Objective: To study the effects of cartilage oligomeric matrix protein (COMP) overexpression on bone morphogenetic protein 2 (BMP-2) induced cell osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs). Methods: MSCs, transfected with plasmid DNA encoding recombinant human COMP, were induced to differentiate into osteocytes and chondrocytes by BMP-2. Western blotting and real-time PCR were used to detect the overexpression of COMP, the expression level changes of osteogenesis related genes collagen type Ⅰ, RUNX2, osteocalcin and chondrogenesis related genes collagen type Ⅱ, SOX9, aggrecan, collagen type X, respectively. Alizarin red staining for osteogenic differentiation and alcian blue staining for chondrogenic differentiation were conducted to evaluate the tendency of cell differentiation. Results: Results of RT-PCR and Western blotting showed that the expression levels of COMP mRNA and protein were significantly increased in MSCs, which were transfected with plasmid DNA encoding recombinant human COMP (P<0.05). The mRNA expression levels of RUNX2, Col1a1 and the protein expression levels of RUNX2, osteocalcin in overexpressed group all decreased significantly compared with that of control group (P<0.05). However, the mRNA expression levels of SOX9, aggrecan and the protein expression levels of SOX9, Col2a1 in overexpressed group all increased significantly compared with that of control group (P<0.05). Alizarin red staining were weakened while alcian blue staining was enhanced. The expression level of Col10a1 gene in overexpressed group was higher than that in control group (P<0.05). Conclusion: Overexpression of COMP gene in MSCs could inhibit BMP-2 induced osteogenic differentiation, promote BMP-2 induced chondrogenic differentiation and supress the chondrocyte hypertrophy and maturation, which may provide new insight for cartilage tissue engineering.
Maize (Zea mays L.) genic male sterility mutant lines are one kind of elite germplasm, which is helpful to genetics and breeding research and hybrid production in maize. A maize genic male sterility mutant, male sterility14 (ms14) was preliminary studied. Firstly, phenotypical analysis and I2-KI dyeing of ms14 tassels and anthers showed that ms14 mutant is completely pollen sterile and exerts no anthers. Secondly, genetic analysis using two F2 segregation populations of ms14 (ms14×zheng58 and ms14×chang7-2), the ratio of fertile to sterile plants is 3 to 1, which shows that the ms14 mutant is controlled by a recessive genic male sterility gene. Thirdly, through the linkage analysis between ms14 locus and molecular markers in the F2 segregation populations of ms14 (ms14×chang7-2), the ms14 gene is roughly mapped to an interval on Chromosome 1 between SSR markers umc2025 and umc1676, with the genetic distance of 2.2 cM and 0.3 cM, respectively. Finally, the potential application value of the ms14 gene in maize hybrid breeding and production is discussed.
A trypsin inhibitor (CoTI1) from Cassia obtusifolia was attributed to the Kunitz-type trypsin inhibitor family. According to the sequence alignment, Arg86, Leu84 and Thr88 might be the key residues of CoTI1. In order to confirm the speculation, the three above residues were replaced as Asp by site-directed mutagenesis, respectively, and analysis the inhibitory activity of the mutants and CoT1 to trypsin and insects' digestive enzyme. Compared with CoT1, the inhibitory activity of the mutant CoTI1R86D to trypsin decreased most obviously, and the inhibitory effect of the original 93% was lost. CoTI1L84D lost 59% of the inhibitory effect; while the inhibitory activity of CoTI1T88D decreased by 64%. The average inhibitory activity decreased 88.7%, 57% and 60.7% to digestive enzymes of Helicoverpa armigera, Beet armyworm and Spodoptera litura, respectively. The result shows that Arg86, Leu84 and Thr88 are the key residues of CoT1, and it was useful for the molecular mechanism and anti-insects study of CoTI1.
To excavate a novel epoxide hydrolase from Phaseolus vulgaris (PvEH1) and explore its enantioconvergent catalytic performance, a PvEH1-encoding gene (pveh1) was amplified from the P. vulgaris total RNA by RT-PCR technique. Then, pveh1 was heterologously expressed in E. coli BL21(DE3) mediated by an expression plasmid pET28a(+). Analysis of primary and three-dimensional structures indicated that the identities of PvEH1 with Vigna radiata and Medicago truncatula epoxide hydrolases are 85.7 and 81.1%. Its catalytic triad is Asp101-His299-Asp264, belonging to the α/β-hydrolase superfamily. When the conversion rate of racemic styrene oxide catalyzed by PvEH1 reached 99.1%, the product, (R)-1-phenyl-1,2-ethanediol, was obtained with an enantiomeric purity of 33.6% e.e.p. PvEH1 possesses the opposite regioselectivity towards (S)-SO and (R)-SO with regioselectivity coefficients (αS and βR) of 91.1 and 53.3%. The discovery of PvEH1 and characterization of its enantioconvergence not only increased the number of plant EHs, but also established a foundation for the study of its catalytic mechanism and the directed modification of its regioselectivity.
Plants are exposed to different abiotic stresses that affect growth, development, and productivity. Previous studies have shown that late embryogenesis abundant (LEA) proteins play protective roles in plant adaptation to abiotic stresses. Based on previous study, CkLEA1 (GenBank accession number: KC309408), a LEA gene isolated from Caragana korshinskii, was transferred into Arabidopsis. The transgene expression was detected by quantitative real-time PCR and three independent transgeniclines with different transgene expression level were selected for further analysis. CkLEA1 transgenic lines germinated more quickly than the wild-type on medium containing 200 mmol/L NaCl or 400 mmol/L mannitol. After drought stress treatment, the CkLEA1 transgenic lines were more resistance to drought stress and exhibited a significantly higher survival ratio. Meanwhile, transgenic lines lost water slow than the wild-type under dehydration treatment, accompanied with less accumulation of malondialdehyde (MDA) and increased superoxide dismutase (SOD) activities as well as glutathione (GSH) content. Taken together, these results indicated that CkLEA1 improved tolerance to salt and osmotic treatments during seed germination and increased tolerance to drought stress during seedling growth.
From the genome of Pseudomonas antitumoralis HUP007, an esterase gene harboring 1041 bp and encoding a protein EstP8 of 377 amino acid was cloned. Esterase EstP8 was efficiently expressed in E. coli BL21(DE3) and further purified. Esterase EstP8 belongs to the IV family of lipases and contains conserved HGGG motif. The optimal substrate of esterase EstP8 was p-NPO, the optimal working temperature of esterase EstP8 was 50℃ and the optimal working pH of esterase EstP8 was 8.0. The hydrolysis activity of esterase EstP8 toward p-NPO was 105.19 U/mg, with Vmax and Km being 89.4 μmol/min and 1.144 mM, respectively. Esterase EstP8 remained high activity at pH7.0~8.0. The relative activity of esterase EstP8 was 41.78% at 4℃ and esterase EstP8 behaved very good thermo-stability at 10~40℃. EstP8 exhibited very good resistance to most metal ions tested. The addition of Cu2+, Mn2+ or Zn2+ of low concentrations could even stimulate the activity of EstP8. Organic solvents such as isooctane, methylbenzene, acetone and DMF could stimulate the activity of EstP8. Esterase EstP8 could generate chiral (R)-1-phenylethanol through kinetic resolution. The addition of organic solvents could well stimulate the stereo-selectivity and conversion during kinetic resolution. The e.e. and conversion of generated (R)-1-phenylethanol could reach 91% and 18%, respectively, in the presence of methylbenzene. The e.e. and conversion of generated (S)-styrallyl acetate could reach 98% and 60%, respectively, in the presence of DMSO. Psychrophilic esterase EstP8 possesses good potential in diverse industries such as stereo-selective biocatalysis.
In order to produce biofuels by the use of microalgae and improve the efficiency of carbon-oxygen transfer in a confined environment, the growth of desert microalgae BG18-3, BE6-2 and freshwater cyanobacterial strain 7924 were thoroughly explored in stressed conditions, and microalgae BG18-3 was found to have the best performance in some stressed conditions. Compared to other two strains of microalgae in static culture, desert microalgae BG1-3 had an advantage on microalgae biomass accumulation and nitrogen and phosphorus removal efficiency, of which the biomass dry weight reached 0.26 g/L, nitrogen and phosphate removal efficiency were 36% and 99%, respectively. In the aeration culture of desert microalgae BG18-3 ventilation, the highest biological dry weight (3% CO2 aeration cultured for 16 days) and the biomass yield reached 2.63 g/L and 164.0 mg/L·d respectively, and the concentration of CO2 at the outlet dropped to 0.04%, while the concentration of O2 increased by 0.68%, which indicates that the BG18-3 has the potential to be a biological medium in a confined environment. Finally, the BG18-3 was identified as Scenedesmus littoralis according to the results of rDNA 18s analysis.
With rapid development of agriculture,genetic engineering as an important means of transformation of the potato traits has been extensively applied.Optimization of potato tissue culture system and exogenous gene conversion conditions are the basis of the work of the transgenic potato.Three potato cultivars having different maturity period(Dongnong 303,Zaodabai,Atlantic)have been tested, with the use of orthogonal experiment,screen,optimize stem segments and Microtuber differentiation system.Stem callus regeneration system and in vitro differentiation of potato direct regeneration system were built.Then applied genetic transformation with Agrobacterium tumefaciens including the reconstruction vector carrying the target gene LYCB and selection marker NPTII.Respectively,the three materials stem section plantlets and microtubers were manipulated,and the transgenic conversion conditions were optimized for each one.Then the three were applyied to the best regeneration system and genetic transformation system,finally positive plants using PCR screening and statistically analysised the data were got.The results show that conversion rate via stem callus of Dongnong 303,Zaodabai and Atlantic respectively were 36%,35%,28%;direct differentiation rate via Microtuber of them separately were 43%,45%,17%.In conclusion,microtuber of Dongnong 303/Zaodabai is suitable for the conversion tissue of test organization,stem of the Atlantic is better conversion one.
A novel strategy for arbitrarily controlling acetone concentration and acetone/butanol ratio in ABE fermentation by C. acetobutylicum was proposed. With this strategy, (1) the residual glucose concentration could reduce to low level close to 0 g/L when ABE fermentation enters the solventogenic phase, by adaptively controlling the initial glucose concentration in the corn-based medium via glucoamylase usage dose, reaction time and temperature regulation; (2) Under the condition of glucose limitation, C. acetobutylicum has the ability to release more glucoamylase for its survival and the secreted glucoamylase could continuously hydrolyze oligosaccharide to maintain glucose at low concentration, and therefore repress the metabolism or synthesis rates of glycolysis and NADH in C. acetobutylicum. At the same time, exogenous addition of acetate creates a glucose/acetate co-substrate environment. The co-substrate system would not deteriorate the energy metabolisms in ABE fermentation, but could adaptively repress butanol synthesis avoiding the early occurrence of butanol inhibition and enhance bio-acetone synthesis by effectively utilizing the exogenously added acetate; (3) On the top of exogenous acetate addition, adaptively adding certain amount of viable S. cerevisiae to form C. acetobutylicum/S. cerevisiae co-culturing system, would enhance C. acetobutylicum tolerant ability against higher butanol concentration environment. The entire system could arbitrarily control acetone concentration and acetone/butanol ratio in the ranges of 5~12 g/L and 0.5~1.0, their maximum values could reach levels of 11.74 g/L and 1.02, while maintaining butanol concentration within normal range of 10~14 g/L, to satisfy different requirements on acetone and butanol products in industrial ABE fermentation.
CRISPR-Cas9 is a novel highly specific and efficient technology of genome editing. CRISPR-Cas9 system consists of single guide RNA (sgRNA) and Cas9 protein, it can produce site-specific DNA double-strand breaks (DSBs), then induces the repair of nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) that gives rise to targeted genome modifications. Since its discovery, with its several advantages of easy operation, low cost, high efficiency and simultaneous targeting of arbitrary number of gene and so on, it has been widely applied. Recent researches show that the genome editing of CRISPR-Cas9 provides a new direction for gene therapy, through accurate disruption of causative endogenous gene or correction of the causative mutation or insertion of a new protective gene.The structure and the mechanism of CRISPR-Cas9, and also its gene therapy in diseases were reviewed and highlighted.
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) is a gene editing technique of which RNA guiding endonuclease to cleavage genome. The discovery of this technology promoted biological and medicinal studies. This simplicity of the CRISPR-Cas9 system has enabled its widespread applications in cell genome editing, animal models generation and gene therapy of disease models. The structure characteristics, mechanism and applications of CRISPR-Cas9 system were focused on.
Endothelial progenitor cells are precursor cells of endothelial cell,it can differentiate into mature endothelial cells and play an important role in vascular endothelial regeneration. According to the recent researches on endothelial progenitor cells, it has wide application prospect in clinical diagnosis, prognosis judgement and treatment of ischemic diseases. However, there is still controversy about standardized criteria and consensus for defining, characterizing, and identifying EPCs.
Transcription factor Rex is a sensor of intracellular NADH/NAD+ redox state by incorporating either NADH or NAD+ in Gram-positive bacteria. In response to NADH/NAD+ ratio, Rex could control many physiological metabolisms by regulating genes expression. NAD[H], the key coenzyme in the metabolic network, could reflect the cellular redox state and influence the activity of Rex. The structures of Rex, either alone or bound with cofactor and/or DNA operator, have been determined in aerobic and anaerobic group. The regulatory mechanism of Rex and the relationship of Rex, NADH/NAD+, and target genes were revealed through comparative analysis on its structure in aerobic and anaerobic bacterial. Meanwhile, the connections of Rex with central carbon, energy metabolism, anaerobic metabolism, fermentation, biofilm formation, oxidative stress as well as cell toxicity were elucidated. In addition, further study trends and applications of Rex were discussed.
Chlamydiae are obligate intracellular Gram-negative bacteria that cause widespread diseases in humans. Vaccination is the most economical and effective way to control chlamydial infections. At present, the main kinds of chlamydial vaccine include subunit vaccine, vector vaccine, DNA vaccine, and so on. These vaccines often require adjuvants to enhance the immune effects. The progress of adjuvants in the applications of chlamydial vaccine are discussed.
Bacterial small non-coding RNAs (sRNAs), an emerging class of regulatory RNAs, interact with their mRNA targets by base-pairing to inhibit or activate their translation, regulate expression of protein, and possess various biological functions. Recently, with the development of RNA-seq, bioinformatics and experimental techniques, hundreds of sRNAs have been explored in bacteria. As regulator factors at the posttranscriptional level, sRNAs have received extensive attention due to their important roles in biological processes. The sRNAs in gram-positive bacteria are focused on. The researches on screening, identification and functions of sRNA are summarized and the inherent relation between sRNAs and virulence factors, quorum sensing, iron metabolism as well as two-component system is elucidated and analyzed, whilst future study trends were proposed.
Cell drugs are eventually used in the human body, it is necessary to establish corresponding standards for quality control. To ensure the safety, effectiveness and stability of the product, the whole process of donor screening,tissue collection, cell separation, cell culture, cell cryopreservation, cell resuscitation, check out, transport and application must be systematically carried out. In recent years, China has gradually changed the management ideas of treating the cell therapy as the third level medical technology. On the one hand, the administrative examination and approval of already existing third level medical technology are currently cancelled. And on the other hand, stem cell transplantations are managed as drug except for autologous stem cell transplantation, and the corresponding standards and management method of quality have already been established.
