Biocontrol potential of endophytic Bacillus subtilis A9 against rot disease of Morchella esculenta.

Introduction: Morchella esculenta is a popular edible fungus with high economic and nutritional value. However, the rot disease caused by Lecanicillium aphanocladii, pose a serious threat to the quality and yield of M. esculenta. Biological control is one of the effective ways to control fungal diseases. Methods and results: In this study, an effective endophytic B. subtilis A9 for the control of M. esculenta rot disease was screened, and its biocontrol mechanism was studied by transcriptome analysis. In total, 122 strains of endophytic bacteria from M. esculenta, of which the antagonistic effect of Bacillus subtilis A9 on L. aphanocladii G1 reached 72.2% in vitro tests. Biological characteristics and genomic features of B. subtilis A9 were analyzed, and key antibiotic gene clusters were detected. Scanning electron microscope (SEM) observation showed that B. subtilis A9 affected the mycelium and spores of L. aphanocladii G1. In field experiments, the biological control effect of B. subtilis A9 reached to 62.5%. Furthermore, the transcritome profiling provides evidence of B. subtilis A9 bicontrol at the molecular level. A total of 1,246 differentially expressed genes (DEGs) were identified between the treatment and control group. Gene Ontology (GO) enrichment analysis showed that a large number of DEGs were related to antioxidant activity related. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the main pathways were Nitrogen metabolism, Pentose Phosphate Pathway (PPP) and Mitogen-Activated Protein Kinases (MAPK) signal pathway. Among them, some important genes such as carbonic anhydrase CA (H6S33_007248), catalase CAT (H6S33_001409), tRNA dihydrouridine synthase DusB (H6S33_001297) and NAD(P)-binding protein NAD(P) BP (H6S33_000823) were found. Furthermore, B. subtilis A9 considerably enhanced the M. esculenta activity of Polyphenol oxidase (POD), Superoxide dismutase (SOD), Phenylal anineammonia lyase (PAL) and Catalase (CAT). Conclusion: This study presents the innovative utilization of B. subtilis A9, for effectively controlling M. esculenta rot disease. This will lay a foundation for biological control in Morchella, which may lead to the improvement of new biocontrol agents for production.

Ecosystem sustainability of rice and aquatic animal co-culture systems and a synthesis of its underlying mechanisms.

Integrated planting and breeding of rice and aquatic animals, including traditional rice-fish co-culture (RF), has been conducted for over 1200 years. It is one of the primary modes of modern ecologically sustainable agriculture. Rice and aquatic animal (RA) co-culture systems reduce risks of environmental pollution, reduce greenhouse gas emissions, maintain soil fertility, stabilize grain incomes, and preserve paddy field biodiversity. Nevertheless, the mechanisms that underlie the ecological sustainability of these systems remain controversial and poorly understood, restricting their practice at a larger scale. Here, the latest advance in understanding the evolution and extension of RA systems is synthesized, in addition to a discussion of the underlying ecological mechanisms of taxonomic interactions, complementary nutrient use, and microbially-driven elemental cycling. Specifically, the aim of this review is to provide a theoretical framework for the design of sustainable agricultural systems by integrating traditional knowledge and modern technologies.

Evaluating response mechanisms of soil microbiomes and metabolomes to Bt toxin additions.

The accumulation and persistence of Bt toxins in soils from Bt plants and Bt biopesticides may result in environmental hazards such as adverse impacts on soil microorganisms. However, the dynamic relationships among exogenous Bt toxins, soil characteristics, and soil microorganisms are not well understood. Cry1Ab is one of the most commonly used Bt toxins and was added to soils in this study to evaluate subsequent changes in soil physiochemical properties, microbial taxa, microbial functional genes, and metabolites profiles via 16S rRNA gene pyrosequencing, high-throughput qPCR, metagenomic shotgun sequencing, and untargeted metabolomics. Higher additions of Bt toxins led to higher concentrations of soil organic matter (SOM), ammonium (NH+4-N), and nitrite (NO2--N) compared against controls without addition after 100 days of soil incubation. High-throughput qPCR analysis and shotgun metagenomic sequencing analysis revealed that the 500 ng/g Bt toxin addition significantly affected profiles of soil microbial functional genes involved in soil carbon (C), nitrogen (N), and phosphorus (P) cycling after 100 days of incubation. Furthermore, combined metagenomic and metabolomic analyses indicated that the 500 ng/g Bt toxin addition significantly altered low molecular weight metabolite profiles of soils. Importantly, some of these altered metabolites are involved in soil nutrient cycling, and robust associations were identified among differentially abundant metabolites and microorganisms due to Bt toxin addition treatments. Taken together, these results suggest that higher levels of Bt toxin addition can alter soil nutrients, probably by affecting the activities of Bt toxin-degrading microorganisms. These dynamics would then activate other microorganisms involved in nutrient cycling, finally leading to broad changes in metabolite profiles. Notably, the addition of Bt toxins did not cause the accumulation of potential microbial pathogens in soils, nor did it adversely affect the diversity and stability of microbial communities. This study provides new insights into the putative mechanistic associations among Bt toxins, soil characteristics, and microorganisms, providing new understanding into the ecological impacts of Bt toxins on soil ecosystems.

Combined metagenomic and metabolomic analyses reveal that Bt rice planting alters soil C-N metabolism.

The environmental impacts of genetically modified (GM) plants remain a controversial global issue. To address these issues, comprehensive environmental risk assessments of GM plants is critical for the sustainable development and application of transgenic technology. In this paper, significant differences were not observed between microbial metagenomic and metabolomic profiles in surface waters of the Bt rice (T1C-1, the transgenic line) and non-Bt cultivars (Minghui 63 (the isogenic line) and Zhonghua 11 (the conventional japonica cultivar)). In contrast, differences in these profiles were apparent in the rhizospheres. T1C-1 planting increased soil microbiome diversity and network stability, but did not significantly alter the abundances of potential probiotic or phytopathogenic microorganisms compared with Minghui 63 and Zhonghua 11, which revealed no adverse effects of T1C-1 on soil microbial communities. T1C-1 planting could significantly alter soil C and N, probably via the regulation of the abundances of enzymes related to soil C and N cycling. In addition, integrated multi-omic analysis of root exudate metabolomes and soil microbiomes showed that the abundances of various metabolites released as root exudates were significantly correlated with subsets of microbial populations including the Acidobacteria, Actinobacteria, Chloroflexi, and Gemmatimonadetes that were differentially abundant in T1C-1 and Mnghui 63 soils. Finally, the potential for T1C-1-associated root metabolites to exert growth effects on T1C-1-associated species was experimentally validated by analysis of bacterial cultures, revealing that Bt rice planting could selectively modulate specific root microbiota. Overall, this study indicate that Bt rice can directly modulate rhizosphere microbiome assemblages by altering the metabolic compositions of root exudates that then alters soil metabolite profiles and physiochemical properties. This study unveils the mechanistic associations of Bt plant-microorganism-environment, which provides comprehensive insights into the potential ecological impacts of GM plants.

Environmental Behaviors of Bacillus thuringiensis (Bt) Insecticidal Proteins and Their Effects on Microbial Ecology.

Bt proteins are crystal proteins produced by Bacillus thuringiensis (Bt) in the early stage of spore formation that exhibit highly specific insecticidal activities. The application of Bt proteins primarily includes Bt transgenic plants and Bt biopesticides. Transgenic crops with insect resistance (via Bt)/herbicide tolerance comprise the largest global area of agricultural planting. After artificial modification, Bt insecticidal proteins expressed from Bt can be released into soils through root exudates, pollen, and plant residues. In addition, the construction of Bt recombinant engineered strains through genetic engineering has become a major focus of Bt biopesticides, and the expressed Bt proteins will also remain in soil environments. Bt proteins expressed and released by Bt transgenic plants and Bt recombinant strains are structurally and functionally quite different from Bt prototoxins naturally expressed by B. thuringiensis in soils. The former can thus be regarded as an environmentally exogenous substance with insecticidal toxicity that may have potential ecological risks. Consequently, biosafety evaluations must be conducted before field tests and production of Bt plants or recombinant strains. This review summarizes the adsorption, retention, and degradation behavior of Bt insecticidal proteins in soils, in addition to their impacts on soil physical and chemical properties along with soil microbial diversity. The review provides a scientific framework for evaluating the environmental biosafety of Bt transgenic plants, Bt transgenic microorganisms, and their expression products. In addition, prospective research targets, research methods, and evaluation methods are highlighted based on current research of Bt proteins.

Molecular cloning, and optimized production and characterization of recombinant cyclodextrin glucanotransferase from Bacillus sp. T1.

Cyclodextrin glucosyltransferase (CGTase) is an enzyme which degrades starch to produce cyclodextrins (CDs). In this study, the ß-CGTase producing strain T1 was identified as Bacillus sp. by its morphological characteristics and 16S rDNA sequence analysis. The cgt-T1 gene was cloned and expressed in Escherichia coli. CGTase-T1 was purified by Ni-nitrilotriacetic acid agarose column and the molecular weight was determined as approximately 75 kDa using SDS-PAGE analysis. For the expression of soluble proteins, the optimal induction conditions were 10 h at 25 °C with OD600 at 0.8. The purified CGTase-T1 exhibited maximum activity with an optimal pH and temperature of 6.0 and 65 °C. The enzyme was stable in a pH range of 7.0-10.0, retaining over 85% relative activity for 1 h. CGTase-T1 activity can be significantly enhanced by adding 1 mM Ba2+. Using a soluble starch substrate, the kinetic parameters were revealed with K M and k cat/K M values of 2.75 mg mL-1 and 1253.97 s-1 mL mg-1, respectively. Additionally, the four enzyme activities of CGTase-T1 were determined. The highest conversion rate to CDs (40.9%) was achieved from soluble starch after 8 h of enzyme reaction, where mainly ß-CD was produced (79.1% of the total CDs yield), indicating that CGTase-T1 potentially has industrial application prospect. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03111-8.

The cold - resistance mechanism of a mutagenic Volvariella volvacea strain VH3 with outstanding traits revealed by transcriptome profiling.

BACKGROUND: The straw mushroom (Volvariella volvacea) is one of the important vegetables that is popular for its delicious taste. However, the straw mushroom is sensitive to low temperature, resulting in economic loss during transportation and storage. We obtained a novel straw mushroom strain, named VH3, via ultraviolet mutagenesis. RESULTS: Our study revealed that VH3 exhibited high cold resistance compared to an ordinary straw mushroom cultivar, V23. We found that the electrolyte leakages of VH3 were always significantly lower than that of V23 treated with 4 °C for 0 h, 2 h,4 h, 8 h, 16 h, and 24 h. Before cold treatment (0 h), there were no difference of MDA contents, SOD activities, and CAT activities between VH3 and V23. At the late stage (8 h, 26 h, and 24 h) of cold treatment, the MDA contents of VH3 were lower while both the SOD and CAT activities were higher than those of V23. To investigate the potential mechanisms of VH3 cold resistance, we performed transcriptome sequencing to detect the transcriptome profiling of VH3 and V23 after 0 h and 4 h cold treatment. Transcriptome sequencing revealed that 111 differentially expressed genes (DEG) between V23 (0 h) and VH3 (0 h) (V23-0_vs_VH3-0), consisting 50 up-regulated and 61 down-regulated DEGs. A total of 117 DEGs were obtained between V23 (4 h) and VH3(4 h) (V23-4_vs_VH3-4), containing 94 up-regulated and 23 down-regulated DEGs. Among these DEGs, VVO_00021 and VVO_00017 were up-regulated while VVO_00003, VVO_00004, VVO_00010, and VVO_00030 were down-regulated in V23-0_vs_VH3-0 and VH3-4_vs_V23-4. KEGG and GO analysis revealed that the 6 DEGs were annotated to pathways related to cold stress. Besides, the GA3 content was also decreased in VH3. CONCLUSIONS: Collectively, our study first revealed that the increased cold resistance of VH3 might be caused by the expression change of VVO_00003, VVO_00004, VVO_00017, VVO_00021, and VVO_00030, and decreased GA3.

Weighted gene co-expression network analysis unveils gene networks regulating folate biosynthesis in maize endosperm.

Folates are essential elements for human growth and development, and their deficiency can lead to serious disorders. Waxy maize is a rich source of folates; however, the regulatory mechanism underlying folate biosynthesis in the endosperm remains unclear. Here, we examined changes in the folate content of maize endosperm collected at 15, 18, 21, 24, and 27 days after pollination (DAP) using liquid chromatograph-mass spectrometry and identified genes related to folate biosynthesis using transcriptome sequencing data. The results showed that 5-methyl-tetrahydrofolate and 5,10-methylene tetrahydrofolate were the main storage forms of folates in the endosperm, and their contents were relatively high at 21-24 days. We also identified 569, 3183, 4365, and 5513 differentially expressed genes (DEGs) in different days around milk stage. Functional annotation revealed 518 transcription factors (TFs) belonging to 33 families exhibiting specific expression in at least one sampling time. The key hub genes involved in folate biosynthesis were identified by weighted gene co-expression network analysis. In total, 24,976 genes were used to construct a co-expression network with 29 co-expression modules, among which the brown and purple modules were highly related to folate biosynthesis. Further, 187 transcription factors in the brown and purple modules were considered potential transcription factors related to endosperm folate biosynthesis. These results may improve the understanding of the molecular mechanism underlying folate biosynthesis in waxy maize and lead to the development of nutritionally fortified varieties. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02974-7.

Biochemical characterization of a GH10 xylanase from the anaerobic rumen fungus Anaeromyces robustus and application in bread making.

Anaeromyces robustus is an anaerobic rumen microorganism which can produce plant cell wall degrading enzymes. In this study, a new GH10 xylanase gene xylAr10 from A. robustus was identified, cloned and expressed in Pichia pastoris GS115. The recombinant protein ArXyn10 was characterized after being purified by Ni-NTA. The optimal pH and temperature of ArXyn10 was determined at 5.5 and 40 °C, respectively. ArXyn10 was stable at the pH range of 4.0-8.0, and could maintain high stability from 35 to 45 °C. The hydrolysis products released from beechwood xylan by ArXyn10 showed chromatographic mobility similar to xylobiose and xylotriose according to thin-layer chromatography analysis. It was shown that the addition of 7.5 mg of ArXyn10 in 100 g high-gluten wheat flour during bread making could increase the reducing sugar content by 10.80%, indicating that xylo-oligosaccharides were produced. With the addition of ArXyn10, the hardness and chewiness of the bread decreased and the quality was improved. The new discovered xylanase ArXyn10 have potential application prospect in bread making.

Composition and diversity of gut microbiota in Pomacea canaliculata in sexes and between developmental stages.

BACKGROUND: The apple snail, Pomacea canaliculata, is one of the world's 100 worst invasive alien species and vector of some pathogens relevant to human health. METHODS: On account of the importance of gut microbiota to the host animals, we compared the communities of the intestinal microbiota from P. canaliculata collected at different developmental stages (juvenile and adult) and different sexes by using high-throughput sequencing. RESULTS: The core bacteria phyla of P. canaliculata gut microbiota included Tenericutes (at an average relative abundance of 45.7 %), Firmicutes (27.85 %), Proteobacteria (11.86 %), Actinobacteria (4.45 %), and Cyanobacteria (3.61 %). The female group possessed the highest richness values, whereas the male group possessed the lowest bacterial richness and diversity compared with the female and juvenile group. Both the developmental stages and sexes had important effects on the composition of the intestinal microbiota of P. canaliculata. By LEfSe analysis, microbes from the phyla Proteobacteria and Actinobacteria were enriched in the female group, phylum Bacteroidetes was enriched in the male group, family Mycoplasmataceae and genus Leuconostoc were enriched in the juvenile group. PICRUSt analysis predicted twenty-four metabolic functions in all samples, including general function prediction, amino acid transport and metabolism, transcription, replication, recombination and repair, carbohydrate transport and metabolism, etc. CONCLUSIONS: This study provided a general understanding of the diversity characteristics of intestinal microbial communities of P. canaliculata, and indicated that developmental stage and gender could both influence the intestinal microbes of P. canaliculata. Further study may focus on the interaction between the gut microbiota and their host.

A sensitive immunosensor based on graphene-PAMAM composites for rapid detection of the CP4-EPSPS protein in genetically modified crops.

A simple electrochemical immunosensor based on nitrogen-doped graphene and polyamide-amine (GN-PAM) composites was proposed for the detection of the CP4-EPSPS protein in genetically modified (GM) crops. In this immunosensor, the amplification of the detection signal was realized through antibodies labeled with gold nanoparticles (AuNPs). The electrochemical responses of the immunosensor were linear (R2 = 0.9935 and 0.9912) when the GM soybean RRS and maize NK603 content ranged from 0.025% to 1.0% and 0.05% to 1.5%, respectively. The limits of detection for the GM soybean RRS and maize NK603 were as low as 0.01% and 0.03%, respectively. The immunosensor also exhibited high specificity, and satisfactory stability, reproducibility, and accuracy. Our findings indicated that the constructed immunosensor provides a new approach for the sensitive detection of the CP4-EPSPS protein. Notably, the sensor may be applied to other proteins or pathogenic bacteria by simply changing the antibodies, and may also be used for multi-component analysis.

Stochastic processes drive bacterial and fungal community assembly in sustainable intensive agricultural soils of Shanghai, China.

Sustainable intensive cropping systems have been implemented for three decades in suburban agricultural districts of Shanghai, China. These human-managed soils have been developed from paleosol or alluvial soils across different regions. However, little is known about the geographical distribution patterns of microbes and microbial community assembly in the sustainable intensive soils after decades of anthropogenic disturbances. Here, we investigated the impact of local geochemical properties and geographic distance on stochastic/deterministic microbial community assembly processes using high-throughput sequencing and phylogenetic null modeling analysis. Our results showed that soil pH was the most important environmental factor determining bacterial and fungal community structure. Importantly, only soil organic matter was positively correlated with fungal α-diversity, suggesting the efficient use of carbon substrates in sustainable agricultural systems, compensating for the lack of chemical fertilization and reduced tillage in these systems. Both bacterial and fungal communities had robust distance-decay patterns, but the rate of turnover of bacterial taxa was faster than that of fungi. Variation in bacterial and fungal communities was mostly attributed to the simultaneous effects of environmental variables and spatial factors. We also mapped the spatial distributions of the dominant bacterial and fungal taxa across the sustainable agricultural fields, making it possible to forecast the responses of agricultural ecosystems to anthropogenic disturbance. Based on the patterns of the ß-nearest taxon index, this study demonstrated that stochastic processes shaped substantial bacterial and fungal community variation in sustainable intensive agricultural soils of the Shanghai suburbs. This variation may be attributed to the increasing microbial dispersal caused by hydrological connectivity in the agricultural fields or the release from environmental stress and weakened environmental filtering across the suitable pH range preferable for most soil microbes. These results unveil assembly mechanisms of soil microbial community after several decades of sustainable intensive management, and contribute to understand the role of microbes in ecosystems in establishing a functional equilibrium which may enable sustainability to be preserved.

Development of a lateral flow test strip for simultaneous detection of BT-Cry1Ab, BT-Cry1Ac and CP4 EPSPS proteins in genetically modified crops.

A colloidal gold immunochromatographic strip (ICS) for simultaneous detection of multiple transgenic proteins, including CP4 EPSPS, BT-Cry1Ab and BT-Cry1Ac, was developed in this study. The sensitivity of the strip to the target protein was 5 ng/mL for CP4 EPSPS, 100 ng/mL for BT-Cry1Ab and Cry1Ac, respectively. Parallel analysis for maize, soybean, sugar beet and cotton showed the strip could detect 1% of transgenic content in crops containing BT-Cry1Ab and Cry1Ac, and, at least, 0.1% of content in crops containing CP4 EPSPS. The detection results for seed samples indicated the multicomponent analysis ICS had good accuracy. The analysis could be completed within 10 min and had the advantages of being high-throughput, easy to operate and visual detection. This is the first report of semi-quantitative ICS for detecting three transgenic proteins simultaneously. The developed approach may provide insights into the development of ICS for analyzing simultaneously multiple components in genetically modified crops.

Identification and Functional Characterization of a Novel Immunomodulatory Protein From Morchella conica SH.

A novel fungal immunomodulatory protein (FIP) was found in the precious medical and edible mushroom Morchella conica SH, defined as FIP-mco, which belongs to the FIP family. Phylogenetic analyses of FIPs from different origins were performed using Neighbor-Joining method. It was found that FIP-mco belonged to a new branch of the FIP family and may evolved from a different ancestor compared with most other FIPs. The cDNA sequence of FIP-mco was cloned and expressed in the yeast Pichia Pastoris X33. The recombinant protein of FIP-mco (rFIP-mco) was purified by agarose Ni chromatography and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The protein rFIP-mco could significantly suppress the proliferation of A549 and HepG2 cells at the concentration of 15 and 5 µg/ml, respectively, and inhibited the migration and invasion of human A549 and HepG2 cells at the concentration of 15 and 30 µg/ml respectively in vitro. Further, rFIP-mco can significantly reduce the expression levels of TNF-α, IL-1ß, and IL-6 in the THP1 cells (human myeloid leukemia mononuclear cells). In order to explore the potential mechanism of the cytotoxicity effect of rFIP-mco on A549 and HepG2 cells, cell cycle and apoptosis assay in the two cancer cells were conducted. The results demonstrated that G0/G1 to S-phase arrest and increased apoptosis may contribute to the proliferation inhibition by rFIP-mco in the two cancer cells. Molecular mechanism of rFIP-mco's reduction effect on the inflammatory cytokines was also studied by suppression of the NF-κB signaling pathway. It showed that suppression of NF-κB signaling is responsible for the reduction of inflammatory cytokines by rFIP-mco. The results indicated the prospect of FIP-mco from M. conica SH as an effective and feasible source for cancer therapeutic studies and medical applications.

Production and characterization of the 13 C/15 N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.

Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope-labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce 13 C/15 N single-labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U-13 C] glucose (5% 13 C-enriched) or [15 N] ammonium chloride (5% 15 N-enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purities of 13 C/15 N-labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13 C and ä 15 N values of 13 C-labeled Cry protein and 15 N-labeled Cry protein were 3,269‰ and 2,854‰, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

Analysis of synonymous codon usage patterns in the edible fungus Volvariella volvacea.

In this study, trends in synonymous codons usage of Volvariella volvecea have been first examined by analysis of complete coding sequences and gene chip data. The results showed that GC content at three codon positions are obviously different and there were several factors shaping the codon usage of V. volvacea genes, including base composition. The comparison of codon usage among four edible fungi such as V. volvacea, Agaricus bisporus, Coprinopsis cinerea, and Pleurotus ostreatus indicated that the similar codon usage pattern was used among V. volvacea, A. bisporus and P. ostreatus, but there was significantly different codon usage pattern of C. cinerea. Two arrays of optimal codons were determined by effective number of codons (ENC) values and gene chip database separately, resulting that most of the ENC-predicted optimal codons were included in the array of gene chip resulted optimal codons. This study can provide useful information for codon usage pattern analysis and gene transformation of V. volvacea.

Edible Safety Assessment of Genetically Modified Rice T1C-1 for Sprague Dawley Rats through Horizontal Gene Transfer, Allergenicity and Intestinal Microbiota.

In this study, assessment of the safety of transgenic rice T1C-1 expressing Cry1C was carried out by: (1) studying horizontal gene transfer (HGT) in Sprague Dawley rats fed transgenic rice for 90 d; (2) examining the effect of Cry1C protein in vitro on digestibility and allergenicity; and (3) studying the changes of intestinal microbiota in rats fed with transgenic rice T1C-1 in acute and subchronic toxicity tests. Sprague Dawley rats were fed a diet containing either 60% GM Bacillus thuringiensis (Bt) rice T1C-1 expressing Cry1C protein, the parental rice Minghui 63, or a basic diet for 90 d. The GM Bt rice T1C-1 showed no evidence of HGT between rats and transgenic rice. Sequence searching of the Cry1C protein showed no homology with known allergens or toxins. Cry1C protein was rapidly degraded in vitro with simulated gastric and intestinal fluids. The expressed Cry1C protein did not induce high levels of specific IgG and IgE antibodies in rats. The intestinal microbiota of rats fed T1C-1 was also analyzed in acute and subchronic toxicity tests by DGGE. Cluster analysis of DGGE profiles revealed significant individual differences in the rats' intestinal microbiota.

A new approach for breeding low-temperature-resistant Volvariella volvacea strains: Genome shuffling in edible fungi.

Volvariella volvacea is difficult to store fresh because of the lack of low-temperature resistance. Many traditional mutagenic strategies have been applied in order to select out strains resistant to low temperature, but few commercially efficient strains have been produced. In order to break through the bottleneck of traditional breeding and significantly improve low-temperature resistance of the edible fungus V. volvacea, strains resistant to low temperature were constructed by genome shuffling. The optimum conditions of V. volvacea strain mutation, protoplast regeneration, and fusion were determined. After protoplasts were treated with 1% (v/v) ethylmethylsulfonate (EMS), 40 Sec of ultraviolet (UV) irradiation, 600 Gy electron beam implantation, and 750 Gy60 Co-γ irradiation, separately, the lethality was within 70%-80%, which favored generating protoplasts being used in following forward mutation. Under these conditions, 16 strains of V. volvacea mutated by EMS, electron beam, UV irradiation, and 60 Co-γ irradiation were obtained. The 16 mutated protoplasts were selected to serve as the shuffling pool based on their excellent low-temperature resistance. After four rounds of genome shuffling and low-temperature resistance testing, three strains (VF1 , VF2 , and VF3 ) with high genetic stability were screened. VF1 , VF2 , and VF3 significantly enhanced fruit body shelf life to 20, 28, and 28 H at 10 °C, respectively, which exceeded 25%, 75%, and 75%, respectively, compared with the storage time of V23, the most low-temperature-resistant strain. Genome shuffling greatly improved the low-temperature resistance of V. volvacea, and shortened the course of screening required to generate desirable strains. To our knowledge, this is the first paper to apply genome shuffling to breeding new varieties of mushroom, and offers a new approach for breeding edible fungi with optimized phenotype.

[Codon usage bias in the straw mushroom Volvariella volvacea].

We analyzed the whole genome coding sequence of Volvariella volvacea to study the pattern utilization of codons by Codon W 1.4.2. As results, 24 optimal codons were identified. Moreover, the frequency of codons usage was calculated by CUSP program. We compared the frequency of codons usage of V. volvacea with other organisms including 6 modal value species (Homo sapiens, Saccharomys cerevisiae, Arabidopsis thalian, Mus musculus, Danio rerio and Drosophila melanogaster) and 4 edible fungi (Coprinopsis cinerea, Agaricus bisporus, Lentinula edodes and Pleurotus ostreatus). We found that there were less differences in 3 edible fungi (excluding Pleurotus ostreatus) than 6 modal value species, comparing with the frequency of codons usage of V. volvacea. With software SPSS16.0, cluster analysis which showed differences in the size of codon bias, reflects the evolutionary relationships between species, which can be used as a reference of evolutionary relationships of species. This was the first time for analysis the codon preference among the whole coding sequences of edible fungi, serving as theoretical basis to apply genetic engineering of V. volvacea.

Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro.

Teth137, a 13.7 kD protein of unknown function from Thermoanaerobacter ethanolicus JW200, is encoded by 360 nucleotides and has been obtained by DNA-coupled column previously. However, no function study of Teth137 has been published. Homologous modeling of Teth137 shows the protein is comprised of a helix-turn-helix motif which is a typical DNA-binding domain. Therefore, it is speculated Teth137 is a DNA-binding protein and involved in transcription of the adhE gene (encodes alcohol dehydrogenase E). To investigate the function of Teth137, recombinant Teth137 is overexpressed in Escherichia coli JM109 and purified by DEAE column. Purified Teth137 exhibits the affinity with the adhE promoter region in gel electrophoresis mobility shift assay (GEMSA). Teth137 at the concentration of 48 µM retards the migration of 5 nM of probe in the presence of the competitor DNA. Mutant analysis indicates that S69, T70, P71 and T72 are critical to protein-DNA interface; Gly substitutions at these residues results in the loss of the binding ability with the adhE promoter region. Moreover, T. ethanolicus JW200 RNA polymerase, σ subunit and template plasmid are prepared for in vitro transcription assay to detect the regulation function of Teth137. The results of the in vitro transcription show that the transcription of 5 nM of the template plasmid is inhibited by 48 µM of Teth137.