Isolation and Identification of Pathogenicity Mutant of Curvularia lunata via Restriction Enzyme-Mediated Integration

In this report, 156 hygromycin-resistant mutants were generated via restriction enzyme-mediated insertional (REMI) mutagenesis. All mutants were subjected to a bioassay on detached leaves. Five mutants (T4, T39, T71, T91, and T135) showed reduced symptom development, whereas one mutant (T120) did not exhibit any symptoms on the leaves compared with the wild type. The pathogenicity of these mutants was further assayed through the spray inoculation of whole seedlings. The results demonstrated that the pathogenicity of the T4, T39, T71, T91, and T135 mutants was reduced, whereas the T120 mutant lost its pathogenicity. Southern blot analysis revealed that the plasmids were inserted at different sites in the genome with different copy numbers. Flanking sequences approximately 550, 860, and 150 bp were obtained from T7, T91, and T120, respectively through plasmids rescue. Sequence analysis of the flanking sequences from T7 and T91 showed no homology to any known sequences in GenBank. The flanking sequence from the T120 mutant was highly homologous to MAPKK kinases, which regulates sexual/asexual development, melanization, pathogenicity from Cochliobolus heterostrophus. These results indicate that REMI and plasmids rescue have great potential for finding pathogenicity genes.     

桑疫病病原拮抗菌的分离、鉴定及发酵条件优化北大核心CSCD

【目的】从健康桑树内生菌中分离获得对桑疫病病原菌(Pseudomonas syringae pv.mori)具有显著拮抗作用的菌株,优化其产生抑菌活性物质的发酵条件,为其生防利用奠定基础。【方法】从严格表面消毒的桑树根茎中分离内生菌,采用平板划线法纯化内生菌,用抑菌圈法筛选拮抗菌;根据菌株的形态与培养特征、生理生化特性、16S rDNA序列分析对其进行鉴定。通过单因素试验和正交设计试验优化培养基组分及发酵条件。【结果】从健康桑树中分离获得内生菌77株,其中,编号为SWg2的菌株对桑疫病病原菌具有强而稳定的抑制作用。菌株SWg2的形态与培养特征、生理生化特性和泛菌属(Pantoea sp.)相符,而16S rDNA序列分析结果显示它与成团泛菌(P.agglomerans)的亲缘关系接近。研究表明其最佳发酵配方和培养条件为:甘油(2.00%)、硝酸铵(2.00%)、KH2PO4(0.10%)和MgSO4·7H2O(0.15%),起始pH为7.5,装瓶量20 mL/100 mL,最适培养温度为28℃,转速为170 r/min,种子液接种量为4%,摇瓶培养5 d。【结论】经鉴定,对桑疫病病原具拮抗作用的桑树内生菌SWg2为成团泛菌(P.agglomerans),命名为成团泛菌SWg2。对其发酵条件进行优化后对桑疫病病原菌显示出更强的拮抗作用。     

Vibrio zhuhaiensis sp. nov., isolated from a Japanese prawn (Marsupenaeus japonicus)

A Gram-negative, oxidase-positive, facultatively anaerobic bacterium, designated strain E20121, was isolated from the digestive tract of a Japanese prawn (Marsupenaeus japonicus) collected from the coastal sea water area of Zhuhai, Guangdong province, China. The new isolate was determined to be closely related to Vibrio ponticus DSM 16217^T, having 97.6 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on recA, pyrH and rpoA also showed low levels of sequence similarities (72.6–96.6 %) with all species of the genus Vibrio. A multigene phylogenetic tree using concatenated sequences of the four genes (16S rRNA, rpoA, recA and pyrH) clearly showed that the new isolate is different from the currently known Vibrio species. DNA–DNA hybridization experiments revealed similarity values below 70 % with the closest related species V. ponticus DSM 16217^T. Several phenotypic traits enabled the differentiation of strain E20121 from the closest phylogenetic neighbours. The DNA G+C content of strain E20121 was determined to be 47.6 mol % and the major fatty acid components identified were C_16:1ω7c and/or C_16:1ω6c (39.8 %), C_18:1ω7c (13.6 %) and C_16:0 (9.6 %). Based on genotypic, phenotypic, chemotaxonomic, phylogenetic and DNA–DNA hybridization analyses, strain E20121 is proposed to represent a novel species of the genus Vibrio for which the name Vibrio zhuhaiensis sp. nov. is proposed. The type strain is E20121^T(=DSM 25602^T = CCTCC AB 2011174^T).     

Ectopic overexpression of porcine DGAT1 increases intramuscular fat content in mouse skeletal muscle

The microsomal enzyme 1, 2-acyl CoA: diacylglyceroltransferase-1 (DGAT1) plays an important role in triglyceride storage in adipose tissue and expresses in skeletal muscle as well. The primary goal of the present study was to investigate the effect of porcine DGAT1 on intramuscular fat (IMF) content of transgenic mice produced by pronuclear microinjection with muscle specific promoter of porcine muscle creatine kinase (MCK). In normal chow-fed diet, 4 month-old male transgenic mice expressed more DGAT1, ACC1, UCP1, and FABP4 mRNAs and proteins in skeletal muscle than control mice by real-time PCR and western blot. No significant changes were detected for ACC2, CD36, ADRP, PPAR gamma and LPL. Triacylglycerol assay and soleus muscle sections showed overexpression of porcine DGAT1 in skeletal muscle increased intramyocellular triglyceride and percent of the total cell surface covered by lipid droplets. Thus, upregulation of porcine DGAT1 in skeletal muscle increases IMF content. The present study may further serve to develop transgenic pigs with higher IMF content and improved meat quality.     

Glutamate transporter type 3 knockout leads to decreased heart rate possibly via parasympathetic mechanism

Parasympathetic tone is a dominant neural regulator for basal heart rate. Glutamate transporters (EAAT) via their glutamate uptake functions regulate glutamate neurotransmission in the central nervous system. We showed that EAAT type 3 (EAAT3) knockout mice had a slower heart rate than wild-type mice when they were anesthetized. We design this study to determine whether non-anesthetized EAAT3 knockout mice have a slower heart rate and, if so, what may be the mechanism for this effect. Young adult EAAT3 knockout mice had slower heart rates than those of their littermate wild-type mice no matter whether they were awake or anesthetized. This difference was abolished by atropine, a parasympatholytic drug. Carbamylcholine chloride, a parasympathomimetic drug, equally effectively reduced the heart rates of wild-type and EAAT3 knockout mice. Positive immunostaining for EAAT3 was found in the area of nuclei deriving fibers for vagus nerve. There was no positive staining for the EAATs in the sinoatrial node. These results suggest that EAAT3 knockout mice have a slower heart rate at rest. This effect may be caused by an increased parasympathetic tone possibly due to increased glutamate neurotransmission in the central nervous system. These findings indicate that regulation of heart rate, a vital sign, is one of the EAAT biological functions.     

Inhibition of Fe-induced colon oxidative stress by lactobacilli in mice

Iron (Fe) can promote hydrogen peroxide (H₂O₂) and hydroxyl radical generation in the colonic surface and promote growth of Fe-dependent bacteria. Some Lactobacillus strains are resistant to oxygen free-radicals, allowing them to survive in a Fe-modulated mucosal environment and influence colon microbial ecology and redox state. Here, we investigated the capacity of lactobacilli with different antioxidant abilities to modify the bacterial profile and prevent oxidative stress in the colon of Fe-overloaded mice. Survival time of Lactobacillus rhamnosus LGG (LGG) in the presence of H₂O₂ and hydroxyl radical was significantly longer compared with the mid- and non-antioxidative strains, Lactobacillus paracasei Fn032 and Lactobacillus plantarum Fn001, respectively. Different Lactobacillus strains are specific in free-radical scavenging activities of their cell-free extracts, which increased to varying extent depending on strains when bacteria were exposed to simulated gastric and pancreatic juice. Fe-overloaded mice showed increased colonic luminal ferrous Fe content, Enterococcus and Escherichia coli concentrations, mucosal malondialdehyde and free-radicals, and decreased mucosal total antioxidative capacity and oxidative enzymatic activity. Translocation of endotoxin to the liver was also significantly increased (P < 0.05). Lactobacilli inhibited ferrous Fe accumulation, especially in LGG and Fn032. LGG significantly inhibited the increase of colonic mucosal free-radicals and malondialdehyde content (P < 0.05). Fn032 only inhibited malondialdehyde (P < 0.05). LGG and Fn032 significantly inhibited increases in colonic Enterococcus (P < 0.05). Fn001 showed no significant antioxidative ability in vivo. The difference of these effects in vivo were well agreed with scavenging activities against reactive oxygen species (ROS) of simulated gastrointestinals fluid pretreated cells in vitro. In conclusion, ROS scavenging activities was essential for Lactobacillus to prevent oxidative stress in vivo and inhibition of ROS-producing bacterial growth and mucosal barrier injury.     

A molecular level prototype for mechanoelectrical transducer in mammalian hair cells

The mechanoelectrical transducer (MET) is a crucial component of mammalian auditory system. The gating mechanism of the MET channel remains a puzzling issue, though there are many speculations, due to the lack of essential molecular building blocks. To understand the working principle of mammalian MET, we propose a molecular level prototype which constitutes a charged blocker, a realistic ion channel and its surrounding membrane. To validate the proposed prototype, we make use of a well-established ion channel theory, the Poisson–Nernst–Planck equations, for three-dimensional (3D) numerical simulations. A wide variety of model parameters, including bulk ion concentration, applied external voltage, blocker charge and blocker displacement, are explored to understand the basic function of the proposed MET prototype. We show that our prototype prediction of channel open probability in response to blocker relative displacement is in remarkable accordance with experimental observation of rat cochlea outer hair cells. Our results appear to suggest that tip links which connect hair bundles gate MET channels.