Identification of Key Novel lncRNAs RP11-400N13.3 and RP11-197K6.1 that Construct ceRNA Networks Associated with Recurrence and Metastasis in Colon Cancer.

BACKGROUND: Colon adenocarcinoma (COAD) is a major cause of cancer mortality worldwide. The occurrence and development of colon cancer is regulated by complex mechanisms that require further exploration. Recently, long non-coding RNAs (lncRNAs) were found to be related to the mortality of colon cancer patients through their participation in competing endogenous RNA (ceRNA) networks. Therefore, screening the lncRNAs involved in colon cancer may contribute to clarifying the complex mechanisms. METHODS: In this study, we explored the potential lncRNAs associated with colon cancer by establishing a ceRNA network using bioinformatics, followed by biological verification. RESULTS: RP11-197K6.1 and RP11-400N13.3 were screened out owing to their involvement in the expression of CDK2NA, a gene that potentially prevents colon cancer cells from high oxygen levels. CONCLUSIONS: Our work explored the mechanisms of recurrence and metastasis in colon cancer and provided potential targets for drug development.

KED gene expression in early response to wounding stress in tomato plants.

The wounding-responsive KED gene, named for its coding for a lysine (K), glutamic acid (E), and aspartic acid (D)-rich protein, is widely present among land plants. However, little is known about its regulation or function. In this study, we found that transcription of the tomato (Solanum lycopersicum) KED gene, SlKED, was rapidly and transiently elevated by wounding or ethephon treatment. Compared to the wild-type plants, the CRISPR/Cas9-mediated SlKED knockout plants did not exhibit altered expression patterns for genes involved in hormone biosynthesis or stress signaling, suggesting a lack of pleiotropic effect on other stress-responsive genes. Conversely, jasmonic acid did not appear to directly regulate SlKED expression. Wounded leaves of the KED-lacking plants exhibited higher binding of Evans blue dye than the wild-type, indicating a possible role for KED in healing damaged tissues. The SlKED knockout plants showed a similar dietary effect as the wild-type on the larval growth of tobacco hornworm. But a higher frequency of larval mandible (mouth) movement was recorded during the first 2 minutes of feeding on the wounded KED-lacking SlKED knockout plants than on the wounded KED-producing wild-type plants, probably reflecting an initial differential response by the feeding larvae to the SlKED knockout plants. Our findings suggest that SlKED may be an ethylene-mediated early responder to mechanical stress in tomato, acting downstream of the wound stress response pathways. Although its possible involvement in response to other biotic and abiotic stresses is still unclear, we propose that SlKED may play a role in plant's rapid, short-term, early wounding responses, such as in cellular damage healing.

Evolutionary analysis of KED-rich proteins in plants.

During the course of evolution, organisms have developed genetic mechanisms in response to various environmental stresses including wounding from mechanical damage or herbivory-caused injury. A previous study of wounding response in the plant tobacco identified a unique wound-induced gene, aptly named KED due to its coding for a protein that has an unusually high content of amino acids lysine (K), glutamic acid (E) and aspartic acid (D). However, by far little is known about this intriguing gene. In this study, we investigated the evolutionary aspects of the KED-rich coding genes. We found that a consistent pattern of wound-induced KED gene expression is maintained across representative species of angiosperm and gymnosperm. KED genes can be identified in species from all groups of land plants (Embryophyta). All the KED proteins from vascular plants (Tracheophyta) including angiosperm, gymnosperm, fern and lycophyte share a conserved 19-amino acid domain near the C-terminus, whereas bryophytes (moss, liverwort and hornwort) possess KED-rich, multi-direct-repeat sequences that are distinct from the vascular plant KEDs. We detected KED-rich sequences in Charophyta species but not in Chlorophyta wherever genome sequences are available. Our studies suggest diverse and complex evolution pathways for land plant KED genes. Vascular plant KEDs exhibit high evolutionary conservation, implicating their shared function in response to wounding stress. The extraordinary enrichment of amino acids K, E and D in these groups of distinct and widely distributed proteins may reflect the structural and functional requirement for these three residues during some 600 million years of land plant evolution.

Prognostic significance of serum inflammation indices for different tumor infiltrative pattern types of gastric cancer.

BACKGROUND: Inflammatory indices are considered to be potential prognostic biomarkers for patients with gastric cancer (GC). However, there is no evidence defining the prognostic significance of inflammatory indices for GC with different tumor infiltrative pattern (INF) types. AIM: To evaluate the significance of inflammatory indices and INF types in predicting the prognosis of patients with GC. METHODS: A total of 962 patients who underwent radical gastrectomy were retrospectively selected for this study. Patients were categorized into the expansive growth type (INFa), the intermediate type (INFb), and the infiltrative growth type (INFc) groups. The cutoff values of inflammatory indices were analyzed by receiver operating characteristic curves. The Kaplan-Meier method and log-rank test were used to analyze overall survival (OS). The chi-square test was used to analyze the association between inflammatory indices and clinical characteristics. The independent risk factors for prognosis in each group were analyzed by univariate and multivariate analyses based on logistic regression. Nomogram models were constructed by R studio. RESULTS: The INFc group had the worst OS (P < 0.001). The systemic immune-inflammation index (P = 0.039) and metastatic lymph node ratio (mLNR) (P = 0.003) were independent risk factors for prognosis in the INFa group. The platelet-lymphocyte ratio (PLR) (P = 0.018), age (P = 0.026), body mass index (P = 0.003), and postsurgical tumor node metastasis (pTNM) stage (P < 0.001) were independent risk factors for prognosis in the INFb group. The PLR (P = 0.021), pTNM stage (P = 0.028), age (P = 0.021), and mLNR (P = 0.002) were independent risk factors for prognosis in the INFc group. The area under the curve of the nomogram model for predicting 5-year survival in the INFa group, INFb group, and INFc group was 0.787, 0.823, and 0.781, respectively. CONCLUSION: The outcome of different INF types GC patients could be assessed by nomograms based on different inflammatory indices and clinicopathologic features.

Expression of a Tardigrade Dsup Gene Enhances Genome Protection in Plants.

DNA damage is one of the most impactful events in living organisms, leading to DNA sequence changes (mutation) and disruption of biological processes. A study has identified a protein called Damage Suppressor Protein (Dsup) in the tardigrade Ramazzotius varieornatus that has shown to reduce the effects of radiation damage in human cell cultures (Hashimoto in Nature Communications 7:12808, 2016). We have generated tobacco plants that express the codon-optimized tardigrade Dsup gene and examined their responses when treated with mutagenic chemicals, ultraviolet (UV) and ionizing radiations. Our studies showed that compared to the control plants, the Dsup-expressing plants grew better in the medium containing mutagenic ethylmethane sulfonate (EMS). RT-qPCR detected distinct expression patterns of endogenous genes involved in DNA damage response and repair in the Dsup plants in response to EMS, bleomycin, UV-C and X-ray radiations. Comet assays revealed that the nuclei from the Dsup plants appeared more protected from UV and X-ray damages than the control plants. Overall, our studies demonstrated that Dsup gene expression enhanced tolerance of plants to genomutagenic stress. We suggest the feasibility of exploring genetic resources from extremotolerant species such as tardigrades to impart plants with tolerance to stressful environments for future climate changes and human space endeavors.

Tissue-Preferential Activity and Induction of the Pepper Capsaicin Synthase PUN1 Promoter by Wounding, Heat and Metabolic Pathway Precursor in Tobacco and Tomato Plants.

A promoter is an essential structural component of a gene that controls its transcription activity in different development stages and in response to various environmental stimuli. Knowledge of promoter functionality in heterologous systems is important in the study of gene regulation and biotechnological application. In order to explore the activity of the pepper capsaicin synthase gene (PUN1) promoter, gene constructs of pPUN1::GUS (for ß-glucuronidase) and pPUN1::NtKED (for a tobacco wound-responsive protein) were introduced into tobacco and tomato, respectively, and their activities were examined. Higher levels of GUS staining intensity and transcription were detected in ovary, anther and pollen than other tissues or organs in tobacco plants. Likewise, transgenic tomato fruits had a higher level of pPUN1::NtKED gene expression than the leaf and flower. The PUN1-driven gene expression can be transiently induced by wounding, heat (40 °C) and the capsaicinoid biosynthetic pathway precursor phenylalanine. When compared to the reported pPUN1::GUS-expressing Arabidopsis, the PUN1 promoter exhibited a more similar pattern of activities among pepper, tobacco and tomato, all Solanaceae plants. Our results suggest the potential utility of this tissue-preferential and inducible promoter in other non-pungent Solanaceae plants for research of gene function and regulation as well as in the biotechnological applications.

Tobacco plants expressing the maize nitrate transporter ZmNrt2.1 exhibit altered responses of growth and gene expression to nitrate and calcium.

BACKGROUND: Nitrate uptake is a highly regulated process. Understanding the intricate interactions between nitrate availability and genetically-controlled nitrate acquisition and metabolism is essential for improving nitrogen use efficiency and increasing nitrate uptake capacity for plants grown in both nitrate-poor and nitrate-enriched environments. In this report, we introduced into tobacco (Nicotiana tabacum) the constitutively expressed maize high-affinity transporter ZmNrt2.1 gene that would bypass the tight control for the endogenous nitrate-responsive genes. By using calcium inhibitors and varying levels of NO3-, Ca2+ and K+, we probed how the host plants were affected in their nitrate response. RESULTS: We found that the ZmNrt2.1-expressing plants had better root growth than the wild type plants when Ca2+ was deficient regardless of the nitrate levels. The growth restriction associated with Ca2+-deficiency can be alleviated with a high level of K+. Furthermore, the transgenic plants exhibited altered expression patterns of several endogenous, nitrate-responsive genes, including the high- and low-affinity nitrate transporters, the Bric-a-Brac/Tramtrack/Broad protein BT2 and the transcription factor TGA-binding protein TGA1, in responding to treatments of NO3-, K+ or inhibitors for the calcium channel and the cytosolic Ca2+-regulating phospholipase C, as compared to the wild type plants under the same treatments. Their expression was not only responsive to nitrate, but also affected by Ca2+. There were also different patterns of gene expression between roots and shoots. CONCLUSION: Our results demonstrate the ectopic effect of the maize nitrate transporter on the host plant's overall gene expression of nitrate sensing system, and further highlight the involvement of calcium in nitrate sensing in tobacco plants.

Theory of ferrimagnetism in the Hubbard model on bipartite lattices with spectral symmetry.

The Hubbard model is one of the most important models in condensed matter physics. In this paper, we developed a theory of ferrimagnetism in the Hubbard model on bipartite lattices with spectral symmetry. By taking three models as examples, we studied the ferrimagnetic orders that emerge from three typical fermionic systems--metal, semi-metal and (Chern) insulator. In particular, we found that there may exist various ferrimagnetic orders and explored the universal features.

Tryptophan and indole analog mediated plastid transformation.

A nonantibiotic/herbicide-resistance selection system for plastid transformation is described here in technical detail. This system is based on the feedback-insensitive anthranilate synthase (AS) α-subunit gene of tobacco (ASA2) as a selective marker and tryptophan (Trp) or indole analogs as selection agents. AS catalyzes the first reaction in the Trp biosynthetic pathway, naturally compartmentalized in the plastids, by converting chorismate to anthranilate and is subjected to feedback inhibition by Trp. In addition to Trp, various Trp analogs and indole compounds that can be converted to Trp analogs can also inhibit AS activity and therefore are toxic to cells. When cells are made to express the feedback-insensitive ASA2, they acquire resistance to these analogs and can be selected for during transformation process. We have demonstrated the feasibility of this selection system in tobacco (Nicotiana tabacum L. cv. Petit Havana). ASA2-expressing transplastomic plants were obtained on medium supplemented with either 7-methyl-DL-tryptophan (7-MT) or 4-methylindole (4-MI). These plants show normal phenotype and fertility and transmit the resistance to the selection agents strictly maternally.

Production of functional native human interleukin-2 in tobacco chloroplasts.

Interleukin-2 (IL-2) is an important T lymphocyte-derived cytokine in the mammalian immune system. Non-native, recombinant IL-2 derived from Escherichia coli is used widely in both medical research and treatment of diseases. Recombinant human IL-2 gene has been expressed in plant nuclear genomes, therefore it can be spread to the environment through pollen. Furthermore, all the plant-produced IL-2 reported thus far had been attached with artificial tags or fusion proteins, which may trigger unintended immunological responses and therefore compromise its full utility as a medicine. To expand the potential of using plant chloroplasts to produce functional native human therapeutic proteins, we inserted an engineered human interleukin-2 (hIL-2)-coding gene, without any tags, into the chloroplast genome of tobacco (Nicotiana tabacum L.). Partially purified hIL-2 protein from the leaves of the transplastomic plants induced in vitro proliferation of IL-2-dependent murine T lymphocytes. Our study demonstrates that plant chloroplasts can serve as a bio-factory for production of an active native human interleukin in a self-contained and therefore environmentally safe manner.

[Study on quality evaluation of cimicifugae rhizoma from different producing areas by HPLC fingerprint].

To establish a fingerprint for Cimicifugae Rhizoma from different producing areas. Column kromasil (4.6 mm x 250 mm, 5 microm) was employed with acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the detection wavelength was 254 nm. Twenty chromatographic peaks were extracted as the common peaks of fingerprint, and 21 batches of samples were compared and classified with such methods as similarity evaluation, cluster analysis and principle component analysis. The results showed 12 common peaks and three categories of samples. The method was so highly reproducible, simple and reliable that it could provide basis for quality control and evaluation of Cimicifugae Rhizoma from different producing areas.

[Optimization of extraction process of Chrysanthemi Flos by multi-index orthogonal experiment].

OBJECTIVE: To explore quantitative assessment indicators of Chrysanthemi Flos, and optimize the extraction process of Chrysanthemi Flos through orthogonal experimental design. METHOD: The concentration of ethanol, amount of ethanol, extraction time and extraction frequency were selected as factors in the L9 (3(4)) orthogonal experiment. A comprehensive assessment was conducted with the peak area of the eight major common peaks in the fingerprint of Chrysanthemi Flos as the indicators. RESULT: The optimum extraction process was selected as follows: using ultrasonic extraction method, adding 30-fold ethanol with 80% concentration, extracting for 2 times for extraction, 40 min for each time. CONCLUSION: The optimized extraction process is reliable, with controllable assessment indicators, which is significant to the standardization of the extraction process and quality control of Chrysanthemi Flos preparations.

[Application of spectrum-effect relationship in Chinese medicine research and related thinking].

Fingerprint technology is the key technology in modern Chinese medicine research, while spectrum-effect relationship research is the advanced stage of fingerprint research. Spectrum-effect relationship research can reveal the relationship between fingerprint and pharmacological effect through multiple statistical analyses, which can be used in Chinese medicine research. Spectrum-effect relationship has been used in many areas of Chinese medicine research, such as effective basis of single and compound Chinese medicine research, component compatibility research, processing mechanism research, pharmacological effect forecast research, technology optimization research, and so on. This paper systematically reviewed the application of spectrum-effect relationship in Chinese medicine research, and indicated some problems in spectrum-effect relationship research. At last, the authors give an outlook of the future of spectrum-effect relationship research.

Climate change and ocean acidification effects on seagrasses and marine macroalgae.

Although seagrasses and marine macroalgae (macro-autotrophs) play critical ecological roles in reef, lagoon, coastal and open-water ecosystems, their response to ocean acidification (OA) and climate change is not well understood. In this review, we examine marine macro-autotroph biochemistry and physiology relevant to their response to elevated dissolved inorganic carbon [DIC], carbon dioxide [CO2 ], and lower carbonate [CO3 (2-) ] and pH. We also explore the effects of increasing temperature under climate change and the interactions of elevated temperature and [CO2 ]. Finally, recommendations are made for future research based on this synthesis. A literature review of >100 species revealed that marine macro-autotroph photosynthesis is overwhelmingly C3 (≥ 85%) with most species capable of utilizing HCO3 (-) ; however, most are not saturated at current ocean [DIC]. These results, and the presence of CO2 -only users, lead us to conclude that photosynthetic and growth rates of marine macro-autotrophs are likely to increase under elevated [CO2 ] similar to terrestrial C3 species. In the tropics, many species live close to their thermal limits and will have to up-regulate stress-response systems to tolerate sublethal temperature exposures with climate change, whereas elevated [CO2 ] effects on thermal acclimation are unknown. Fundamental linkages between elevated [CO2 ] and temperature on photorespiration, enzyme systems, carbohydrate production, and calcification dictate the need to consider these two parameters simultaneously. Relevant to calcifiers, elevated [CO2 ] lowers net calcification and this effect is amplified by high temperature. Although the mechanisms are not clear, OA likely disrupts diffusion and transport systems of H(+) and DIC. These fluxes control micro-environments that promote calcification over dissolution and may be more important than CaCO3 mineralogy in predicting macroalgal responses to OA. Calcareous macroalgae are highly vulnerable to OA, and it is likely that fleshy macroalgae will dominate in a higher CO2 ocean; therefore, it is critical to elucidate the research gaps identified in this review.

Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa.

Secondary Wall-Associated NAC Domain 1s (SND1s) are transcription factors (TFs) known to activate a cascade of TF and pathway genes affecting secondary cell wall biosynthesis (xylogenesis) in Arabidopsis and poplars. Elevated SND1 transcriptional activation leads to ectopic xylogenesis and stunted growth. Nothing is known about the upstream regulators of SND1. Here we report the discovery of a stem-differentiating xylem (SDX)-specific alternative SND1 splice variant, PtrSND1-A2(IR), that acts as a dominant negative of SND1 transcriptional network genes in Populus trichocarpa. PtrSND1-A2(IR) derives from PtrSND1-A2, one of the four fully spliced PtrSND1 gene family members (PtrSND1-A1, -A2, -B1, and -B2). Each full-size PtrSND1 activates its own gene, and all four full-size members activate a common MYB gene (PtrMYB021). PtrSND1-A2(IR) represses the expression of its PtrSND1 member genes and PtrMYB021. Repression of the autoregulation of a TF family by its only splice variant has not been previously reported in plants. PtrSND1-A2(IR) lacks DNA binding and transactivation abilities but retains dimerization capability. PtrSND1-A2(IR) is localized exclusively in cytoplasmic foci. In the presence of any full-size PtrSND1 member, PtrSND1-A2(IR) is translocated into the nucleus exclusively as a heterodimeric partner with full-size PtrSND1s. Our findings are consistent with a model in which the translocated PtrSND1-A2(IR) lacking DNA-binding and transactivating abilities can disrupt the function of full-size PtrSND1s, making them nonproductive through heterodimerization, and thereby modulating the SND1 transcriptional network. PtrSND1-A2(IR) may contribute to transcriptional homeostasis to avoid deleterious effects on xylogenesis and plant growth.

MicroRNAs in trees.

MicroRNAs (miRNAs) are 20-24 nucleotide long molecules processed from a specific class of RNA polymerase II transcripts that mainly regulate the stability of mRNAs containing a complementary sequence by targeted degradation in plants. Many features of tree biology are regulated by miRNAs affecting development, metabolism, adaptation and evolution. MiRNAs may be modified and harnessed for controlled suppression of specific genes to learn about gene function, or for practical applications through genetic engineering. Modified (artificial) miRNAs act as dominant suppressors and are particularly useful in tree genetics because they bypass the generations of inbreeding needed for fixation of recessive mutations. The purpose of this review is to summarize the current status of information on miRNAs in trees and to guide future studies on the role of miRNAs in the biology of woody perennials and to illustrate their utility in directed genetic modification of trees.

Poly(T) adaptor RT-PCR.

Reverse transcription PCR (RT-PCR) is one of the most important techniques for analyzing RNA abundance. MicroRNAs (miRNAs) are a group of 20- to 24-nucleotide regulatory small RNAs which play an important role in plants and animals. However, the small size of miRNAs makes them difficult to be detected and quantified by conventional RT-PCR techniques. Here, we describe a poly(T) adaptor RT-PCR method specifically designed for quantifying miRNAs. In this method, total RNAs, including miRNAs, are extended by a poly(A) tailing reaction using poly(A) polymerase and ATP. The miRNA with a poly(A) tail is converted into cDNA through reverse transcription primed by a poly(T) adaptor, and then PCR-amplified using a miRNA-specific forward primer and a universal poly(T) adaptor reverse primer. The RT-PCR amplification can be monitored by real-time detection or by end-point detection for quantifying the miRNA transcript level. The PCR amplicons can be sequenced for validating the expression of the specific miRNA gene.

Hybrid Rubisco of tomato large subunits and tobacco small subunits is functional in tobacco plants.

Biogenesis of functional ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants requires specific assembly in the chloroplast of the imported, cytosol-synthesized small subunits (SS) with the chloroplast-made large subunits (LS). Accumulating evidence indicates that chloroplasts (plastids) generally have a low tolerance for assembling foreign or modified Rubisco. To explore Rubisco engineering, we created two lines of transplastomic tobacco plants whose rbcL gene was replaced by tomato-derived rbcL: plant LLS2 with Rubisco composed of tobacco SS and Q437R LS and plant LLS4 with a hybrid Rubisco of tobacco SS and tomato LS (representing four substitutions of Y226F, A230T, S279T and Q437R from tobacco LS). Plant LLS2 exhibited similar phenotypes as the wild type. Plant LLS4 showed lower chlorophyll and Rubisco levels particularly in young emerging leaves, lower photosynthesis rates and biomass during early stages of development, but was able to reach reproductive maturity and somewhat wild type-like phenotype under ambient CO2 condition. In vitro assays detected similar carboxylase activity and RuBP affinity in LLS2 and LLS4 plants as in wild type. Our studies demonstrated that tomato LS was sufficiently assembled with tobacco SS into functional Rubisco. The hybrid Rubisco of tomato LS and tobacco SS can drive photosynthesis that supports photoautotrophic growth and reproduction of tobacco plants under ambient CO2 and light conditions. We discuss the effect of these residue substitutions on Rubisco activity and the possible attribution of chlorophyll deficiency to the in planta photosynthesis performance in the hybrid Rubisco plants.

The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue L-azetidine-2-carboxylic acid as a selectable marker system for plant transformation.

The yeast N-acetyltransferase MPR1 gene has previously been shown to confer resistance to the toxic proline analogue azetidine-2-carboxylic acid (A2C) in yeast and transgenic tobacco. Here experiments were carried out to determine if MPR1 and A2C can work as a selectable marker system for plant transformation. The MPR1 gene was inserted into a binary vector under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and transformed into tobacco via the Agrobacterium tumefaciens-mediated leaf disc method. A2C was applied in the selection medium to select for putative transformants. PCR analysis showed that 28.4% and 66.7% of the plantlets selected by 250 muM and 300 muM A2C were positive for the MPR1 gene, respectively. Southern and northern blot analysis and enzyme activity assay confirmed the stable gene incorporation, transcription, and translation of the MPR1 transgene in the transgenic plants. The transgene-carrying T(1) progeny could be distinguished from the recessive progeny when grown on 400, 450, or 500 muM A2C. Examination of the metabolism of 22 transgenic plants by gas chromatography-mass spectrometry profiling did not reveal any significant changes. In conclusion, the results demonstrate that MPR1/A2C is a safe and efficient selection system that does not involve microbial antibiotic or herbicide resistance genes. Recent studies showed that MPR1 can protect yeast against oxidative stresses by decreasing the accumulation of the proline catabolite Delta(1)-pyrroline-5-carboxylate (P5C). However, H(2)O(2) treatment resulted in contradictory responses among the five transgenic lines tested. Further experiments are required to assess the response of MPR1 transgenic plants under oxidative stress.

A gyrB-targeted PCR for rapid identification of Paenibacillus mucilaginosus.

Paenibacillus mucilaginosus, one of the typical silicate bacteria, has long been used as a biofertilizer in agriculture and has recently shown potential in bioleaching and wastewater engineering. There has been considerable research involving the isolation of P. mucilaginosus for various utilizations; therefore, rapid identification of this species is of great interest. Herein, we describe a specific polymerase chain reaction (PCR) method developed for a rapid identification of P. mucilaginosus, which might provide potential utilization in the investigation of populations, detection of biofertilizers, and identification of novel isolates on a large scale. A gyrB-targeted species-specific primer pair, F2 (5'-ACG GAT ATC TCC CAG ACG TTC AT-3') and R5 (5'-ACG GGC ACG CTG CGC CTG TAC G-3'), was successfully designed to selectively amplify a 519-bp amplicon from P. mucilaginosus. Good specificity was demonstrated by both reference strains and total soil deoxyribonucleic acid, from which only the gyrB gene of P. mucilaginosus was amplified. The detection limit was 4-10 cells per assay. Using the culture-PCR method, 20 of 26 soil isolates on a nitrogen-free medium were rapidly identified as P. mucilaginosus, which was confirmed by sequencing of the gyrB gene.