Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare

The virulence of eight Flavobacterium columnare strains was studied to find correlations between several virulence-related factors and virulence. Virulence was tested in vivo using rainbow trout, Oncorhynchus mykiss (Walbaum). Suggested virulence-related factors such as production of the degradative enzyme chondroitin lyase, plasmid occurrence and adhesion capability were studied in vitro. Infection with the four most virulent strains resulted in 95-100% mortality within 114 h. Chondroitin lyase activity was found to be significantly related to the virulence of the strains at 25 degrees C and it was also shown to be temperature-dependent, being higher at 25 degrees C than at 20 degrees C. Virulence was not plasmid associated. The adhesion capability of the strains in vitro varied substantially when tested on crude mucus-coated slides and no statistical relationship between adhesion and virulence was found using this method.     

茄科雷尔氏菌IMSA-LAMP检测方法的建立

【目的】桑树青枯病是由茄科雷尔氏菌Ralstonia solanacearum引起的一种危害严重的细菌性病害,建立一种快速、灵敏的茄科雷尔氏菌检测方法,对桑树青枯病的有效控制有重要意义。【方法】本研究以茄科雷尔氏菌果胶裂解酶基因（Pectate lyase gene）为靶标,基于等温多自配引发扩增技术（Isothermal multiple self-matching-initiated amplification,IMSA）的引物设计原理,结合环介导等温扩增技术（Loop-mediated isothermal amplification,LAMP）的反应体系,建立一种快速有效检测茄科雷尔氏菌的IMSA-LAMP法,并对该方法的最佳反应参数进行了筛选。【结果】基于果胶裂解酶基因建立的IMSA-LAMP检测方法在64.5℃条件下,45 min内可完成对阳性样品的特异检测,对茄科雷尔氏菌的模板DNA检测灵敏度达200 fg/μL （对应菌为1×10²CFU/mL）;对生产上收集的疑似桑树青枯病病样的检出率为87.5%。【结论】IMSA-LAMP检测方法具有良好的实用...     

Potassium phosphate induces tolerance against the Russian wheat aphid (Diuraphis noxia,Homoptera: Aphididae) in wheat

Diuraphis noxia Kurdjumov is an aphid pest that hampers the global production of wheat (Triticum aestivum L.). Infestation of wheat by D. noxia reduces crop yield and sometimes leads to the death of susceptible cultivars. To date resistant cultivars have been used to control infestations. However, the development of resistance-breaking aphid biotypes is complicating this management strategy. This study aimed to ascertain the effect that 25 mM potassium phosphate (K₃PO₄) treatment has on inducing resistance in wheat against D. noxia. The effects of potassium phosphate on induced resistance against D. noxia were determined using gene regulation and enzymatic levels of lipoxygenase (EC 1.13.11) and phenylalanine ammonia lyase (EC 4.3.1.24). The results indicated that the treatment of both susceptible (Scheepers) and resistant (Tugela DN) wheat cultivars with potassium phosphate induces genes differentially. A phenotypic study showed delayed development and less severity of symptoms with decreased numbers of aphids feeding on plants treated with potassium phosphate in comparison with untreated plants for both resistant and susceptible cultivars. The results from the gene expression and enzyme activity studies indicated that both of the induced systemic resistance-linked genes were similarly regulated after potassium phosphate treatment. The data strongly support treatment of wheat with potassium phosphate to induce tolerance against D. noxia.     

海洋细菌来源低温褐藻胶裂解酶的分泌表达和酶学性质研究

为了筛选稳定性较好的低温褐藻胶裂解酶,本研究进行了海洋细菌分离鉴定、酶的编码基因克隆与分析、酶分泌表达与纯化、不同因素对酶活力和稳定性的影响以及酶解产物分析实验。结果显示,用以褐藻胶为唯一碳源的平板,筛选出一株能分泌低温褐藻胶裂解酶的海洋细菌SJ-H-12,基于16S rDNA序列构建进化树,该菌株鉴定为Yangia sp. SJ-H-12。进而克隆酶的编码基因Alyya,ALYYA属于PL5家族褐藻胶裂解酶。将基因在食品级宿主解脂耶氏酵母(Yarrowia lipolytica)中进行分泌表达,重组ALYYA的活力达到34.2 U/mL,分子量约为39.0 kDa,具有较强的PolyM偏好性。ALYYA在25℃~35℃时表现出80%以上的活力,且在30℃时表现出最高活力;在pH为5.0~10.0的范围内稳定性较好,孵育后剩余超过60%的活力;0~2.0 mol/L的NaCl能明显激活ALYYA的活力。ALYYA降解褐藻胶的产物主要是二糖,另有少部分单糖和三糖,该酶是一种内切褐藻胶裂解酶。综上所述,本研究筛选到一株产低温褐藻胶裂解酶细菌,所产ALYYA是典型的低温褐藻胶裂解酶,具有优良的酶活力和稳定性。本研究为低温褐藻胶裂解酶的筛选和开发利用提供了参考数据。     

Calcium-sensitive extracellular poly(α-L-guluronate)lyase from a marine bacterium Pseudomonas sp. strain F6: Purification and some properties

ABSTRACT: Poly(α- L -guluronate)lyase, as one of alginate lyases, was purified from the culture medium of a marine bacterium, Pseudomonas sp. strain F6, to an electrophoretically homogeneous state. The enzyme was shown to have a molecular mass of 36 kDa by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and was most active at around pH 7.5 and was stable between pH 6.5 and pH 8.5. In the thermal stability experiments, the enzyme's activity diminished through an intermediate state with increasing incubation temperatures and was finally lost when heated at 100°C for 15 min. The addition of hen egg-white lysozyme to the enzyme decreased thermal stability dramatically. The apparent retention of enzyme activity (approximately 50%) was observed after the addition of 6 M guanidine hydrochloride and 8 M urea. Enzyme activity was lost completely with 10 mMSDS, while the ordered structure, which is considered likely to be β-structure, was markedly created. The similar conformational feature has also been created in marine bacterial and mollusc enzymes and the β-structure is commonly observed in polyuronate lyases. The divalent cation (Ca²⁺) promoted the activity of the calcium chelator-treated enzyme significantly, suggesting that Ca²⁺ is involved in the formation of the active intermediate between the acidic uronate(s) and amino acid side-chain(s) of the enzyme.     

The effect of different nitrogen sources on the symbiotic interaction between Sorghum bicolor and Glomus intraradices: Expression of plant and fungal genes involved in nitrogen assimilation

In the arbuscular mycorrhizal (AM) symbiosis, plants take up part of the nitrogen (N) through a mycorrhizal pathway. In this study, we assessed the effect of different N sources on the expression of genes coding for enzymes and transporters of the mycorrhizal N uptake pathway, using Sorghum bicolor and Glomus intraradices as a model. Some of the genes investigated were differentially regulated in the intraradical and in the extraradical mycelium depending on the N source. In AM roots, some fungal and plant genes were co-regulated, suggesting an interdependence of both partners in the mycorrhizal N uptake pathway. Mycorrhizal N transfer may have a preference for glycine (plant growth and N uptake stimulation).     

苜蓿斑蚜为害对5种苜蓿品种(系)PAL、POD、PPO酶活性的影响

在室内将苜蓿斑蚜人工接种在不同抗性级别的苜蓿品种（系）幼苗上,苜蓿斑蚜为害后的3、7、10 d分别测定了5个品种（系）叶片中苯丙氨酸解氨酶（PAL）、多酚氧化酶(PPO)、过氧化物酶（POD）3种酶活性。结果表明,在试验期内,所有品种（系）叶片中的PAL活性在接虫后随着时间的增加而升高; PPO活性随天数增加呈下降趋势; POD与PAL酶的情况相同,随着为害时间的延长不断升高。HA 3感蚜后3种酶活性明显高于其他品种。     

香蕉枯萎病菌2个生理小种PL基因真核表达载体的构建与表达

【目的】构建香蕉枯萎病菌1号小种（FOC1）和4号小种（FOC4）果胶裂解酶（Pectate lyases, PL）基因的真核表达载体,并进行诱导表达,为进一步研究PL在病原菌致病过程中的作用奠定基础。【方法】将质粒pMD18-pl-foc1、pMD18-pl-foc4和真核表达穿梭载体pPICZαA进行EcoRⅠ和XbaⅠ双酶切反应,回收目的片段连接,转化至DH5α进行筛选扩繁,对菌落进行PCR鉴定后抽提质粒进行PCR和EcoRⅠ、XbaⅠ双酶切鉴定。将阳性载体线性化后电击转化毕赤酵母 SMD1168感受态细胞,用甲醇进行诱导表达,对表达产物进行SDS-PAGE分析。【结果】成功构建了FOC1和FOC4 PL基因的真核表达载体pPICZαA-pl-foc1和pPICZαA-pl-foc4,经甲醇诱导表达后分别获得了重组的PL蛋白,其分子质量约为23.4 ku。【结论】FOC1和FOC4 2个香蕉枯萎病菌小种的PL基因在酵母中得到了成功表达。     

Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524

An alginate lyase-producing bacterial strain, Pseudoalteromonas sp. SM0524, was screened from marine rotten kelp. In an optimized condition, the production of alginate lyase from Pseudoalteromonas sp. SM0524 reached 62.6 U/mL, suggesting that strain SM0524 is a good producer of alginate lyases. The bifunctional alginate lyase aly-SJ02 secreted by strain SM0524 was purified. Aly-SJ02 had an apparent molecular mass of 32 kDa. The optimal temperature and pH of aly-SJ02 toward sodium alginate was 50 °C and 8.5, respectively. The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C. Aly-SJ02 was most stable at pH 8.0. N-terminal sequence analysis suggested that aly-SJ02 may be an alginate lyase of polysaccharide lyase family 18. Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-D-mannuronic acid), indicating that it is a bifunctional alginate lyase. Aly-SJ02 had lower K(m) toward polyG than toward polyM and sodium alginate. Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.     

弱毒菌株尤Ⅱ对小麦条锈病的诱导抗性 Ⅱ.诱导抗性的病生理变化观察

弱小种尤诱导接种后,PAL活性始终大于同期其他处理;先诱导接种尤再挑战接种条中29号处理的POD活性于72h和120h时产生两个高峰;用条中29号小种挑战接种后PPO活性的变化趋势与单独接种尤的处理相同,均高于单独接种条中29号小种和对照处理;挑战接种条中29处理的SOD活性远比同期其他处理的低。