High-Efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR) for Determination of a Highly Degenerated Prophage WO Genome in a Wolbachia Strain Infecting a Fig Wasp Species

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of Wolbachia sequencing. Only one partial genome of a WO phage has been determined directly from isolated particles. We determine the complete genome sequence of prophage WO (WOSol) in Wolbachia strain wSol, which infects the fig wasp Ceratosolen solmsi (Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. The genome of WOSol is highly degenerated and disrupted by a large region (14,267 bp) from Wolbachia. Consistent with previous molecular studies of multiple WO genomes, the genome of WOSol appears to have evolved by single nucleotide mutations and recombinations.     

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis–C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen–host.     

Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene

Like other filamentous fungi, the medicinal ascomycete Cordyceps militaris frequently degenerates during continuous maintenance in culture by showing loss of the ability to reproduce sexually or asexually. Degeneration of fungal cultures has been related with cellular accumulation of reactive oxygen species (ROS). In this study, an antioxidant glutathione peroxidase (Gpx) gene from Aspergillus nidulans was engineered into two C. militaris strains, i.e., the Cm01 strain which can fruit normally and the Cm04 strain which has lost the ability to form fruiting bodies on different media through subculturing. The results showed that the mitotically stable mutants had higher Gpx activities and stronger capacity to scavenge cellular ROS than their parental strains. Most significantly, the fruiting ability of Cm04 strain was restored by overexpression of the antioxidant enzyme. However, after being successively transferred for up to ten generations, two of three Cm04 mutants again lost the ability to fruit on insect pupae while Cm01 transformants remained fertile. This study confirms the relationship between fungal culture degeneration and cellular ROS accumulation. Our results indicate that genetic engineering with an antioxidant gene can be an effective way to reverse fungal degeneration during subculturing.     

Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli

Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.     

Dynamic expression of miR-126 and its effects on proliferation and contraction of hepatic stellate cells

In our previous study, miR-126 was identified as one of the leading miRNAs that is downregulated during activation of hepatic stellate cells (HSCs). However, the roles and related mechanisms of miR-126 in HSCs are not understood. In this study, we compared expression of miR-126 during HSC activation both in vitro and in vivo. We also applied RNA interference to analyze the role and mechanism of miR-126^∗ in the activation of HSCs. Restoring HSCs with Lv-miR-126^∗ resulted in decreased proliferation, accumulation of extracellular matrix components, and cell contraction, while also negatively regulating the vascular endothelial growth factor (VEGF) signal transduction pathways by partially targeted VEGF-A. Thus, we postulate that miR-126 may be a biological marker for the activation of HSCs, and useful for reducing intrahepatic vascular resistance and improving the sinusoidal microcirculation in chronic liver diseases.     

钝齿棒杆菌argR基因缺失株构建及其缺失对精氨酸生物合成途径相关基因转录水平的影响

[目的]钝齿棒杆菌AS 1.542中argR基因编码的蛋白ArgR在精氨酸生物合成途径中扮演负调控的角色,但其对相关基因在转录水平的影响还未见报道.因此,本课题组构建了钝齿棒杆菌argR基因缺失株,并在转录水平上比较野生株与缺失株精氨酸生物合成途径相关基因的变化.[方法]采用无痕敲除的方法构建了钝齿棒杆菌argR基因缺失株,并采用荧光定量PCR方法分析缺失株和野生株精氨酸生物合成途径相关基因在转录水平的变化.[结果]利用pK18mobsacB质粒中蔗糖致死基因sacB反向筛选标记及PCR方法成功筛选到钝齿棒杆菌argR基因缺失株;荧光定量PCR结果表明,argR基因缺失株精氨酸生物合成途径中相关基因在转录水平获得大量提高,平均约上调162.13倍.[结论]钝齿棒杆菌精氨酸生物合成途径的相关基因受负调控蛋白ArgR的显著调控,但其基因的敲除并没有引起精氨酸产量发生明显的变化.     

Molecular Profiling of Genetic Variability in Domesticated Groundnut (Arachis hypogaea L.) Based on ISJ, URP, and DAMD Markers

To detect DNA polymorphisms in the peanut, we screened 26 polymorphic primers using intron–exon splice junction (ISJ), universal rice primer (URP), and directed amplification of minisatellite region DNA (DAMD) techniques. Amplification of genomic DNA of 16 peanut accessions yielded 121 ISJ, 50 URP, and 25 DAMD fragments, of which 34, 25 and 16 were polymorphic, respectively. The range of polymorphism was 10.0–62.5%, averaging 27.7%, for ISJ; 20–80%, averaging 49.5%, for URP; and 28.6–50.0%, averaging 36.3%, for DAMD. In comparisons of multiplex ratio, average polymorphism information content, and marker index, the URP markers were relatively more efficient than ISJ and DAMD markers. Clustering results remained more or less the same with ISJ and URP markers. To the best of our knowledge, this is the first report on the study of the genetic diversity of the peanut using ISJ, URP, and DAMD markers.     

Genomic Resources for Gene Discovery,Functional Genome Annotation,and Evolutionary Studies of Maize and Its Close Relatives

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.     

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.