[Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata].

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

itol.toolkit accelerates working with iTOL (Interactive Tree of Life) by an automated generation of annotation files.

SUMMARY: iTOL is a powerful and comprehensive phylogenetic tree visualization engine. However, adjusting to new templates can be time-consuming, especially when many templates are available. We developed an R package namely itol.toolkit to help users generate all 23 types of annotation files in iTOL. This R package also provides an all-in-one data structure to store data and themes, accelerating the step from metadata to annotation files of iTOL visualizations through automatic workflows. AVAILABILITY AND IMPLEMENTATION: The manual and source code are available at https://github.com/TongZhou2017/itol.toolkit.

PuCRZ1, an C2H2 transcription factor from Polyporus umbellatus, positively regulates mycelium response to osmotic stress.

Polyporus umbellatus is an edible and medicinal mushroom with the capacity to produce sclerotia. However, the mechanism of P. umbellatus sclerotia formation is unclear. CRZ1 is a C2H2 family transcription factor involved in the Ca2+-calcineurin signaling pathway, which has the function of regulating sclerotia formation, maintaining ion homeostasis, and responding to stress. In this study, we identified 28 C2H2 transcription factors in P. umbellatus genome, 13 of which are differentially expressed between mycelium and sclerotia, including PuCRZ1. Combining DNA affinity purification and sequencing (DAP-seq) and quantitative real-time PCR (qRT-PCR), three genes (PuG10, PuG11, PuG12) were identified as putative PuCRZ1 target genes containing a putative binding motif (GTGGCG) within their promoter. Yeast single hybridization (Y1H) and EMSA further confirmed that PuCRZ1 can bind to the promoter region of PuG10, PuG11, and PuG12. PuCRZ1 gene could reduce the sensitivity of NaCl in yeast cells. Furthermore, overexpression of the PuCRZ1 target gene, especially the FVLY domain containing gene PuG11, could improve the mycelia growth rate and mannitol tolerance in P. umbellatus. These results demonstrate that PuCRZ1 in the Ca2+-calcineurin signaling pathway plays an important role in mycelia growth, as well as osmotic stress tolerance.

Genome-wide analysis of Panax MADS-box genes reveals role of PgMADS41 and PgMADS44 in modulation of root development and ginsenoside synthesis.

Panax root is an important material used in food and medicine. Its cultivation and production usually depend on root shape and ginsenoside content. There is limited understanding about the synergistic regulatory mechanisms underlying root development and ginsenoside accumulation in Panax. MADS-box transcription factors possibly play a significant role in regulation of root growth and secondary metabolites. In this study, we identified MADS-box transcription factors of Panax, and found high expression levels of SVP, ANR1 and SOC1-like clade genes in its roots. We confirmed that two SOC1-like genes, PgMADS41 and PgMADS44, bind to expansion gene promoters (PgEXLB5 and PgEXPA13), which contribute to root growth, and to SE-4, CYP716A52v2-4, and ß-AS-13 promoters, which participate in ginsenoside Ro biosynthesis. These two genes were found to increase lateral root number and main root length in transgenic Arabidopsis thaliana by improving AtEXLA1, AtEXLA3, AtEXPA5, and AtEXPA6 gene expression. As a non-phytohormone regulatory tool, Ro can stimulate adventitious root growth by influencing their expression and ginsenoside accumulation. Our study provides new insights into the coordinated regulatory function of SOC1-like clade genes in Panax root development and triterpenoid accumulation, paving the way towards understanding root formation and genetic improvement in Panax.

Accurate identification of taxon-specific molecular markers in plants based on DNA signature sequence.

Accurate identification of plants remains a significant challenge for taxonomists and is the basis for plant diversity conservation. Although DNA barcoding methods are commonly used for plant identification, these are limited by the low amplification success and low discriminative power of selected genomic regions. In this study, we developed a k-mer-based approach, the DNA signature sequence (DSS), to accurately identify plant taxon-specific markers, especially at the species level. DSS is a constant-length nucleotide sequence capable of identifying a taxon and distinguishing it from other taxa. In this study, we performed the first large-scale study of DSS markers in plants. DSS candidates of 3899 angiosperm plant species were calculated based on a chloroplast data set with 4356 assemblies. Using Sanger sequencing of PCR amplicons and high-throughput sequencing, DSSs were validated in four and 165 species, respectively. Based on this, the universality of the DSSs was over 79.38%. Several indicators influencing DSS marker identification and detection have also been evaluated, and common criteria for DSS application in plant identification have been proposed.

Isolation, genomic characterization, and mushroom growth-promoting effect of the first fungus-derived Rhizobium.

Polyporus umbellatus is a well-known edible and medicinal mushroom, and some bacteria isolated from mushroom sclerotia may have beneficial effects on their host. These mushroom growth-promoting bacteria (MGPBs) are of great significance in the mushroom production. In this work, we aimed to isolate and identify MGPBs from P. umbellatus sclerotia. Using the agar plate dilution method, strain CACMS001 was isolated from P. umbellatus sclerotia. The genome of CACMS001 was sequenced using PacBio platform, and the phylogenomic analysis indicated that CACMS001 could not be assigned to known Rhizobium species. In co-culture experiments, CACMS001 increased the mycelial growth of P. umbellatus and Armillaria gallica and increased xylanase activity in A. gallica. Comparative genomic analysis showed that CACMS001 lost almost all nitrogen fixation genes but specially acquired one redox cofactor cluster with pqqE, pqqD, pqqC, and pqqB involved in the synthesis of pyrroloquinoline quinone, a peptide-derived redox participating in phosphate solubilization activity. Strain CACMS001 has the capacity to solubilize phosphate using Pikovskaya medium, and phnA and phoU involved in this process in CACMS001 were revealed by quantitative real-time PCR. CACMS001 is a new potential Rhizobium species and is the first identified MGPB belonging to Rhizobium. This novel bacterium would play a vital part in P. umbellatus, A. gallica, and other mushroom cultivation.

Increasing Expression of PnGAP and PnEXPA4 Provides Insights Into the Enlargement of Panax notoginseng Root Size From Qing Dynasty to Cultivation Era.

Root size is a key trait in plant cultivation and can be influenced by the cultivation environment. However, physical evidence of root size change in a secular context is scarce due to the difficulty in preserving ancient root samples, and how they were modified during the domestication and cultivation stays unclear. About 100 ancient root samples of Panax notoginseng, preserved as tribute in the Palace Museum (A.D. 1636 to 1912, Qing dynasty), provided an opportunity to investigate the root size changes during the last 100 years of cultivation. The dry weight of ancient root samples (~120 tou samples, tou represents number of roots per 500 g dry weight) is 0.22-fold of the modern samples with the biggest size (20 tou samples). Transcriptome analysis revealed that PnGAP and PnEXPA4 were highly expressed in 20 tou samples, compared with the 120 tou samples, which might contribute to the thicker cell wall and a higher content of lignin, cellulose, and callose in 20 tou samples. A relatively lower content of dencichine and higher content of ginsenoside Rb1 in 20 tou samples are also consistent with higher expression of ginsenoside biosynthesis-related genes. PnPHL8 was filtrated through transcriptome analysis, which could specifically bind the promoters of PnGAP, PnCYP716A47, and PnGGPPS3, respectively. The results in this study represent the first physical evidence of root size changes in P. notoginseng in the last 100 years of cultivation and contribute to a comprehensive understanding of how the cultivation environment affected root size, chemical composition, and clinical application.

Mycorrhizosphere Bacteria, Rahnella sp. HPDA25, Promotes the Growth of Armillaria gallica and Its Parasitic Host Gastrodia elata.

Gastrodia elata is an entirely heterotrophic plant, the growth of which is completely reliant on Armillaria gallica, an orchid mycorrhizal fungus. To avoid damaging ecosystems, G. elata cultivation is shifting from woodland to farmland. However, whether the microbial community structure remains stable during this conversation is unknown. Here, we cultivated G. elata in woodland or farmland and found that woodland-cultivated G. elata produced a greater yield and larger tuber size. The relative abundance of Rahnella was 22.84- and 122.25-fold higher in woodland- and farmland-cultivated soil samples, respectively, than that in uncultivated soil samples. To investigate how Rahnella impacts the growth of G. elata and establishes symbiosis with Armillaria gallica, three Rahnella spp. strains (HPDA25, SBD3, and SBD11) were isolated from mycorrhizosphere soil samples. It was found that these strains, especially HPDA25, promoted the growth of A. gallica. Ultra-performance liquid chromatography coupled to a triple quadrupole mass spectrometry analysis detected the indole-3-acetic acid with 16.24 ng/ml in HPDA25 fermentation solution. Co-culturing with the strain HPDA25 or exogenous indole-3-acetic acid increased the branching and fresh weight of rhizomorphs and the growth rate and extracellular laccase activity of A. gallica, compared with A. gallica cultured alone. The results of RNA-seq and quantitative real-time polymerase chain reaction analysis showed that co-culturing A. gallica with HPDA25 increased the expression level of the genes including hydrophobin, SUR7/PalI family, and pectin methylesterase, whereas decreased the expression levels of glycolysis-related genes. Furthermore, co-culturing with the strain HPDA25, A. gallica promotes the growth of G. elata and enhances the tuber size of G. elata. These results provide new insights into an orchid mycorrhizal symbiosis and the cultivation of G. elata.

[Differences between male and female leaves of Schisandra sphenanthera: based on RNA-Seq].

Schisandra sphenanthera is dioecious and only the fruits of female plants can be used as medicine and food. It is of great significance for the cultivation and production of S. sphenanthera to explore the differences between male and female plants at the non-flowering stage and develop the identification markers at non-flowering or seedling stage. In this study, the transcriptome of male and female leaves of S. sphenanthera at the non-flowering stage was sequenced by Illumina high-throughput sequencing technology and analyzed based on bioinformatics. A total of 236 682 transcripts were assembled by Trinity software and 171 588 were chosen as unigenes. Finally, 1 525 differentially expressed genes(DEGs) were identified, with 458 up-regulated and 1 067 down-regulated in female lea-ves. The down-regulated genes mainly involve photosynthesis, photosynthesis-antenna protein, carbon fixation in photosynthetic or-ganisms, and other pathways. Real-time quantitative PCR(qPCR) identified two genes between male and female leaves and one of them was a HVA22-like gene related to floral organ development and abscisic acid(ABA). Enzyme linked immunosorbent assay(ELISA) was applied to determine the content of ABA, auxin, gibberellin, and zeatin riboside(ZR) in leaves of S. sphenanthera. The results showed that the content of ABA and ZR in male leaves was significantly higher than that in female leaves. The involvement of down-regulated genes in female leaves in the photosynthesis pathway and the significant differences in the content of endogenous hormones between male and female leaves lay a scientific basis for analyzing the factors affecting sex differentiation of S. sphenanthera.

[Bioinformatics analysis and function prediction of ginseng TCP transcription factor family].

The longevity mechanism of ginseng(Panax ginseng) is related to its strong meristematic ability. In this paper, this study used bioinformatic methods to identify the members of the ginseng TCP gene family in the whole genome and analyzed their sequence characteristics. Then, quantitative real-time fluorescent PCR was performed to analyze the TCP genes containing elements rela-ted to meristem expression in the taproots, fibrous roots, stems, and leaves. According to the data, this study further explored the expression specificity of TCP genes in ginseng tissues, which facilitated the dissection of the longevity mechanism of ginseng. The ginseng TCP members were identified and analyzed using PlantTFDB, ExPASy, MEME, PLANTCARE, TBtools, MEGA and DNAMAN. The results demonstrated that there were 60 TCP gene family members in ginseng, and they could be divided into two classes: Class Ⅰ and Class Ⅱ, in which the Class Ⅱ possessed two subclasses: CYC-TCP and CIN-TCP. The deduced TCP proteins in ginseng had the length of 128-793 aa, the isoelectric point of 4.49-9.84 and the relative molecular mass of 14.2-89.3 kDa. They all contained the basic helix-loop-helix(bHLH) domain. There are a variety of stress response-related cis-acting elements in the promoter regions of ginseng TCP genes, and PgTCP20-PgTCP24 contained the elements associated with meristematic expression. The transcription levels of PgTCP20-PgTCP24 were high in fibrous roots and leaves, but low in stems, indicating the tissue-specific expression of ginseng TCP genes. The Class Ⅰ TCP members which contained PgTCP20-PgTCP23, may be important regulators for the growth and development of ginseng roots.

The third complete chloroplast genome of Melicope pteleifolia (Rutaceae): a widely used folk medicinal herb.

Melicope pteleifolia commonly known as thin evodia, is an herb used to therapy eczema, dermatitis, and other ailments in traditional Chinese medicine. Here, we reported the third complete chloroplast genome of M. pteleifolia based on next-generation sequencing. The third chloroplast genome of M. pteleifolia is 158,933 bp in length consisting of large and small single-copy regions of length 85,020 and 18,607 bp, separated by two IR regions of 27,683 bp. The overall GC content was 38.30%. De novo assembly and annotation showed the chloroplast genome of M. pteleifolia encodes 134 genes, including 89 protein-coding genes, 37 tRNA genes, and eight rRNA genes. A huge intraspecies variation was found with 248 SNPs and 97 INDELs among three assemblies of M. pteleifolia. Phylogenetic tree indicated that three assemblies of M. pteleifolia form a clade, sister to the genus Phellodendron and Casimiroa.

Complete chloroplast genome sequence of Murraya paniculata (Rutaceae): a widely used folk medicinal herb.

Murraya paniculate, is traditionally used for management of gut, air way and cardiovascular disorders. In this study, we sequenced the complete chloroplast genome of M. paniculata based on next-generation sequencing and used the data to assess genomic resources. The chloroplast genome of M. paniculata is 160,280 bp in length consisting of large and small single-copy regions of length 87,605 and 18,609 bp, separated by two IR regions of 27,033 bp. The overall GC content was 38.61%. De novo assembly and annotation showed the presence of unique genes with 85 protein-coding genes, 29 tRNA genes, and eight rRNA genes. A maximum-likelihood phylogenomic analysis showed that M. paniculata was closely related to M. caloxylon.

[Quantitative taxonomic study on agronomic traits of cultivated Gastrodia elata].

To better understand the formation mechanism of Gastrodia elata traits, the agronomic traits of aboveground tissues and tubers were measured and analyzed in this study. It has shown that the color and thickness of the stems of the 39 samples of the G. elata collected are affected by the germplasm and variation. Clustering analysis of 39 agronomic traits of G. elata was conducted with Ward's method and Euclidean distance. The threshold of 11.0 was divided into three groups, namely hybrid G. elata, G. elata f. elata and G. elata f. glauca. Simultaneously, the correlation analysis, coefficient of variation analysis, factor analysis and cluster analysis of 13 agronomic traits of 105 G. elata tuber samples were carried out. The results showed that the weight of G. elata was significantly positively correlated with tuber length and width. The agronomic traits of tuber were highly variable, and the depth of variability of the scar was the largest and 13 agronomic traits could be divided into 6 types of factors and the contribution up to 89.348%, furthermore, tuber length factor, width and weight factor contributed more than 20%, indicating that it is of great significance for distinguishing G. elata germplasm. Cluster analysis was performed by Ward's method and Euclidean distance, with 8.0 as the threshold can be divided into three categories in the light of the origin of the source, 33 samples from Shanxi and Hubei are clustered into one category, and 19 samples from Yunnan and Guizhou are clustered into one group, and the remaining samples are grouped into one category. This study will provide a basis for the identification and purification of G. elata germplasm and germplasm resources.

[Effect of fresh Gastrodia elata on gut microbiota in mice].

Gastrodia elata B1.,a traditional Chinese medicine,was frequently applied as a cure for headache or migraine. Its effects include suppressing hyperactive liver,calming endogenous wind,dredging collateralsand relieving spasm. There has been a proportion that G. elata should be added to The List of Substances That Are Traditionally Both Food and Chinese Medicinal Materials. The dry G. elata was commonly used in clinic,which have some fundamental study on efficacy and mechanism. However,fresh G. elata,which was added to herbal cuisine very often,lacks corresponding research. The interaction of diet,microbiota and human is a hot issue and lots of scholars are focusing on it. This research sequenced the 16 S rRNA of mouse cecal contents on Mi Seq platform to understand the effect of taking fresh G. elata. As the results showing,multiple probiotics grew after taking fresh G. elata extract,including Ruminiclostridium,Butyricicoccus,and Parvibacter. To contrast,some pathogens or potential pathogens,such as Escherichia/Shigella,Parasutterella,decreased. This manifests that fresh G. elata performs a positive regulation on mouse gut microbiota,especially the low-dose fresh G. elata extraction could restructure the microbiota apparently. Our result reveals that microbiota might be a new target for G. elata extract and provides an important basis for further research on the interaction between gut microbiota and pharmacological activity of G. elata.

Ultrafast all-optical switching in one-dimensional photonic crystal with two defects.

One-dimensional (1D) photonic crystals (PC) containing two-layer CdS defects are proposed and fabricated by using electron beam evaporation. Ultrafast nonlinear optical responses were characterized with the ultrafast pump-probe method in both time and spectral domains. Two-photon absorption coefficient enhancement and pump-beam-induced defect mode shift were reported. Both effects are attributed to the light localization in the defect layer of the multilayer structures. Our results demonstrated that defective photonic crystals are good candidates for fabrication of ultrafast all-optical switching devices.

Defect-mode dependence of two-photon-absorption enhancement in a one-dimensional photonic bandgap structure.

A one-dimensional photonic crystal containing a single CdS defect layer of various thicknesses was fabricated. The dependence of the two-photon-absorption (TPA) coefficient on the defect mode was investigated by use of a femtosecond pump-probe method. Experimental results show that the TPA coefficient of the CdS defect layer depends strongly on the defect mode in the photonic bandgap. This is consistent with the predicted dependence of light intensity within the defect layer.