using a high-speed and high-resolution active pointing stabilization control system. The maximum frequency of the pointing fluctuations was less than 50 Hz, and the amplitude was approximately 2.8 µrad (RMS). An online test of pointing fluctuations with active stabilization control demonstrated that pointing fluctuations could be reduced to 0.63 µrad (RMS), approximately one-quarter of that without active stabilization control. The benefits of reduced pointing fluctuation were estimated using a multi-beamlet petawatt laser system; the results demonstrated that peak power could be increased by 51.7% when active stabilization control was used in an eight-beamlet picosecond–petawatt laser system

ESTs Analysis of Putative Genes Engaged in Polyporus umbellatus Sclerotial Development

Polyporus umbellatus is one of the most widely used and precious medicinal fungi and the underground sclerotia are known to be with great medicinal value. However, the molecular mechanisms involved in sclerotial development are poorly understood. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of Polyporus umbellatus to identify genes expressing differently between mycelium and sclerotia. In this library, a total of 1202 clones were sequenced, assembled into 222 contigs and 524 singletons which were further searched against the NCBI nonredundant (NR) protein database (E-value cutoff, 10−5). Based on sequence similarity with known proteins, 378 sequences between mycelium and sclerotial were identified and classified into different functional categories through Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs). We have finally identified a majority of differentially expressed genes (constituting 5.6% of the present library) between the two different periods. An expression level of 32 selected expressed sequence tags (ESTs) generated from the above SSH cDNA library was studied through RT-PCR. This study provides the first global overview of genes putatively involved in Polyporus umbellatus sclerotial development and provides a preliminary basis for further functional research in terms of regulated gene expression in sclerotial production

Revealing proteins associated with symbiotic germination of Gastrodia elata by proteomic analysis

Abstract Background Gastrodia elata, a mycoheterotrophic orchid, is a well-known medicinal herb. In nature, the seed germination of G. elata requires proper fungal association, because of the absence of endosperm. To germinate successfully, G. elata obtains nutrition from mycorrhizal fungi such as Mycena. However, Mycena is not able to supply nutrition for the further development and enlargement of protocorms into tubers, flowering and fruit setting of G. elata. To date, current genomic studies on this topic are limited. Here we used the proteomic approach to explore changes in G. elata at different stages of symbiotic germination. Results Using mass spectrometry, 3787 unique proteins were identified, of which 599 were classified as differentially accumulated proteins. Most of these differentially accumulated proteins were putatively involved in energy metabolism, plant defense, molecular signaling, and secondary metabolism. Among them, the defense genes (e.g., pathogenesis-/wound-related proteins, peroxidases, and serine/threonine-protein kinase) were highly expressed in late-stage protocorms, suggesting that fungal colonization triggered the significant defense responses of G. elata. Conclusions The present study indicated the metabolic change and defensive reaction could disrupt the balance between Mycena and G. elata during mycorrhizal symbiotic germination

Identification of Anoectochilus based on rDNA ITS sequences alignment

The internal transcribed spacer (ITS) sequences alignment and proteomic difference of Anoectochilus interspecies have been studied by means of ITS molecular identification and surface enhanced laser desorption ionization time of flight mass spectrography. Results showed that variety certification on Anoectochilus by ITS sequences can not determine species, and there is proteomic difference among Anoectochilus interspecies. Moreover, proteomic finger printings of five Anoectochilus species have been established for identifying species, and genetic relationships of five species within Anoectochilus have been deduced according to proteomic differences among five species

Combined Metabolome and Transcriptome Analyses Reveal the Effects of Mycorrhizal Fungus Ceratobasidium sp. AR2 on the Flavonoid Accumulation in Anoectochilus roxburghii during Different Growth Stages

Anoectochilus roxburghii is a traditional Chinese herb with high medicinal value, with main bioactive constituents which are flavonoids. It commonly associates with mycorrhizal fungi for its growth and development. Moreover, mycorrhizal fungi can induce changes in the internal metabolism of host plants. However, its role in the flavonoid accumulation in A. roxburghii at different growth stages is not well studied. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in mycorrhizal A. roxburghii (M) and non-mycorrhizal A. roxburghii (NM) growth for six months. An association analysis revealed that flavonoid biosynthetic pathway presented significant differences between the M and NM. Additionally, the structural genes related to flavonoid synthesis and different flavonoid metabolites in both groups over a period of six months were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The results showed that Ceratobasidium sp. AR2 could increase the accumulation of five flavonol-glycosides (i.e., narcissin, rutin, isorhamnetin-3-O-beta-d-glucoside, quercetin-7-O-glucoside, and kaempferol-3-O-glucoside), two flavonols (i.e., quercetin and isorhamnetin), and two flavones (i.e., nobiletin and tangeretin) to some degrees. The qRT-PCR showed that the flavonoid biosynthetic genes (PAL, 4CL, CHS, GT, and RT) were significantly differentially expressed between the M and NM. Overall, our findings indicate that AR2 induces flavonoid metabolism in A. roxburghii during different growth stages, especially in the third month. This shows great potential of Ceratobasidium sp. AR2 for the quality improvement of A. roxburghii

Antimicrobial activities of endophytic fungi isolated from <it>Ophiopogon japonicus</it> (Liliaceae)

Abstract Background Drug resistance in bacteria has become a global concern and the search for new antibacterial agents is urgent and ongoing. Endophytes provide an abundant reservoir of bioactive metabolites for medicinal exploitation, and an increasing number of novel compounds are being isolated from endophytic fungi. Ophiopogon japonicus, containing compounds with antibacterial activity, is a traditional Chinese medicinal plant used for eliminating phlegm, relieving coughs, latent heat in the lungs, and alleviating diabetes mellitus. We investigated the antimicrobial activities of 30 strains of O. japonicus. Methods Fungal endophytes were isolated from roots and stems of O. japonicus collected from Chongqing City, southwestern China. Mycelial extracts (MC) and fermentation broth (FB) were tested for antimicrobial activity using peptide deformylase (PDF) inhibition fluorescence assays and MTT cell proliferation assays. Results A total of 30 endophytic strains were isolated from O. japonicus; 22 from roots and eight from stems. 53.33% of the mycelial extracts (MC) and 33.33% of the fermentation broths (FB) displayed potent inhibition of PDF. 80% of MC and 33.33% of FB significantly inhibited Staphylococcus aureus. 70% of MC and 36.67% of FB showed strong activities against Cryptococcus neoformans. None showed influence on Escherichia coli. Conclusion The secondary metabolites of endophytic fungi from O. japonicus are potential antimicrobial agents.</p