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Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, β-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%-11.3% and 5.6%-7.7%, respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

Background Production of vitamin C has been traditionally based on the Reichstein process and the two-step process. However, the two processes share a common disadvantage: vitamin C cannot be directly synthesized from D-glucose. Therefore, significant effort has been made to develop a one-step vitamin C fermentation process. While, 2-KLG, not vitamin C, is synthesized from nearly all current one-step fermentation processes. Vitamin C is naturally synthesized from glucose in Arabidopsis thaliana via a ten-step reaction pathway that is encoded by ten genes. The main objective of this study was to directly produce vitamin C from D-glucose in Escherichia coli by expression of the genes from the A. thaliana vitamin C biosynthetic pathway. Results Therefore, the ten genes of whole vitamin C synthesis pathway of A. thaliana were chemically synthesized, and an engineered strain harboring these genes was constructed in this study. The direct production of vitamin C from D-glucose based on one-step fermentation was achieved using this engineered strain and at least 1.53 mg/L vitamin C was produced in shaking flasks. Conclusions The study demonstrates the feasibility of one-step fermentation for the production of vitamin C from D-glucose. Importantly, the one-step process has significant advantages compared with the currently used fermentation process: it can save multiple physical and chemical steps needed to convert D-glucose to D-sorbitol; it also does not involve the associated down-streaming steps required to convert 2-KLG into vitamin C.

Nitrobenzene is widely present in industrial wastewater and soil. Biodegradation has become an ideal method to remediate organic pollutants due to its low cost, high efficiency, and absence of secondary pollution. In the present study, 10 exogenous genes that can completely degrade nitrobenzene were introduced into Escherichia coli, and their successful expression in the strain was verified by fluorescence quantitative polymerase chain reaction and proteomic analysis. The results of the degradation experiment showed that the engineered strain could completely degrade 4 mM nitrobenzene within 8 h. The formation of intermediate metabolites was detected, and the final metabolites entered the E. coli tricarboxylic acid cycle smoothly. This process was discovered by isotope tracing method. Results indicated the integrality of the degradation pathway and the complete degradation of nitrobenzene. Finally, further experiments were conducted in soil to verify its degradation ability and showed that the engineered strain could also degrade 1 mM nitrobenzene within 10 h. In this study, engineered bacteria that can completely degrade nitrobenzene have been constructed successfully. The construction of remediation-engineered bacteria by synthetic biology laid the foundation for the industrial application of biological degradation of organic pollutants.

Carotenoids are a group of natural pigments that are widely distributed in various plants. Carrots are plants rich in carotenoids and have fleshy roots with different colors. Carotenoid accumulation is a complex regulatory process with important guiding significance for carrot production. In this work, three carrot cultivars with different taproot colors, Hongxinqicun (orange), Benhongjinshi (red), and Tianzi (purple) were chosen as experimental materials to explore the molecular mechanism of carotenoid accumulation in carrot. Results showed that the three carotenoids, namely, α-carotene, β-carotene, and lutein, had accumulated in orange carrot cultivar Hongxinqicun. Lycopene was only detected in the taproots of Benhongjinshi. Lutein was the main carotenoid in Tianzi. Comparison of the carotenoid contents in different tissues of carrot showed that leaf blade was the tissue with the highest carotenoid accumulation. Expression analysis of carotenoid biosynthesis genes and its correlation with carotenoid accumulation confirmed the regulatory role of structural genes in carrots. The high expression of five lycopene synthesis-related genes, DcPSY2, DcPDS, DcZDS1, DcCRT1, DcCRT2, and low expression of DcLCYE may result in the lycopene accumulation in Benhongjinshi. However, the function of certain genes, such as DcPSY1 that was lowly expressed in red carrot, requires further investigation. Our results provided potential insights into the mechanism of carotenoid accumulation in three carrot cultivars with different taproot colors.

Organophosphate compounds are widely used in pesticides to control weeds, crop diseases, and insect pests. Unfortunately, these synthetic compounds are hazardous and toxic to all types of living organisms. In the present work, Escherichia coli was bioengineered to achieve methyl parathion (MP) degradation via the introduction of six synthetic genes, namely, opdS, pnpAS, pnpBS, pnpCS, pnpDS, and pnpES, to obtain a new transformant, BL-MP. MP and its subsequent decomposition intermediates were completely degraded by this transformant to enter the metabolites of multiple anabolic pathways. The MP-degraded strain created in this study may be a promising candidate for the bioremediation of MP and potential toxic intermediates.

Industrial thiocyanate (SCN

Carrot is an important root vegetable crop abundant in bioactive compounds including carotenoids, vitamins, and dietary fibers. Carrot intake and its products are gradually growing owing to its high antioxidant activity. Auxins are a class of plant hormones that control many processes of plant growth and development. Yet, the effects of exogenous application of auxin on lignin biosynthesis and gene expression profiles of lignin-related genes in carrot taproot are still unclear. In order to investigate the effect of exogenous indole-3-butyric acid (IBA) on lignin-related gene profiles, lignin accumulation, anatomical structures and morphological characteristics in carrot taproots, carrots were treated with different concentrations of IBA (0, 50, 100, and 150 µM). The results showed that IBA application significantly improved the growth parameters of carrot. The 100 or 150 µM IBA treatment increased the number and area of xylem vessels, whereas transcript levels of lignin-related genes were restricted, resulting in a decline in lignin content in carrot taproots. The results indicate that taproot development and lignin accumulation may be influenced by the auxin levels within carrot plants.

Although glutathione S-transferase (GST, EC 2.5.1.18) is thought to play important roles in abiotic stress, limited information is available regarding the function of its gene in grapes. In this study, a GST gene from grape, VvGSTF13, was cloned and functionally characterized. Transgenic Arabidopsis plants containing this gene were normal in terms of growth and maturity compared with control plants but had enhanced resistance to salt, drought, and methyl viologen stress. The increased tolerance of the transgenic plants correlated with changes in activities of antioxidative enzymes. Our results indicate that the gene from grape plays a positive role in improving tolerance to salinity, drought, and methyl viologen stresses in Arabidopsis.

The high toxicity of persistent pollutants limits the phytoremediation of pollutants-contaminated soil. In this study, heterologous expressing Halorhodospira halophila single-stranded DNA binding protein gene (HhSSB) improves tolerance to 2,4,6-trinitrotoluene (TNT), 2,4,6-trichlorophenol (2,4,6-TCP), and thiocyanate (SCN−) in A. thaliana and tall fescue (Festuca arundinacea). The HhSSB transformed Arabidopsis, and tall fescue also exhibited enhanced phytoremediation of TNT, 2,4,6-TCP, and SCN− separately contaminated soil and co-contaminated soil compared to control plants. TNT assay was selected to explore the mechanism of how HhSSB enhances the phytoremediation of persistent pollutants. Our result indicates that HhSSB enhances the phytoremediation of TNT by enhancing the transformation of TNT in Arabidopsis. Moreover, transcriptomics and comet analysis revealed that HhSSB improves TNT tolerance through three pathways: strengthening the defense system, enhancing the ROS scavenging system, and reducing DNA damage. These results presented here would be particularly useful for further studies in the remediation of soil contaminated by organic and inorganic pollutants.

Thermostability is an important property of xylanase because high temperature is required for its applications, such as wood pulp bleaching, baking, and animal feedstuff processing. In this study, XynB from Thermobacillus composti, a moderately thermophilic gram-negative bacterium, was modified via site-directed mutagenesis (based on its 3D structure) to obtain thermostable xylanase, and the properties of this enzyme were analyzed. Results revealed that the half-life of xylanase at 65°C increased from 10 to 50 min after a disulfide bridge was introduced between the α-helix and its adjacent β-sheet at S98 and N145. Further mutation at the side of A153E named XynB-CE in the C-terminal of this α-helix enhanced the half-life of xylanase for 60 min at 65°C. Therefore, the mutant may be utilized for industrial applications.

Plant glutathione S-transferases (GSTs) are important for protecting plants against oxidative damage. We studied the function of a glutathione S-transferase family protein in Arabidopsis, AtGSTF2. Our results indicate the transgenic plants showed increased tolerance to oxidative stress caused by application of phenol. Under phenol stress, the lipid hydroperoxidation [the production of malondialdehyde (MDA)] of the leaves in overexpressing lines was suppressed compared with that of control plants. The antioxidative enzyme activities (SOD and POD) were higher in transgenic plants than in control. Furthermore, the residual phenol in medium was decreased more in transgenic plants than in control plants. These results indicate overexpressing GST protein reduce the damage of lipid hydroperoxidation and oxidative damage caused by phenol. Our findings also provide a suitable remediation strategy for sites contaminated by phenol.

Abscisic acid-responsive element (ABRE)-binding factors (ABFs) are important transcription factors involved in various physiological processes in plants. Stomata are micro channels for water and gas exchange of plants. Previous researches have demonstrated that ABFs can modulate the stomatal development in some plants. However, little is known about stomata-related functions of ABFs in carrots. In our study, DcABF3, a gene encoding for ABF transcription factor, was isolated from carrot. The open reading frame of DcABF3 was 1329 bp, encoding 442 amino acids. Expression profiles of DcABF3 indicated that DcABF3 can respond to drought, salt or ABA treatment in carrots. Overexpressing DcABF3 in Arabidopsis led to the increase of stomatal density which caused severe water loss. Expression assay indicated that overexpression of DcABF3 caused high expression of stomatal development-related transcription factor genes, SPCH, FAMA, MUTE and SCRMs. Increased antioxidant enzyme activities and higher expression levels of stress-related genes were also found in transgenic lines after water deficit treatment. Changes in expression of ABA synthesis-related genes and AtABIs indicated the potential role of DcABF3 in ABA signaling pathway. Under the treatment of exogenous ABA, DcABF3-overexpression Arabidopsis seedlings exhibited increased root length and germination rate. Our findings demonstrated that heterologous overexpression of DcABF3 positively affected stomatal development and also reduced ABA sensitivity in transgenic Arabidopsis.

The ADI1 Arabidopsis plants enhanced tolerance and degradation efficiency to naphthalene and had great potential for phytoremediation of naphthalene in the plant material before composting or harvesting and removal. Naphthalene is a global environmental concern, because this substance is assumed to contribute considerably to human cancer risk. Cleaning up naphthalene contamination in the environment is crucial. Phytoremediation is an efficient technology to clean up contaminants. However, no gene that can efficiently degrade exogenous recalcitrant naphthalene in plants has yet been discovered. Ferredoxin (Fd) is a key player of biological electron transfer reaction in the PAH degradation process. The biochemical pathway for bacterial degradation of naphthalene has been well investigated. In this study, a rice gene, ADI1, which codes for a putative photosynthetic-type Fd, has been transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants enhanced tolerance and degradation efficiency of naphthalene. Compared with wild-type plants, transgenic plants assimilated naphthalene from the culture media faster and removed more of this substance. When taken together, our findings suggest that breeding plants with overexpressed ADI1 gene is an effective strategy to degrade naphthalene in the environment.

To date, only aroA variants derived from Agrobacterium tumefaciens CP4 have been used to generate the commercial glyphosate-resistant crops currently available in the market. Therefore, it is of interest to increase aroA gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In this study, a novel aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase from Paracoccus denitrificans was isolated using the genomic library construction and complementary screening. Furthermore, the transgenic Arabidopsis plants with the aroA gene from P. denitrificans were obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

The plant-specific tau class of glutathione S-transferases (GSTs) is often highly stress-inducible and expressed in a tissue-specific manner, thereby suggesting its important protective roles. Although activities associated with the binding and transport of reactive metabolites have been proposed, little is known about the regulatory functions of GSTs. Expression of AtGSTU19 is induced by several stimuli, but the function of this GST remains unknown. In this study, we demonstrated that transgenic over-expressing (OE) plants showed enhanced tolerance to different abiotic stresses and increased percentage of seed germination and cotyledon emergence. Transgenic plants exhibited an increased level of proline and activities of antioxidant enzymes, along with decreased malonyldialdehyde level under stress conditions. Real-time polymerase chain reaction (PCR) analyses revealed that the expression levels of several stress-regulated genes were altered in AtGSTU19 OE plants. These results indicate that AtGSTU19 plays an important role in tolerance to salt/drought/methyl viologen stress in Arabidopsis.

Thermostability is an important property of xylanase because high temperature is required for its applications, such as wood pulp bleaching, baking, and animal feedstuff processing. In this study

Trichlorophenol (TCP) is a widely used and persistent environmentally toxic compound that poses a carcinogenic risk to humans. Phytoremediation is a proficient cleanup technology for organic pollutants. In this study, we found that the disulfide isomerase-like protein AtPDIL1–2 in plants is a good candidate for enhancing 2,4,6-TCP phytoremediation. The expression of AtPDIL1-2 in Arabidopsis was induced by 2,4,6-TCP. The heterologously expressed AtPDIL1-2 in Escherichia coli exhibited both oxidase and isomerase activities as protein disulfide isomerase and improved bacteria tolerance to 2,4,6-TCP. Further research revealed that transgenic tobacco overexpressing AtPDIL1-2 was more tolerant to high concentrations of 2,4,6-TCP and removed the toxic compound at far greater rates than the control plants. To elucidate the mechanism of action of AtPDIL1-2, we investigated the chemical interaction of AtPDIL1-2 with 2,4,6-TCP for the first time. HPLC analysis implied that AtPDIL1-2 exerts a TCP-binding activity. A suitable configuration of AtPDIL1-2-TCP binding was obtained by molecular docking studies using the AutoDock program. It predicted that the TCP binding site is located in the b-b′ domain of AtPDIL1-2 and that His254 of the protein is critical for the binding interaction. These findings imply that AtPDIL1-2 can be used for TCP detoxification by the way of overexpression in plants.

The CO2 concentration in the atmosphere has increased significantly in recent decades and is projected to rise in the future. The effects of elevated CO2 concentrations on morphological and anatomical characteristics, and nutrient accumulation have been determined in several plant species. Carrot is an important vegetable and the effects of elevated CO2 on carrots remain unclear. To investigate the effects of elevated CO2 on the growth of carrots, two carrot cultivars (‘Kurodagosun’ and ‘Deep purple’) were treated with ambient CO2 (a[CO2], 400 μmol⋅mol-1) and elevated CO2 (e[CO2], 3000 μmol⋅mol-1) concentrations. Under e[CO2] conditions, taproot and shoot fresh weights and the root/shoot ratio of carrot significantly decreased as compared with the control group. Elevated CO2 resulted in obvious changes in anatomy and ascorbic acid accumulation in carrot roots. Moreover, the transcript profiles of 12 genes related to AsA biosynthesis and recycling were altered in response to e[CO2]. The ‘Kurodagosun’ and ‘Deep purple’ carrots differed in sensitivity to e[CO2]. The inhibited carrot taproot and shoot growth treated with e[CO2] could partly lead to changes in xylem development. This study provided novel insights into the effects of e[CO2] on the growth and development of carrots.

A novel Fibre Bragg grating displacement sensor based on the cantilever beam structure is designed in this paper. Two fiber gratings with different central wavelengths are symmetrically pasted on the both sides of the cantilever beam. When the free end of the cantilever beam is changed, the two fiber gratings are respectively subjected to tension and pressure, which lead to the drift of the gratings’ center wavelength to the opposite direction. At the same time, the problem of cross sensitivity between temperature and displacement is solved. In addition, a smart device using to change the measuring range of the sensor is designed and it is also easy to assemble and disassemble, so the whole sensor can be widely used. The experimental results show that when the range is 30mm, the average sensitivity of the displacement sensor is 92.4pm/mm and the correlation coefficient is 0.999. While the range is 60mm, the average sensitivity of the displacement sensor is 47.7pm/mm and the correlation coefficient is 0.998.

The widespread presence of polycyclic aromatic hydrocarbons (PAHs) and their potential harm to various organisms has generated interest in efficiently eliminating these compounds from the environment. Phytoremediation is an efficient technology for cleaning up pollutants. However, unlike microorganisms, plants lack the catabolic pathway for complete degradation of these dangerous groups of compounds. One way to enhance the potential of plants for remediation of these compounds is by transferring genes involved in xenobiotic degradation from microbes to plants. In this paper, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as NahAa and NahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were transferred and ectopically expressed in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing the heterozygous naphthalene dioxygenase system exhibited enhanced tolerance toward 2-4 rings PAHs. Transgenic plants assimilated PAHs from the culture media faster and accumulated less in vivo than wild-type plants. Furthermore, examination of metabolic intermediates by gas chromatography-mass spectrometry revealed that the naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Taken together, our findings suggest that grafting the naphthalene dioxygenase complex into plants is a possible strategy to breed PAH-tolerant plants to efficiently degrade PAHs in the environment.

Although a large number of AroA enzymes (EPSPS: 5-enopyruvylshikimate-3-phosphate synthase) have been identified, cloned, and tested for glyphosate resistance, only two AroA variants, derived from Agrobacterium tumefaciens strain CP4 and Zea mays, have been utilized to produce the commercial glyphosate-resistant crops. Here, we have used a PCR-based twostep DNA synthesis method to synthesize an aroA gene (aroAA. metalliredigens) from Alkaliphilus metalliredigens, encoding a new EPSPS. Furthermore, transgenic Arabidopsis with the new aroAA. metalliredigens gene was obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

The widespread presence and potential harm conferred by polycyclic aromatic hydrocarbons (PAHs) to various organisms have raised increased research interest on these pollutants. Plants lack of efficient PAH degradation genes so that they had to suffer from PAH stress. To improve the ability of plants to tolerate various PAHs, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as nahAa and nahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were ectopically expressed in rice. The hybrid multicomponent naphthalene dioxygenase can assemble correctly and break down PAHs powerfully. Transgenic rice plants exhibited enhanced tolerance toward two- to four-ring PAHs. PAH tolerance in transgenic plants was associated with high chlorophyll levels and photosynthesis efficiency, normal development, and minimal ion leakage under PAH stress. The naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Our results indicate that grafting the naphthalene dioxygenase system into plants is useful to degrade PAHs efficiently in vivo and improve the plant tolerance to the pollutants. This study had introduced a suitable strategy to accelerate the bioremediation process of PAH-contaminated soil under waterlogged conditions and improve the food safety in contaminated area.

Glyphosate is the active component of the top-selling herbicide, the phytotoxicity of which is due to its inhibition of the shikimic acid pathway. 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the shikimic acid pathway. Glyphosate tolerance in plants can be achieved by the expression of a glyphosate-insensitive aroA gene (EPSPS). In this study, we used a PCR-based two-step DNA synthesis method to synthesize a new aroA gene (aroAR. leguminosarum) from Rhizobium leguminosarum. In vitro glyphosate sensitivity assays showed that aroAR. leguminosarum is glyphosate tolerant. The new gene was then expressed in E. coli and key kinetic values of the purified enzyme were determined. Furthermore, we transformed the aroA gene into Arabidopsis thaliana by the floral dip method. Transgenic Arabidopsis with the aroAR. leguminosarum gene was obtained to prove its potential use in developing glyphosate-resistant crops.

Environmental levels of bisphenol A (BPA) are a global concern because the compound can cause damage to reproductive organs, the thyroid gland, and brain tissues at developmental stages. Plants are important in removing BPA from the atmosphere, soil, and water. However, knowledge on the mechanism by which plants respond to this compound is limited. To determine the response mechanism of plants to BPA, we used a microarray system to analyze the gene expression patterns of Arabidopsis thaliana after irrigation with 3.0 mM BPA. We identified 651 genes that were differentially expressed upregulated and 470 genes that were downregulated by BPA. These genes may specifically contribute to BPA uptake, transformation, conjugation, and compartmentation in plants. The potential function of upregulated genes in plant defense against BPA was also determined.

Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Proteus mirabilis was identified and isolated. Furthermore, the transgenic Arabidopsis with the novel aroA gene was obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

Catalase is well known to eliminate H2O2 in cells and reduces the toxicity of peroxide compounds. A catalase gene HpCAT1 of methylotrophic yeast Hansenula polymorpha without the part coding the native signal peptide was cloned into expression vector pYM3165 and then integrated into genome of Pichia pastoris GS115 by electroporation. The result of the enzyme activity assay and SDS-PAGE demonstrated that the recombinant protein (HpCAT1) of H. polymorpha was extracellularly expressed in P. pastoris. The expressed catalase was recovered from the culture supernatant of P. pastoris GS115 and purified by (NH4)2SO4 fractionation and Ni-NTA affinity chromatography. The main biochemical properties of the recombinant protein HpCAT1, such as thermodependence and thermostability, pH optimum and pH stability, as well as the effect of metal ions and chemicals, were characterized. With H2O2 as the substrate, HpCAT1 displayed pH and temperature optima of ∼2.6 and 45°C, respectively. The recombinant HpCAT1 activity was inhibited by 1 mM Hg2+ and Cu2+, but was highly enhanced by 1.0 mM Fe2+.

A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.

Pichia pastoris is a suitable host for selecting mutant enzymes, since it can secret many gene products to the medium. However, poor transformation efficiency is often encountered. This makes it difficult to obtain a large number of transformants necessary to thoroughly cover a large library. We report here that pre-coating DNA with polyethyleneimine and dextran sulfate increased gene integration significantly in Pichia electroporation. This improvement on integrative efficiency has been proved in different voltage, resistance, cell phase, and DNA concentrations. The condensed DNA nanoparticles make Pichia available as a host for screening large libraries of random mutants.

A full-length cDNA sequence encoding a thiolase II of Betula platyphylla was identified from a constructed cDNA-AFLP library and obtained by the rapid amplification of cDNA ends (RACE) method. The cDNA gene, designated as BplTHI2, is 1 203 bp in length and encodes an enzyme of 400 amino acid residues with a calculated MW of 40.9 kDa. The BplTHI2 displays high similarity to the thiolase II of Camellia oleifera (90 % sequence identity) and Populus trichocarpa (89 %). It was predicted to possess two main domains, an N-terminal domain (10–268) and a C-terminal domain (278–398), which contain three predicted active sites (Cys96, His356 and Cys386). Its three-dimensional structure was predicted based on the crystal structure of human mitochondria thiolase (2ib8B). Expression analysis of the BplTHI2 gene showed that it was highly expressed in female inflorescences and not expressed in male ones. We suggest that BplTHI2 is a female inflorescence-specific gene and may be responsible for the development of female inflorescence.

Tomato yellow leaf curly virus (TYLCV), transmitted by the whitefly (), causes leaf curling and yellowing, plant dwarfism, and growth inhibition in tomato ( L.). The APETALA2 (AP2) and ethylene response factor (ERF) transcription factor (TF) family, the largest plant-specific TF family, was identified to function in plant development and pathogen defense. Our study aimed to analyze the mechanism underlying the function of ERF (SlERF) TFs in response to TYLCV infection and improve useful information to increase the resistance to TYLCV in tomato. A total of 22 tomato AP2/ERF TFs in response to TYLCV were identified according to transcriptome database. Five ERF-B3 TFs were identified in cultivars Hongbeibei (highly resistant), Zheza-301, Zhefen-702 (both resistant), Jinpeng-1, and Xianke-6 (both susceptible). Interaction network indicated that SlERF TFs could interact with mitogen-activated protein kinase (MAPK). Expression profiles of five ERF-B3 genes (, , , , and ) were detected by quantitative real-time–polymerase chain reaction (qRT-PCR) after TYLCV infection in five tomato cultivars. expression was upregulated in five tomato cultivars. The expressions of three genes (, , and ) were upregulated in Zheza-301 and Zhefen-702. and expressions were downregulated in Hongbeibei and Xianke-6, respectively. Yeast one-hybrid showed that the GCC-box binding ability of ERF-B3 TFs differed in resistant and susceptible tomato cultivars. Expression profiles were related to the GCC-box binding ability of SlERF TFs in resistant and susceptible tomato cultivars. The defense mechanism underlying the tomato’s response to TYLCV involved a complicated network, which provided important information for us in breeding and genetic analysis.

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized. The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K m of 0.15 mM and V max of 62 μmol min−1 mg−1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate. LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

Triphenylmethane dyes are extensively utilized in textile industries, medicinal products, biological stains, and food processing industries, etc. They are generally considered as xenobiotic compounds, which are very recalcitrant to biodegradation. The widespread persistence of such compounds has generated concerns with regard to remediation of them because of their potential carcinogenicity, teratogenicity, and mutagenicity. In this study, we present a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of triphenylmethane reductase (TMR) from the Citrobacter sp. The morphology and growth of TMR transgenic Arabidopsis plants showed significantly enhanced tolerances to crystal violet (CV) and malachite green (MG). Further, HPLC and HPLC-MS analyses of samples before and after dye decolorization in culture media revealed that TMR transgenic plants exhibited strikingly higher capabilities of removing CV from their media and high efficiencies of converting CV to non-toxic leucocrystal violet (LCV). This work indicates that microbial degradative gene may be transgenically exploited in plants for bioremediation of triphenylmethane dyes in the environment.

Trichlorophenol (TCP) and its derivatives are introduced into the environment through numerous sources, including wood preservatives and biocides. Environmental contamination by TCPs is associated with human health risks, necessitating the development of cost-effective remediation techniques. Efficient phytoremediation of TCP is potentially feasible because it contains a hydroxyl group and is suitable for direct phase II metabolism. In this study, we present a system for TCP phytoremediation based on sugar conjugation by overexpressing a Populus putative UDP-glc-dependent glycosyltransferase (UGT). The enzyme PtUGT72B1 displayed the highest TCP-conjugating activity among all reported UGTs. Transgenic Arabidopsis demonstrated significantly enhanced tolerances to 2,4,5-TCP and 2,4,6-TCP. Transgenic plants also exhibited a strikingly higher capacity to remove TCP from their media. This work indicates that Populus UGT overexpression in Arabidopsis may be an efficient method for phytoremoval and degradation of TCP. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TCP.

Birch (Betula platyphylla) has been widely applied in furniture, paper-making, and architecture for its special properties. One of the major problems is it has a long juvenile phase which limits the possibilities for production and application in these industries. To date, the molecular mechanism and biochemical pathways of the long juvenile phase are poorly understood. In this study, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was used to analyze genes specifically expressed in the male or female inflorescence of birch. More than 7,000 TDFs (Transcript Derived Fragments) were screened by using 100 primer combinations. Of them, 168 male and 103 female inflorescence specific TDFs were isolated, subcloned and sequenced. About 54% of these TDFs were found to be similar with known genes in NCBI databases. Through GO annotation and KEGG pathway analysis, the results indicated that the male inflorescence of B. platyphylla possesses nearly two-fold more transcripts than the female i...

A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

Coke powder (CP) added as the substitution of lean coal for coal blending was carried out in 5 kg coke oven. The influences of the particle size, the particle size interval and the amount of CP on the coke quality were investigated. The interfaces in which CP added inset the coke were discussed by means of microplariscope and SEM to confirm the effect of the microstructure on the coke quality. The results showed that the optimal particle size interval, the particle size and the amount of CP were 0.1~0.2 mm

In this paper, a research on adding dusts of blast furnace (DBF) which contains iron and carbon to the coal blend was carried out on 5 Kg coke oven. The influences of addition amount (0.25%~1.25%) and size distribution (0~2.5 mm) for DBF on the quality of the coke were investigated. The results showed that the influence on the quality of the coke was little when amount of DBF was less than 0.25% and particle size was from 0.5 mm to 1 mm, but the quality of the coke decreased obviously when addition amount was more than 0.25% and the size was larger than 1 mm.

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Plants are able to take up a range of harmful organic compounds, including some of the most abundant environmental pollutants like TCE. In this study, complementary DNA microarrays were constructed to have a better view of transcript expression in Arabidopsis thaliana during TCE-induced stress. The microarray analysis demonstrated the complexity of gene expression patterns resulting from TCE. A total of 1,020 transcripts were differentially up-regulated by TCE. Those genes might specifically contribute to the TCE transformation, conjugation, and compartmentation in plant. This study provides informative preliminary data for more in-depth analyses of TCE tolerance in Arabidopsis thaliana.

The gene encoding a cold-active and xylose-stimulated β-glucosidase of Marinomonas MWYL1 was synthesized and expressed in Escherichia coli. The recombinant enzyme (reBglM1) was purified and characterized. The molecular mass of the purified reBglM1 determined by SDS-PAGE agree with the calculated values (50.6 Da). Optima of temperature and pH for enzyme activity were 40°C and 7.0, respectively. The enzyme exhibited about 20% activity at 5°C and was stable over the range of pH 5.5–10.0. The presence of xylose significantly enhanced enzyme activity even at higher concentrations up to 600 mM, with maximal stimulatory effect (about 1.45-fold) around 300 mM. The enzyme is active with both glucosides and galactosides and showed high catalytic efficiency (k cat = 500.5 s−1) for oNPGlc. These characterizations enable the enzyme to be a promising candidate for industrial applications.

Polychlorinated biphenyls (PCBs) are considered as persistent organic pollutants. Their long term accumulation in soils and progressive bioaccumulation in the food chain makes PCBs a risk for human health. Phytoremediation, or the use of plants to enhance pollutant dissipation, is an efficient technique for the removal of organic compounds, including PCBs. However, the molecular basis of plant responses to PCBs has not been clearly elucidated. Here we studied the effects on growth and the transcriptional profile of Arabidopsis thaliana after exposure to 2,2′,3,3′-tetrachlorobiphenyl, a representative PCB. A comprehensive survey of global gene expression response to this PCB was done by using Arabidopsis (V4) gene expression microarray (4 × 44 K) to quantify the spatio-temporal variations in transcript abundance of 42,000 genes. The results revealed a coordinated induction or suppression of 146 or 148 genes, respectively. Of these, expression of 40 genes was validated by RT-PCR analysis. The functional classification of these PCB-responsive genes indicated their involvement in various metabolic pathways, such as ion transport, signal transduction, transcriptional regulation, and other processes related to growth and development.

In recent years, the biotechnological use of xylanases has grown remarkably. To efficiently produce xylanase for food processing and other industry, a codon-optimized recombinant xylanase gene from Streptomyces sp. S38 was synthesized and extracellularly expressed in Pichia pastoris under the control of AOX1 promoter. SDS-PAGE and activity assay demonstrated that the molecular mass of the recombinant xylanase was estimated to be 25 kDa, the optimum pH and optimum temperature were 5.5 and 50°C, respectively. In shake flask culture, the specific activity of the xylanase activity was 5098.28 U/mg. The K ( m ) and V ( max ) values of recombinant xylanase were 11.0 mg/ml and 10000 μmol min(-1) mg(-1), respectively. In the presence of metal ions such as Ca(2+), Cu(2+), Cr(3+) and K(+), the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of Hg(2+). This is the first report on the expression properties of a recombinant xylanase gene from the Streptomyces sp. S38 using Pichia pastoris. The attractive biochemical properties of the recombinant xylanase suggest that it may be a useful candidate for variety of commercial applications.

As one of China's great metropolises, Shanghai is vulnerable to various forms of industrial and agricultural contamination associated with its development. Polychlorinated biphenyls (PCBs) are man-made chemicals that never existed in nature until the 1900s when they started to be released into the environment. PCBs are hazardous environmental contaminants that bind strongly to soil. In this study, four soil samples were screened for the presence of PCB-degrading bacteria. The 16 S rDNAs were amplified from those genomes and the products (~1.5 kb) were purified and sequenced for the isolation and identification of bacterial species. Four Pseudomonas strains (strain 1-212 from sample 1; strain 2-241 from sample 2; strain 3-318 from sample 3; and strain 4-150 from sample 4) were selected for analysis by HPLC. Setting the content of the biphenyl in CK as 100%, the biphenyl contents was 2.32% in 1-212, 73.11% in 2-241, 69.83% in 3-318, and 86.16% in 4-150. The results of this study suggest directions for future research, including genetic screening, cloning and restructuring, and provide guidance for the cultivation of PCBs-degrading bacteria.

We applied in vitro mutagenesis and colony screening, using the wild type phyI1s gene from Aspergillus niger 113 as the template, and obtained two mutant phyI1s (gene products) after one round of screening. The two mutants had mutations at two nucleic acid sites, resulting in changes in two amino acids: K41E, E121F. None of the amino acid substitutions in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants indicated that the substitutions gave rise to 2.5- and 3.1-fold increased specific activity, and a 1.78- and 3.24-fold reduced affinity for sodium phytate. In addition, the overall catalytic efficiency (k cat/K m) of the two mutants was changed by 0.52-fold and 0.68-fold compared to that of the wild type. Such mutants will be instrumental for the structure–function study of the enzyme and for industrial application.

Zinc is essential but toxic in excess. A bacterial metallothionein, SmtA from Synechococcus PCC 7942, has high affinity for Zn2+ and the intracellular exclusively handling of Zn2+. In this study, we report a functional analysis of SmtA in Arabidopsis thaliana and its response to zinc stress. After high zinc stress, the transgenic plants over-expressing SmtA showed higher survival rate than the wild type. We also found that over-expression of SmtA in Arabidopsis increased the activities of SOD and POD, and enhanced the tolerance to zinc stress. Together, our results indicate that SmtA may play an important role in the response to zinc stress in Arabidopsis.

The coatings consisting of Fe0.64 Ni0.36 dendrites as matrix and (Fe, Cr)7 (C, B)3 interdendritic compounds as reinforcement were fabricated on the medium steel substrate by laser cladding using the Fe-based powders with different Co content, and Co is uniformly dissolved in the coatings. Compared with the coatings containing 1% and 5% Co, the coating containing 3% Co increases in the area fraction of the interdendritic region by about 5%, in the microhardness nearby the interface by about HV 55 and in the interfacial bond strength by about 30 MPa. The interface fracture morphology exhibits the tear appearance, which can be characterized as the ductile fracture.