采前壳寡糖处理对杏果实黑斑病的抗性诱导

以新疆赛买提杏为试验材料,分别在果实坐果期、膨大期、转色期及采收前48h,采用分子量为5 000、浓度为0.05%的壳寡糖(GOS)溶液对杏果实进行喷施处理,以喷施清水为对照(CK);采收后的杏果实在机械损伤接种链格孢菌后置于4℃、相对湿度90%~95%的条件下贮藏,定期统计接种链格孢菌杏果实的病斑直径和发病率,测定抗病相关酶苯丙氨酸解氨酶(PAL)、β-1,3-葡聚糖酶(GLU)和几丁质酶(CHT)的活性及木质素、富含羟脯氨酸糖蛋白(HRGP)的含量,探讨采前壳寡糖处理对杏果实黑斑病的抗性诱导及其生理机制。结果显示,贮藏结束时,采前壳寡糖处理的果实发病率与病斑直径分别比对照显著降低了16.37%和17.57%。随着贮藏期间的延长,壳寡糖处理杏果实PAL、GLU、CHT的活性和木质素、HRGP的含量均表现出先上升后下降的变化趋势,且始终显著高于同期对照,并分别在接种后第21、28、21、28和14天达到峰值,峰值比同期对照分别显著提高12.17%、78.22%、31.41%、34.81%和77.44%。研究表明,采前壳寡糖处理能通过诱导提高杏果实病程相关蛋白及细胞壁HRGP和木质素的含量来增强杏果实对黑斑病的抗性。     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.     

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT₅₀ of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60–75 g/kg rice), faster sedimentation time (ST₅₀) (2.3–2.65 h in 0.1% (w/v) Tween 80) and lower LC₅₀ (1.4–5.7×10³ conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).     

发酵透明质酸寡糖兽疫链球菌工程菌株的构建

透明质酸(HA)广泛应用于医学、化妆品、食品等领域。HA的生物活性取决于其分子量(M_w)。透明质酸寡糖由于具有重要的生理活性与特殊生理功能,在医药领域具有重要的应用前景。兽疫链球菌因其发酵周期短、生产强度较强的特点,在商业生产HA上具有广泛的应用。为了高效发酵合成透明质酸寡糖和解决发酵过程的溶氧问题,文中通过在兽疫链球菌WSH-24中过表达透明质酸合酶HasA以及优化表达水蛭来源的透明质酸酶LHAase。重组菌株摇瓶发酵24h,透明质酸寡糖积累至0.97g/L,比野生菌提高了182.0%。在3L发酵罐中发酵24 h,透明质酸寡糖生产强度为294.2 mg/(L·h),HA积累至7.06 g/L,比野生菌的罐上水平提高了112.4%。文中所构建的发酵合成透明质酸寡糖的兽疫链球菌重组菌株具有重要的应用前景。     

Production of chitinolytic enzymes from a novel species ofAeromonas

A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source. The isolated bacterium, designated asAeromonas sp No. 16 exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml^–1. A slightly higher enzymatic activity of 1.5 U ml^–1 was obtained when cultivation was carried out in a 5-liter jar fermentor using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of -N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.     

Structural Analysis of N-Glycans of Storage Glycoproteins in Soybean (Glycine max. L) Seed

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz ¹H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man₃Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 3.4%).     

Large-scale production of GDP-fucose and Lewis X by bacterial coupling

A large-scale production system of GDP-fucose (GDP-Fuc) and fucosylated oligosaccharides was established by the combination of recombinant Escherichia coli cells overexpressing GDP-Fuc biosynthetic genes and Corynebacterium ammoniagenes cells. E. coli cells overexpressed the genes for glucokinase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose (GDP-Man) dehydratase, and GDP-4-keto-6-deoxy-mannose (GKDM) epimerase/reductase as well as phosphoglucomutase and phosphofructokinase. C. ammoniagenes contributed to the formation of GTP from GMP. GDP-Fuc accumulated to 29 mM (18.4 g l⁻¹) after a 22-h reaction starting with GMP and mannose through introducing the two-step reaction to overcome the inhibition of GDP-Fuc on GDP-Man dehydratase activity. When E. coli cells overexpressing the α1,3-fucosyltransferase gene of Helicobacter pylori were put into the GDP-Fuc production system, Lewis X [Galβ1–4(Fucα1–3)GlcNAc] was produced at an amount of 40 mM (21 g l⁻¹) for 30 h from GMP, mannose, and N-acetyl lactosamine. The production system through bacterial coupling can be applied to the industrial manufacture of fucosylated oligosaccharides. Journal of Industrial Microbiology & Biotechnology (2000) 25, 213–217. Received 01 May 2000/ Accepted in revised form 20 July 2000     

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.