Cotton (Gossypium hirsutum) 14‐3‐3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling

Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14‐3‐3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14‐3‐3L, Gh14‐3‐3e and Gh14‐3‐3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14‐3‐3 RNAi transgenic plants were significantly shorter than those of wild type. This ‘short fibre’ phenotype of the 14‐3‐3 RNAi cotton could be partially rescued by application of 2,4‐epibrassinolide (BL). Expression levels of the BR‐related and fibre‐related genes were altered in the Gh14‐3‐3 transgenic fibres. Furthermore, we identified Gh14‐3‐3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14‐3‐3L/e/h were required for Gh14‐3‐3‐GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14‐3‐3 proteins. Additionally, 14‐3‐3‐regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14‐3‐3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Multi-centre testing and validation of current protocols for the identification of Gyrodactylus salaris (Monogenea)

Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss’ Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a “G. salaris-like” (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select “G. salaris-like” specimens, which are subsequently identified by molecular-based techniques.     

Identification of a fourth subunit of mammalian DNA polymerase delta

A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase delta by conventional chromatography. A 16-mer peptide sequence was obtained from the 12-kDa polypeptide which matched a new open reading frame from a human EST () encoding a hypothetical protein of unknown function. The protein was designated as p12. Human EST was identified as the putative human homologue of Schizosaccharomyces pombe Cdm1 by a tBlastn search of the EST data base using S. pombe Cdm1. The open reading frame of human EST encoded a polypeptide of 107 amino acids with a predicted molecular mass of 12.4 kDa, consistent with the experimental findings. p12 is 25% identical to S pombe Cdm1. Both of the 25-kDa polypeptide sequences matched the hypothetical KIAA0039 protein sequence, recently identified as the third subunit of pol delta. Western blotting of immunoaffinity purified calf thymus pol delta revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12. With the identification of p12 mammalian pol delta can now be shown to consist of four subunits. These studies pave the way for more detailed analysis of the possible functions of the mammalian subunits of pol delta.     

Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production

Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the bla_CMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare bla_CMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying bla_CMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying bla_CMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of bla_CMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing bla_CMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.     

植物泛素/26S蛋白酶体途径的研究进展

泛素/26S蛋白酶体途径（ubiquitin/26S proteasome pathway,UPP）是目前已知最有效的、最具特异性的蛋白质降解途径。该途径介导了真核生物80%-85%的蛋白质降解,参与了细胞多项生命活动过程,对于维持细胞正常生理功能具有重要意义。研究结果表明,植物生长发育的诸多方面以及干旱胁迫响应等过程都受到该途径的调控。概述了泛素/26S蛋白酶体途径及其在植物生长发育过程中的作用,并着重阐述了由泛素-蛋白连接酶E3介导的植物干旱胁迫响应及其作用机制的研究进展。     

Biology, life table and predation of Feltiella acarisuga (Diptera: Cecidomyiidae) feeding on Tetranychus cinnabarinus eggs (Acari: Tetranychidae)

Feltiella acarisuga (Vallot) is a common predatory gall midge, which feeds on many species of spider mites. All major life history and life table parameters of F. acarisuga were determined using the carmine spider mite, Tetranychus cinnabarinus (Boisduval) eggs as prey under laboratory conditions [26.7 ± 2 °C, 75 ± 5% RH, and a photoperiod of l4:10 (L:D) h]. Developmental times of F. acarisuga were 2.6, 7.1, and 6.7 d for eggs, larvae, and pupae, respectively, with an average of 16.4 d from egg to adult emergence. Female immatures development took ≈1 d longer than male immatures did. Adult F. acarisuga lived an average of 12.8 d, and the female adults (13.3 d) lived significantly longer than male adults did (11.9 d). After an average of 1.5 d preoviposition period, each female laid an average of 27.3 eggs in its life span with an average of 2.1 eggs per day and an average of 2.8 eggs on each of its oviposition day. The raw data were analyzed using an age-stage, two-sex life table method that takes into consideration of the variable developmental rates among individuals and between sexes. The intrinsic rate of natural population increase (r), net reproductive rates (R_o), gross reproductive rate (∑m_x), generation time (T), and doubling time (DT), and the finite rate of increase (λ) of F. acarisuga were estimated using the age-stage, two-sex (male and female) life table analysis as 0.122 d⁻¹, 16.19 eggs per female, 20.81 eggs per female, 22.81 d, 1.1298 d, and 5.7 d⁻¹, respectively. Each of the first, second, and third instar larvae of F. acarisuga consumed an average of 35.5, 54.0 and 86.9 T. cinnabarinus eggs per day, respectively. Larvae of F. acarisuga could consume an average of 175.4, T. cinnabarinus eggs, and female larvae consumed 14% more spider mite eggs (187.6 eggs) than male larvae (165.1 eggs). The significance of other life table parameters related to the population and the potential of using F. acarisuga as a biological control agent are discussed.