Abstract:

Abstract:Various artificial riboswitches have been constructed by utilization of designed aptamers or by modification of natural riboswitch systems, because they can regulate gene expression in a highly efficient, precise and fast way, and promise to supply simple cis-acting, modular, and non-immunogenic system for use in future gene therapy applications. In this review, we present an overview of currently available technologies to design and select engineered riboswitches, and discuss some possible technologies that would allow them highly responsive to non-natural ligands, and dynamic control of gene expression in mammalian cells. Though how to bring custom-designed riboswitches as a novel and versatile tool box to gene control system is still a great challenge, the combination of structure-activity relationship information, computer based molecular design, in vitro selection, and high-through screening will serve as powerful tools for further development of riboswitch based gene regulatory systems.

Abstract:Plant height is one of the most important agronomic traits that could affect both crop yield and quality. Among all the hormones, gibberellins are crucial to regulate plant height. Cloning and molecular mechanism research of the plant height genes associated gibberellins have extremely important value for the regulation of crop growth and agricultural production, and have been widely used in rice, wheat and other grain crops breeding. In order to promote utilization of gibberellins in fruit trees, flowers and other horticultural crops breeding, we reviewed the regulation of plant height by gibberellins biosynthesis and signal transduction at the molecular level in this paper.

Abstract:Phosphatases play a key role not only in cell physiological functions of an organism, but also in host-pathogen interactions. Many studies demonstrated that some Gram-negative pathogenic bacteria could evade host immunity and promote pathogenicity by injecting phosphatases into host cells through type III secretion system. However, there were few reports about pathogenic fungi evading the immunity of hosts. Our researches indicated that the entomogenic fungus Metarhizium anisopliae could dephosphorylate the signal transduction substance of locust humoral immunity specifically in vitro by secreting extracellular protein tyrosine phosphatase, which implied that the fungus might interfere with the immune defense of locust. To provide reference for further studies of the functions of phosphatases, we reviewed the types of phosphatases and their roles in pathogen infection.

Abstract:Porcine interferon-α (pIFN-α) fermentative production by recombinant Pichia pastoris was carried out in a 10-L bioreactor to study its metabolism changes and effects on fermentation under different inducing strategies, by analyzing the change patterns of the corresponding metabolism and energy regeneration. The results show that the specific activities of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) largely increased when reducing temperature from 30 °C to 20 °C under pure methanol induction, leading significant enhancements in methanol metabolism, formaldehyde dissimilatory energy metabolism and pIFN-α antiviral activity. The highest pIFN-α antiviral activity reached 1.4×106 IU/mL, which was about 10-folds of that obtained under 30 °C induction. Using methanol/sorbitol co-feeding strategy at 30 °C, the major energy metabolism energizing pIFN-α synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle, formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved, and methanol flux distribution towards to pIFN-α synthesis was enhanced. Under this condition, the highest pIFN-α antiviral activity reached 1.8×107 IU/mL which was about 100-folds of that obtained under pure methanol induction at 30 °C. More important, enhanced pIFN-α production with methanol/sorbitol co-feeding strategy could be implemented under mild conditions, which greatly reduced the fermentation costs and improved the entire fermentation performance.

Abstract:Filamentous fungi have been extensively used in industrial fermentation processes. One of the most interesting topics of filamentous fungi is their bothersome morphology, which closely correlates with the productivity and broth rheology. Aiming at the optimization of the microbial process, works mainly focused on the relationships between morphology, productivity and environment in the last decades. Based on those works, we tried to interpret the mechanism of filamentous fungi growth from physiological aspects, and reviewed the mathematical models describing the hyphal growth, differentiation during the formation of target products. Above all, efficient and effective morphology controlling strategies were addressed from the engineering view.

Abstract:To investigate the mechanism of high product specificity of γ-clodextrin glucanotransferase (CGTase) from alkalophilic Bacillus clarkii 7364, we aligned protein sequence and structure model, found out that loss of 6 amino acids at subsite ?7 probably affected its product specificity. Using overlapping PCR method, we inserted 6 amino acids into subsite ?7 of CGTase. The mutant CGTase gene was ligated with pET-20b (+) and expressed in Escherichia coli BL21 (DE3). The extracellular recombinant enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild CGTase, the γ-CDs produced by mutant enzyme decreased from 76.0% to 12.5%, whereas the ratio of α- and β-CDs increased from 8.7% and 15.2% to 37.5% and 50%. The possible mechanism was that, compared to α-, β-CGTase, wild γ-CGTase lacks 6 amino acids in its subsite ?7. This conformation provided more space for glucose combination and was thus advantageous for forming γ-CD. When the 6 amino acids were inserted into the subsite ?7 of wild γ-CGTase, the space to bind with glucose reduced and consequently resulted in less γ-CD production.

Abstract:We describe a genetic transformation method of secondary somatic embryogenesis in alfalfa through cotyledon-stage somatic embryos of alfalfa infected by Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary vector pCAMBIA2301 that had gus gene as reporter and nptⅡ gene as selectable marker. The infected primary embryos were induced through series of medium under 75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS expression in different tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, the stable integration and transformation efficiency were tested by polymerase chain reaction and Southern blotting hybridization. The result showed that GUS expression was different in different organs of transgenic alfalfa; the copy number of integrated nptⅡgene was from 1 to 4; the transformation efficiency via primary somatic embryogenesis was 65.82%.

Abstract:In this study, the natural biological inducer, rat regenerating pancreatic extract (RPE), was used to induce human amniotic mesenchymal stem cells (hAMSCs) into insulin-secreting cells. We excised 60% of rat pancreas in order to stimulate pancreatic regeneration. RPE was extracted and used to induce hAMSCs at a final concentration of 20 μg/mL. The experiment methods used were as follows: morphological-identification, dithizone staining, immumofluorescence analysis, reverse transcription-PCR (RT-PCR) and insulin secretion stimulated by high glucose. The results show that the cell morphology of passge3 hAMSCs changed significantly after the induction of RPE, resulting in cluster shape after induction for 15 days. Dithizone staining showed that there were scarlet cell masses in RPE-treated culture. Immumofluorescence analysis indicated that induced cells were insulin-positive expression. RT-PCR showed the positive expression of human islet-related genes Pdx1 and insulin in the induced cells. The result of insulin secretion stimulated by high glucose indicated that insulin increasingly secreted and then kept stable with prolongation of high glucose stimulation. In conclusion, hAMSCs had the potential to differentiate into insulin-secreting cells induced by RPE in vitro.

Abstract:We reconstructed the erythromycin macrocyclic lactone (6-deoxyerythronolide B, 6dEB) synthesis pathway in Escherichia coli. We first cloned all the genes needed to synthesize the 6dEB into multi-gene co-expressed vectors. Then using the recognition sequences of isoschizomers XbaⅠ/SpeⅠ of vectors, we assembled the related genes into a series multiple-genes recombinant plasmids pBJ144, pBJ130. The recombinant plasmids pBJ144, pBJ130 were cotransformed into BAP1 to get the recombinant BAP1(pBJ144/pBJ130). SDS-PAGE analysis showed that individual genes were expressed correctly. After inducing at low temperature, adding propionate as substrate, we validated the crude product by mass spectrometry and the 6dEB yield was about 10 mg/L. These results indicated that the synthetic pathway of 6dEB was successfully assembled and reconstructed in Escherichia coli, which will greatly facilitate the reconstruction of whole erythromycin synthesis pathway and finally help to establish a stable research platform for developing of new derivatives of erythromycin and combinatorial biosynthesis of polyketide-type antibiotics.

Abstract:Proteasome is a multi-subunit protease complex in eukaryocytes, and plays an important role in ubiquitin-proteosome pathway. Recombinant proteasome can be used to screen proteasome inhibitors. In this study, recombinant plasmid of pET28a-PSMB1 was constructed by inserting human proteasome catalytic subunit (PSMB1) cDNA (726bp) into the prokaryotic expression vector pET28a(+), and transforming the plasmid into E. coli BL21(DE3) cells for expression. After overnight induction (1 mmol/L IPTG, 20 °C), an expected protein band with molecular weight of 27 kDa was observed on SDS-PAGE gel. The recombinant protein was then purified through affinity chromatography, and the purity is more than 95%. The amino acid sequence of the recombinant protein was validated by NanoLC-MS/MS. The data from in vitro BIAcore analysis showed that the recombinant PSMB1 could bind to celastrol. The binding affinity between PSMB1 and 10 μmol/L celastrol was more than 27RU.

Abstract:In the current study, we synthesized a 16-residue-long peptide VR with the aim of inspecting the feasibility to design β-hairpin-like antimicrobial peptide. The peptide was designed by alternating arrangement of arginine and valine and linking two stranded antiparallel β-sheet with a short loop segment (DPG) and a disulfide bridge. Antimicrobial and hemolytic activities were investigated. Melittin was chosen as a control peptide. We also tested bactericidal kinetics and salt sensitivity. Results show that VR had similar antibacterial activity compared with melittin. However, VR displayed much less hemolytic activity than melittin. These results suggest that VR had higher cell selectivity than melittin. The antibacterial activity of VR was not inhibited in the presence of 25 and 50 mmol/L NaCl. VR still possessed antibacterial activity in the presence of 100 mmol/L NaCl. Collectively, the de novo peptide VR displayed high antimicrobial activity, low hemolytic activity, and salt resistant, indicating that VR was a promising candidate for novel antimicrobial applications.