应用基因敲除快速构建大肠杆菌突变体改造脂肪酸代谢途径

【目的】基因敲除技术是研究基因功能的重要手段。我们试图建立一种快速、高效的大肠杆菌基因敲除方法。【方法】利用大肠杆菌(Escherichia coli)BW25113单基因缺失体Keio文库,将经典的Red同源重组技术与P1噬菌体转导技术相结合,对E.coli MG1655脂肪酸代谢基因进行快速敲除。【结果】获得了大肠杆菌β-氧化途径的缺失菌株△fadD、△fadE和△fadD-△fadE;脂肪酸合成途径缺失菌株△fabH、△fabF和△fabH-△fabF。敲除fadD和fadE对生长情况没有影响;敲除fabH后,生长速度明显减慢;敲除fabF对生长几乎没有影响。FadD、FadE及双敲缺失体的脂肪酸含量18.2 mg/L、20.0mg/L和19.2 mg/L,略高于野生型17.5 mg/L;FabH、FabF及双敲缺失体的含量分别为12.6 mg/L、15.2 mg/L和11.2 mg/L,明显低于野生型。【结论】在单基因突变体文库基础上,利用P1噬菌体转导、Red同源重组和抗性基因消除进行基因敲除,简化了构建大肠杆菌单基因和多重突变体的方法。     

Actinoplanes hulinensis sp. nov., a novel actinomycete isolated from soybean root (Glycine max (L.) Merr)

A novel actinomycete, designated strain NEAU-M9^T, was isolated from soybean root (Glycine max (L.) Merr) and characterized using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain NEAU-M9^T belonged to the genus Actinoplanes, being most closely related to Actinoplanes campanulatus DSM 43148^T (98.85 %), Actinoplanes capillaceus DSM 44859^T (98.70 %), Actinoplanes lobatus DSM 43150^T (98.30 %), Actinoplanes auranticolor DSM 43031^T (98.23 %) and Actinoplanes sichuanensis 03-723^T (98.06 %); similarity to other type strains of the genus Actinoplanes ranged from 95.87 to 97.56 %. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that the isolate formed a distinct phyletic line with A. campanulatus DSM 43148^T and A. capillaceus DSM 44859^T. This branching pattern was also supported by the tree constructed with the maximum-likelihood method. However, the low level of DNA–DNA relatedness allowed the isolate to be differentiated from the above-mentioned two Actinoplanes species. Moreover, strain NEAU-M9^T could also be distinguished from the most closely related species by morphological, physiological and characteristics. Therefore, it is proposed that strain NEAU-M9^T represents a novel Actinoplanes species, Actinoplanes hulinensis sp. nov. The type strain of Actinoplanes hulinensis is NEAU-M9^T (= CGMCC 4.7036^T = DSM 45728^T).     

Molecular Analysis of a Deletion Hotspot in the NRXN1 Region Reveals the Involvement of Short Inverted Repeats in Deletion CNVs

NRXN1 microdeletions occur at a relatively high frequency and confer increased risk for neurodevelopmental and neurobehavioral abnormalities. The mechanism that makes NRXN1 a deletion hotspot is unknown. Here, we identified deletions of the NRXN1 region in affected cohorts, confirming a strong association with the autism spectrum and other neurodevelopmental disorders. Interestingly, deletions in both affected and control individuals were clustered in the 5′ portion of NRXN1 and its immediate upstream region. To explore the mechanism of deletion, we mapped and analyzed the breakpoints of 32 deletions. At the deletion breakpoints, frequent microhomology (68.8%, 2–19 bp) suggested predominant mechanisms of DNA replication error and/or microhomology-mediated end-joining. Long terminal repeat (LTR) elements, unique non-B-DNA structures, and MEME-defined sequence motifs were significantly enriched, but Alu and LINE sequences were not. Importantly, small-size inverted repeats (minus self chains, minus sequence motifs, and partial complementary sequences) were significantly overrepresented in the vicinity of NRXN1 region deletion breakpoints, suggesting that, although they are not interrupted by the deletion process, such inverted repeats can predispose a region to genomic instability by mediating single-strand DNA looping via the annealing of partially reverse complementary strands and the promoting of DNA replication fork stalling and DNA replication error. Our observations highlight the potential importance of inverted repeats of variable sizes in generating a rearrangement hotspot in which individual breakpoints are not recurrent. Mechanisms that involve short inverted repeats in initiating deletion may also apply to other deletion hotspots in the human genome.     

Effects of disperser abundance, seed type, and interspecific seed availability on dispersal distance

Seed dispersal distance is influenced by a variety of seed properties and functional responses of dispersers. However, to our knowledge, how and why seed dispersal distances are determined remains poorly understood. In the present study, seeds of sympatric tree species, Pinus koraiensis, Corylus mandshurica, Corylus heterophylla, and Quercus mongolica were released to investigate the effects of rodent abundance, seed type, and seed availability on seed dispersal. Our results showed that seeds of P. koraiensis were dispersed further than those of C. heterophylla and C. mandshurica regardless of the ambient rodent and seed abundances, reflecting a consistent effect of seed type on seed dispersal distances. Seed dispersal distance was greatly facilitated by lower per-capita seed abundance (the ratio of seeds to rodents); however, seed caching and cache survival were benefited from higher per-capita seed abundance. Although seed dispersal and seed caching of a particular tree species can be enhanced by its own seed availability, no consistent influence was detected at interspecific levels, reflecting different interspecific effects of seed availability on seed dispersal of sympatric seed species. Our results provide evidences that the effect of seed availability on seed dispersal should be evaluated in terms of per-capita seed abundance and interspecific effects, rather than the independent influence of seed or disperser abundances.     

Potato purple top phytoplasma‐induced disruption of gibberellin homeostasis in tomato plants

Phytoplasmas are phloem‐inhabiting, cell wall‐less bacteria that cause numerous plant diseases worldwide. Plants infected by phytoplasmas often exhibit various symptoms indicative of hormonal imbalance. In this study, we investigated the effects of potato purple top (PPT) phytoplasma infection on gibberellin homeostasis in tomato plants. We found that PPT phytoplasma infection caused a significant reduction in endogenous levels of gibberellic acid (GA₃). The decrease in GA₃ content in diseased plants was correlated with down regulation of genes responsible for biosynthesis of bioactive GAs ( GA20ox1 and GA3ox1) and genes involved in formation of GA precursors [geranyl diphosphate synthase (GPS) and copalyldiphosphate synthase (CPS)]. Exogenous application of GA₃ at 200 µmol L^?1 was able to restore the GA content in infected plants to levels comparable to those in healthy controls, and to attenuate the characteristic ‘big bud’ symptoms induced by the phytoplasma. The interesting observation that PPT phytoplasma‐infected plants had prolonged low expression of key GA biosynthesis genes GA20ox1 and GA3ox1 under GA deficiency conditions led us to hypothesise that there was a diminished sensitivity of the GA metabolism feedback regulation, especially GA biosynthesis negative feedback regulation, in those affected plants, and such diminished sensitization in early stages of infection may represent a central element of the phytoplasma‐induced disruption of GA homeostasis and pathogenesis.     

Diversity of prokaryotes associated with soils around coal-fire gas vents in MaNasi county of Xinjiang, China

Bacterial and archaeal diversity in surface soils of three coal-fire vents was investigated by T-RFLP analysis and clone libraries of 16S rRNA genes. Soil analysis showed that underground coal fires significantly influenced soil pH, moisture and NO₃ ^? content but had little effect on other elements, organic matter and available nutrients. Hierarchical cluster analysis showed that bacterial community patterns in the soils were very similar, but abundance varied with geographic distance. A clone library from one soil showed that the bacterial community was mainly composed of Firmicutes, Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, and unidentified groups. Of these, Firmicutes was the most abundant, accounting for 71.4 % of the clones, and was mainly represented by the genera Bacillus and Paenibacillus. Archaeal phylotypes were closely related to uncultivated species of the phyla Crenarchaeota (97.9 % of clones) and Thaumarchaeota (2.1 %). About 28 % of archaeal phylotypes were associated with ammonia oxidization, especially phylotypes that were highly related to a novel, ammonia-oxidizing isolate from the phylum Thaumarchaeota. These results suggested that microbial communities in the soils were diverse and might contain a large number of novel cultivable species with the potential to assimilate materials by heterotrophic metabolism at high temperature.     

Protection against cardiac injury by small Ca2 +-sensitive K+ channels identified in guinea pig cardiac inner mitochondrial membrane

We tested if small conductance, Ca^2 +‐sensitive K⁺ channels (SK_Ca) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O₂‐derived free radicals are required to initiate protection via SK_Ca channels, and, importantly, if SK_Ca channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O₂^?), and m[Ca^2 +] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK_Ca and IK_Ca channel opener DCEBIO (DCEB) was given for 10 min and ended 20 min before IR. Either TBAP, a dismutator of O₂^?, NS8593, an antagonist of SK_Ca isoforms, or other K_Ca and K_ATP channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O₂^? and m[Ca^2 +], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK_Ca channels to mitochondria and IMM was evidenced by a) identification of purified mSK_Ca protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca^2 +]‐dependence of mSK_Ca channels in planar lipid bilayers, and d) matrix K⁺ influx induced by DCEB and blocked by SK_Ca antagonist UCL1684. This study shows that 1) SK_Ca channels are located and functional in IMM, 2) mSK_Ca channel opening by DCEB leads to protection that is O₂^? dependent, and 3) protection by DCEB is evident beginning during ischemia.     

Bone morphogenetic protein-7 (BMP-7) mediates ischemic preconditioning-induced ischemic tolerance via attenuating apoptosis in rat brain

A mild cerebral ischemic insult, also known as ischemic preconditioning (IPC), confers transient tolerance to a subsequent ischemic challenge in the brain. This study was conducted to investigate whether bone morphogenetic protein-7 (BMP-7) is involved in neuroprotection elicited by IPC in a rat model of ischemia. Ischemic tolerance was induced in rats by IPC (15 min middle cerebral artery occlusion, MCAO) at 48 h before lethal ischemia (2 h MCAO). The present data showed that IPC increased BMP-7 mRNA and protein expression after 24 h reperfusion following ischemia in the brain. In rats of ischemia, IPC-induced reduction of cerebral infarct volume and improvement of neuronal morphology were attenuated when BMP-7 was inhibited either by antagonist noggin or short interfering RNA (siRNA) pre-treatment. Besides, cerebral IPC-induced up-regulation of B-cell lymphoma 2 (Bcl-2) and down-regulation of cleaved caspase-3 at 24 h after ischemia/reperfusion (I/R) injury were reversed via inhibition of BMP-7. These findings indicate that BMP-7 mediates IPC-induced tolerance to cerebral I/R, probably through inhibition of apoptosis.     

Combined small RNA and degradome sequencing reveals microRNA regulation during immature maize embryo dedifferentiation

Genetic transformation of maize is highly dependent on the development of embryonic calli from the dedifferentiated immature embryo. To better understand the regulatory mechanism of immature embryo dedifferentiation, we generated four small RNA and degradome libraries from samples representing the major stages of dedifferentiation. More than 186 million raw reads of small RNA and degradome sequence data were generated. We detected 102 known miRNAs belonging to 23 miRNA families. In total, we identified 51, 70 and 63 differentially expressed miRNAs (DEMs) in the stage I, II, III samples, respectively, compared to the control. However, only 6 miRNAs were continually up-regulated by more than fivefold throughout the process of dedifferentiation. A total of 87 genes were identified as the targets of 21 DEM families. This group of targets was enriched in members of four significant pathways including plant hormone signal transduction, antigen processing and presentation, ECM-receptor interaction, and alpha-linolenic acid metabolism. The hormone signal transduction pathway appeared to be particularly significant, involving 21 of the targets. While the targets of the most significant DEMs have been proved to play essential roles in cell dedifferentiation. Our results provide important information regarding the regulatory networks that control immature embryo dedifferentiation in maize.