凝结芽孢杆菌磷酸盐响应启动子的研究

耐热凝结芽孢杆菌因其对营养要求简单、发酵产物浓度高以及耐高温等特点,已成为乳酸发酵的主要菌种。在前期的研究中,我们发现磷酸盐可以激活凝结芽孢杆菌l-乳酸脱氢酶基因的转录,从而提高乳酸产量。然而,磷酸盐如何激活乳酸脱氢酶的基因表达,目前还不清楚,也未有类似的研究报道。【目的】对凝结芽孢杆菌响应磷酸盐的调控机制进行研究。【方法】通过RT-PCR分析磷酸盐添加时凝结芽孢杆菌乳酸脱氢酶转录水平变化,确定响应磷酸盐的关键元件区域,进一步通过分子生物学手段,分析凝结芽孢杆菌响应磷酸盐的关键基因片段。【结果】确定了响应磷酸盐的关键元件位于乳酸脱氢酶基因上游启动子区,解析了响应磷酸盐的l-乳酸脱氢酶启动子核心区,利用该启动子及核心区能够有效驱动外源d-乳酸脱氢酶基因的表达,实现在凝结芽孢杆菌中d-乳酸的合成。【结论】本研究有望获得一种新的响应磷酸盐的调控元件,为提高其他生物化学品的合成效率改造提供参考。     

响应面模型分析高温蛋白酶菌株增殖和产酶关系

【目的】【方法】采用Box-Behnken法设计三因素三水平响应面实验,对比分析了高温蛋白酶高产菌株枯草芽孢杆菌BY25发酵过程中细菌生长与产酶之间的关系。【结果】实验表明,细菌生长与产酶的关系在各因素交互影响下变化显著。在中低水平的有机氮源添加条件下,细菌的大量繁殖显著抑制了蛋白酶产量,而在高水平的有机氮源添加条件下,细菌量的增长对产酶的作用由抑制转为促进。【结论】该结果表明,产酶诱导物有机氮源豆制品废渣的添加很可能显著提高了枯草芽孢杆菌群体中产酶菌的比率,而在没有合适诱导物的情况下通过增加细菌量并不能有效提高蛋白酶产量。     

基于盲肠菌群和回肠粘膜基因表达探讨解淀粉芽孢杆菌提高蛋鸡生产性能的潜在机制

【背景】我们的前期工作表明,在蛋鸡饲料中添加解淀粉芽孢杆菌BLCC1-0238可显著提高产蛋性能和蛋品质,但其益生机制还不清楚。【目的】从基因组学和转录组学的视角去揭示益生菌提高蛋鸡生产性能的潜在机制。【方法】利用高通量测序技术比较分析基础日粮组(C组)和解淀粉芽孢杆菌BLCC1-0238添加组(0.06%,2.0×1010 CFU/g,T组)盲肠微生物菌群和回肠粘膜基因表达的差异。【结果】基因组学分析发现,与对照组相比,益生菌添加组的盲肠菌群Chao1和Shannon指数升高;在门水平上,厚壁菌门/拟杆菌门(Firmicutes/Bacteroidetes)的比例显著增加,梭杆菌门(Fusobacteria)和变形菌门(Proteobacteria)的相对丰度降低;在属水平上,厚壁菌门中的考拉杆菌属(Phascolarctobacterium)、乳酸杆菌属(Lactobacillus)、瘤胃球菌科UCG-014属(Ruminococcaceae UCG-014)、厌氧棍状菌属(Anaerotruncus)、瘤胃梭菌属(Ruminiclostridium9)、克里斯滕森氏菌(Christensenellaceae_R-7_group)相对丰度明显增加。转录组学结果表明,回肠粘膜组织中共有356个差异表达基因,其中,上调基因199个,下调基因157个。生物信息学分析提示,上调基因参与了淀粉和蔗糖代谢、半乳糖代谢以及甘氨酸、丝氨酸和苏氨酸代谢等促进物质代谢和营养吸收的过程。【结论】日粮中添加解淀粉芽孢杆菌BLCC1-0238,可通过改善盲肠菌群多样性和肠道营养吸收能力提高蛋鸡的饲料利用率和产蛋性能,具有潜在的实用价值。     

酱醪细菌菌株的分离及功能分析

【目的】分离获得来源于酱醪的细菌,考察菌株与酱油品质相关的特性,初步评价其应用于酱油发酵的潜力。【方法】从日式酱油发酵的酱醪体系中分离和筛选优势或特征细菌菌株,比较它们的耐盐性及其在高盐条件下产蛋白酶、有机酸、挥发性物质和氨基酸等的能力。【结果】从日式酱油酱醪中共分离得到9株细菌,分别属于魏斯氏菌(Weissella)、乳酸足球菌(Pediococcus)、乳酸杆菌(Lactobacillus)、芽孢杆菌(Bacillus)、四联球菌(Tetragenococcus)和葡萄球菌(Staphylococcus)属。其中耐盐的细菌有类肠膜魏斯氏菌(Weissella paramesenteroides)CQ03、嗜酸乳酸足球菌(Pediococcus acidilactici)JY07、戊糖乳酸足球菌(Pediococcus pentosaceus)JY08、葡萄球菌(Staphylococcus sp.)JY09和嗜盐四联球菌(Tetragenococcus halophilus)MRS1。在高盐条件下,对它们的特性分析表明:解淀粉芽孢杆菌(Bacillus amyloliquefaciens)B2产蛋白酶和糖化酶的能力较强,W.paramesenteroides CQ03可水解原料产生较多鲜味氨基酸,T.halophilus MRS1产有机酸能力较强,它和S.sp.JY09代谢产生的挥发性物质较多。【结论】筛选得到9株在促进原料水解和提高风味物质合成方面有潜力的菌株,如果应用到酱油工业生产中,将有利于缩短发酵周期,提高酱油品质。     

发酵乳杆菌CSC-19胞外粗多糖提取物抑制单核细胞增生李斯特菌生物被膜形成能力的研究

【目的】筛选能有效抑制单核细胞增生李斯特菌(Listeria monocytogenes,LM)形成生物被膜的乳酸菌,分析其活性成分并进行功能表征。【方法】采用结晶紫染色法筛选抑制LM形成生物被膜的不同乳酸菌提取物;通过酸中和、蛋白酶处理及热处理,推测抑制生物被膜活性物质以胞外多糖(extracellular crude polysaccharide,ECP)为主;乙醇沉淀法提取目标乳酸菌分离株胞外粗多糖,分析其抑制生物被膜形成活性和对LM生长的影响;运用激光共聚焦扫描显微镜(laser confocal scanning microscopy,LCSM)和扫描电子显微镜(scanning electron microscopy,SEM)观察胞外粗多糖对生物被膜细胞形态和结构的影响。【结果】发酵乳杆菌CSC-19发酵上清液对1516-2LM生物被膜的抑制率为81.7%;经热和蛋白酶处理后,发酵上清抑制生物被膜形成的活性未发生显著变化(P>0.05),表明发酵上清液中抑制生物被膜形成的物质可能为胞外多糖;在不抑制LM生长的条件下所提取的胞外粗多糖抑制生物被膜形成能力具有浓度依赖性。激光共聚焦扫描显微镜和扫描电子显微镜结果显示,胞外粗多糖显著抑制了生物被膜的形成能力,生物被膜三维、有组织的蜂窝状结构被破坏,仅有少量的粘附细胞分散于细胞爬片表面。【结论】发酵乳杆菌CSC-19胞外粗多糖能有效抑制LM生物被膜的形成,有望应用于高效防控该菌污染食品。     

芝麻香型白酒微生物多样性及其与胁迫因子相关性

【背景】近年来芝麻香型白酒的生产工艺日臻成熟,然而相应的科学研究却没有同步发展起来。高通量测序技术越来越多地应用于物种多样性的研究,但偏重于研究物种的相对丰度,没有关注物种的生物数量。【目的】深度解析芝麻香型白酒发酵过程微生物群落结构变化及其与胁迫因子相关性,并研究主要酵母菌与细菌的相关性,为揭示芝麻香型白酒发酵机理和控制发酵质量提供理论支撑。【方法】使用Thermofisher的Ion S5~(TM)XL测序平台进行16S rDNA和ITS rDNA扩增子高通量测序,结合微生物传统的定量方法,测定芝麻香型白酒发酵过程微生物群落结构的变化,同时监测发酵过程乳酸、乙酸、乙醇的含量变化,通过样品复杂度分析、多样品比较分析、环境因子关联性分析探究发酵过程微生物群落及其与胁迫因子的关系。通过Pearson相关性分析酵母菌与细菌的相关性。【结果】发酵前期纤维素菌、魏斯氏菌和芽孢杆菌占主要优势,发酵中后期乳杆菌占绝对优势,其次是纤维素菌、魏斯氏菌和芽孢杆菌。整个发酵过程伊萨酵母占绝对优势,其次是维克霉菌、酿酒酵母、假丝酵母。大部分微生物与胁迫因子呈负相关,只有乳杆菌与乙酸呈极显著性正相关。酵母菌与部分细菌呈正相关性。【结论】白酒发酵过程胁迫因子和微生物间的相互作用促进了群落演替过程,发酵后期乳杆菌和芽孢杆菌发酵产酸抑制了大部分不耐酸菌,有机酸是影响群落结构变化的主要胁迫因子。微生物数量结合相对丰度揭示了发酵过程群落结构演替及其与环境因子相关性的更多信息。     

反复冻融对枯草芽孢杆菌St-zn-34抑制链格孢菌活性的影响北大核心CSCD

【目的】以从冬枣分离的内生枯草芽孢杆菌(Bacillus subtilis St-zn-34)为供试菌株,明确反复冻融对菌体形态及其发酵滤液抑制枣缩果病初侵染病菌(Alternaria alternata)活性的变化。【方法】对供试菌株分批发酵后进行反复冻融,采用梯度稀释计数法、滤纸片法分别测定活菌数量和芽孢含量、发酵滤液抑菌活性,对供试菌株的形态变化进行电镜观察。【结果】发酵培养中pH值、活菌量、芽孢量及抑菌活性随发酵时间的增加均呈先上升后下降的趋势,其中发酵60 h发酵滤液抑菌活性最大,对此时的发酵液反复冻融,冻融3次枯草芽孢杆菌活菌量和发酵滤液抑菌活性依次减少,以后再冻融差异不显著(P>0.05),电镜观察发现随冻融次数增加,菌体变小,表面凹陷、扭曲,胶状物流出。抑菌谱检测发现发酵滤液对12种植物病原菌具有抑菌能力。不同温度和蛋白酶处理发酵滤液表明,低于60℃以下,抑菌活性与对照相比差异不显著;80℃以上抑菌活性随温度上升而下降,与对照相比差异显著;蛋白酶K可降低抑菌活性。【结论】反复冻融影响细菌形态并降低发酵滤液的抑菌活性。     

一株凝结芽孢杆菌的分离筛选及产孢条件优化

【背景】凝结芽孢杆菌除了具有一般乳酸菌的益生功能外,还具较强的耐酸、耐胆盐、耐高温、易贮存等生物特性。【目的】从泡菜中筛选一株性能优良的凝结芽孢杆菌用于微生态制剂的制备,并对其产孢率进行优化,为该菌株的进一步工业化生产提供参考依据。【方法】采用选择性培养基通过特定的培养条件,筛选到一株抑菌效果良好的产酸芽孢杆菌,并对其进行特异性引物的鉴定、16S rRNA基因序列分析及生理生化实验。通过单因素及正交试验对菌株的产芽孢条件进行优化。【结果】筛选得到一株凝结芽孢杆菌BC01,该菌株对大肠杆菌(Escherichia coli CVCC 1527)、鼠伤寒沙门氏菌(Salmonella typhimurium CVCC 2228)、产气荚膜梭菌(Clostridium perfringens CVCC 46)、猪霍乱沙门氏菌(Salmonella choleraesuis CVCC 503)等均有较强的抑制作用;模拟胃液处理120 min存活率达到94%;0.3%的胆盐存活率达到84.3%。单因素及正交试验优化后的最适培养基配方:糖蜜10.0 g/L,酵母浸出粉20.0 g/L,NaCl 5.0 g/L,K_2HPO_4 5.0 g/L,MnSO_4 10.0 mg/L;最适培养条件:接种量4%,温度45°C,初始pH 7.0,转速200 r/min,培养时间36 h。在该优化条件下,其活菌数最高达到6.7×10~9 CFU/mL,产孢率达到89.2%。【结论】筛选得到一株可用于微生态制剂的菌株——凝结芽孢杆菌BC01,对其产孢率进行了优化,为工业化生产奠定了基础。     

酱香型白酒发酵中地衣芽孢杆菌与酿酒酵母的相互作用

【目的】为解析酱香型白酒酿造群体微生物的发酵过程, 研究了酱香型白酒酿造中重要微生物地衣芽孢杆菌与酿酒酵母之间的相互作用, 并对它们之间的作用机制进行初步探讨。【方法】通过地衣芽孢杆菌与酿酒酵母共培养体系的构建, 认识了两者的相互作用, 初步分析了酿酒酵母产生抑制物的分子量, 耐热性及对蛋白酶敏感性等特性。【结果】研究表明, 酿酒酵母发酵造成的酸性环境以及某些代谢物质能够抑制地衣芽孢杆菌的生长, 这些物质分子量大于10 kD, 对热和蛋白酶敏感。【结论】白酒酿造中酿酒酵母通过产酸以及大分子的蛋白质类物质对地衣芽孢杆菌生长形成抑制, 该研究促进了对白酒酿造群体微生物发酵过程的解析。     

微生物制剂对奥尼罗非鱼生长及消化酶活性的影响

选用192尾初始体重(34.500.25) g的健康奥尼罗非鱼(Oreochromis niloticusO. aureu), 研究在基础饲料中分别添加相同剂量(活菌含量为3.01011 cfu/kg饲料)的汉逊德巴利酵母、枯草芽孢杆菌和凝结芽孢杆菌对奥尼罗非鱼生长及消化酶活性的影响, 试验期56d。试验结果表明, 与对照组相比, 添加枯草芽孢杆菌和凝结芽孢杆菌, 奥尼罗非鱼的增重率分别提高12.27%和8.56%(P0.05), 饵料系数分别降低10.92%和8.18%(P0.05)。饲料干物质表观消化率分别提高10.54%和10.07%(P0.05), 蛋白质表观消化率分别提高4.18%和3.63%(P0.05)。添加枯草芽孢杆菌和凝结芽孢杆菌组, 奥尼罗非鱼肠道、肝胰脏和胃蛋白酶活性显著高于对照组和汉逊德巴利酵母组(P 0.05), 而添加三种微生物制剂对奥尼罗非鱼肠道、肝胰脏和胃的淀粉酶和脂肪酶没有显著影响(P0.05)。结果显示, 三种微生物制剂相互比较, 饲料中添加剂量为3.01011 cfu/kg饲料的枯草芽孢杆菌和凝结芽孢杆菌能显著提高肠道、肝胰脏和胃的蛋白酶活性, 满足最佳生长。       

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

甘菊ClHSP70与ClHSP90基因的克隆及表达分析

该研究以甘菊(Chrysanthemum lavandulifolium)为实验材料,通过RT-PCR方法从甘菊转录组数据中分离出热激蛋白合成相关基因,命名为ClHSP70和ClHSP90。序列分析表明,ClHSP70基因ORF全长为2 559bp,编码852个氨基酸,蛋白功能区预测表明含有典型的HSP70蛋白NBD和SBD保守结构域;ClHSP90基因ORF全长为2 094bp,编码697个氨基酸,含有HATPase结构域和HSP90保守结构域。生物信息学分析表明,甘菊ClHSP70与大豆(Glycine max)和烟草(Nicotiana tomentosiformis)HSP70蛋白有较高的一致性,ClHSP90基因编码的氨基酸序列与紫茎泽兰(Ageratina adenophora)HSP90高度相似;实时荧光定量表达分析表明,在42℃处理不同时间,甘菊叶片中ClHSP70和ClHSP90基因表达均在0.5h时显著增加,1h达到最大值,2h后缓慢下降;不同组织表达分析表明,甘菊在42℃处理1h后,ClHSP70在成熟叶中的表达量显著高于嫩叶和根等其他组织;ClHSP90在成熟茎中的表达量最高。研究说明,ClHSP70和ClHSP90基因具有热激蛋白特征,参与了甘菊热胁迫应答过程,该研究结果为以后深入研究其基因功能奠定了基础。     

Molecular and morphological characterization of Leveillula (Ascomycota: Erysiphales) on monocotyledonous plants

Leveillula on monocotyledonous plants have been recorded as L. taurica by several authors, whereas the fungus on Allium has been described as an independent species, namely L. allii, by some authors. We sequenced ca 600 bp of the rDNA ITS region for two Leveillula specimens from Allium and Polianthes (both from monocotyledons) and compared them with several already published sequences from Leveillula isolates from dicotyledons. Pair-wise percentages of sequence divergences were calculated for all Leveillula isolates. The ITS sequence of the Polianthes isolate was identical to L. taurica on Helianthus and Vicia. The sequence of the Allium isolate was 99.5 % identical to L. taurica on Euphorbia, Haplophylum, Peganum, etc. These results suggest close relationships between monocot and dicot pathogenic Leveillula species. The identity between two monocot isolates was 98.4 %. Phylogenetic analysis revealed that the two monocot isolates do not group into a clade together. This result suggests that Leveillula acquired parasitism to monocots at least twice independently.     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.     

下延叉蕨和芽胞叉蕨配子体发育的研究

利用光学显微镜详细观察了叉蕨属(Tectaria)下延叉蕨(Tectaria decurrens)和芽胞叉蕨(T.fauriei)的配子体发育过程,记录了配子体各发育阶段的特征。结果表明:(1)下延叉蕨和芽胞叉蕨的孢子均为单裂缝,具周壁,由周壁形成纹饰,孢子极面观椭圆形,赤道面观豆形或肾形。(2)孢子萌发方式为向心型。(3)原叶体发育方式为三叉蕨型。(4)成熟原叶体心脏形,两翼向斜上方扩展。(5)均具单细胞和多细胞毛状体,在丝状体或片状体阶段出现。研究认为,从配子体发育角度看,叉蕨属是较进化的陆生真蕨类;毛状体的类型、位置和出现时间等特征在叉蕨属种间存在差异,可作为该属种间分类的特征。     

Evolutionary relationships among aquatic anamorphs and teleomorphs: Lemonniera, Margaritispora, and Goniopila

The hypothesis that similar conidial morphologies in aquatic hyphomycetes are a result of convergent evolution was tested using molecular sequence data. Cladistic analyses were performed on partial sequences of 28S rDNA of seven species of Lemonniera, one species of Margaritispora and one species of Goniopila. Lemonniera has tetraradiate conidia with long arms, whereas Margaritispora and Goniopila have typically globose (isodiametric) conidia, with short conical protuberances in a stellate or quadrangular arrangement. Lemonniera and Margaritispora have phialidic conidiogenesis and both produce dark, minute sclerotia in culture whereas Goniopila has holoblastic conidiogenesis and does not produce sclerotia in culture. Goniopila produces a microconidial phialidic synanamorph in culture. All three genera have schizolytic conidial secession. Molecular analyses demonstrate that Lemonniera species are placed in two distinct clades: one within Leotiomycetes; the other within Pleosporales, Dothideomycetes. Margaritispora is placed with Lemonniera species within Leotiomycetes. Goniopila and Lemonniera pseudofloscula are placed within Dothideomycetes. No morphological character was entirely congruent with the molecular derived phylogeny. This suggests that for the group of species studied, conidial shape is not a reliable indicator of phylogeny but more likely the result of convergent evolution in response to the aquatic environment.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

New species of Phaeoramularia, Pseudocercospora and Stenella from Western Ghats of India

Four new species of the hyphomycete genera Phaeoramularia viz. Ph. caesalpinae, Pseudocercospora viz., Ps. tiliacearum, Stenella, viz. S. argyreiae and S. grewiae occurring on Caesalpinia bonducella Fleming (Caesalpiniaceae), Grewia sp. (Tiliaceae), Argyrea sp. Lour (Convolvulaceae) and Grewia sp. L. (Tiliaceae), respectively are described and illustrated here. All these fungi were collected from Western Ghats of India.