乳酸乳球菌双组分系统调控有氧呼吸的研究

【背景】乳酸乳球菌作为食品行业的代表性菌株,如何通过双组分系统响应环境因子与代谢调控的分子机制研究,对发酵食品产业和益生菌制剂行业有着重要的意义。【目的】探究乳酸乳球菌双组分系统对有氧呼吸代谢调控的相关网络,为乳酸菌适应性代谢研究提供新思路。【方法】采用生物信息学方法,系统性地分析乳酸乳球菌双组分系统组氨酸激酶和反应调节因子的结构域组成及预测双组分系统功能,筛选出与有氧呼吸有潜在联系的双组分,并进一步通过基因转录表达和非靶向代谢组学验证。【结果】以乳酸乳球菌的代表菌株NZ9000为例构建相互作用蛋白网络,显示双组分系统与丙酮酸代谢网络关键连接点为丙酮酸铁氧还蛋白氧化还原酶(nifJ)。在不同的生长时期,Lactococcus lactis NZ9000双组分转录表达在延滞期变化显著。与厌氧培养相比,有氧培养和有氧呼吸培养的菌体双组分呈现下调趋势。双组分系统参与乳酸菌氧化应激和血红素胁迫过程。【结论】明确乳酸乳球菌参与有氧呼吸的双组分系统以及代谢通路,有助于提高发酵剂、益生菌剂的存活率和竞争力。     

酸适应对一株乳酸乳球菌膜脂肪酸组成和膜蛋白表达的影响

[目的]筛选具有较强酸适应能力的菌株,研究酸适应对其膜脂肪酸组成和膜蛋白表达的影响.[方法]从20株菌中筛选出一株具有较强酸适应能力的乳酸乳球菌KLDS4.0312,以GC-MS法测定该菌酸适应前后膜脂肪酸组成变化;对酸适应前后该菌膜蛋白的差异表达进行双向电泳分析.[结果]酸适应后,该菌膜不饱和脂肪酸含量从30.77%上升到42.93%,饱和脂肪酸含量从69.23%下降到57.07%,且有一种新的长链单不饱和脂肪酸C<,19:1>-n6被诱导产生.酸适应过程中至少有65个蛋白质点表达出现显著差异,其中上调的蛋白质点有43个,减弱表达的蛋白质点有22个.而添加氯霉素后,菌株的酸适应能力消除,可能与氯霉素抑制新蛋白的合成有关.[结论]说明细胞膜脂肪酸组成的适应性改变和应激蛋白的诱导产生是该菌主要的酸适应机制.     

乳酸乳球菌中冷休克蛋白基因的高效表达及其抗冻保护作用

[目的]微生物在适应外界环境急剧降温的条件下都会发生应激反应,产生一系列蛋白质被称为冷休克蛋白.冷休克蛋白对乳酸菌适应低温环境和增强抗冻能力方面发挥着重要作用.本文目的是为了研究乳酸乳球菌中冷休克蛋白CspC、CspD的作用.[方法]将冷休克蛋白CspC、CspD基因分别重组到质粒pNZ8148,转化乳酸乳球菌NZ9000后,加入Nisin诱导,对表达产物进行SDS-PAGE电泳分析,比较重组菌与空白菌在30℃条件下菌体生长差异及反复冻融活菌数的差异.[结果]得出CspC、CspD的相对分子量分别为7.0、6.2 kDa.[结论]CspC使菌体更加迅速的恢复了生长;冷休克蛋白CspD增强了菌体的抗冻存活率(增加了30～40倍).     

乳杆菌代谢产物对化脓性链球菌的抑制作用

【目的】分析乳杆菌代谢产物对化脓性链球菌的抑制作用。【方法】基于双层平板打孔法,通过测量抑菌圈大小来检测乳杆菌代谢产物对化脓性链球菌的抑菌作用;然后分别采用高效液相色谱法和4-氨酰安替比林法检测乳杆菌代谢产物中的有机酸和H2O2含量;最后,检测乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)。【结果】对化脓性链球菌的抑菌效果以植物乳杆菌KLDS1.0667最好,副干酪乳杆菌KLDS1.0342-1次之,瑞士乳杆菌KLDS1.0203抑菌效果最差;乳酸和乙酸产量KLDS1.0667>KLDS1.0342-1>KLDS1.0203;H2O2产量KLDS1.0203>KLDS1.0667>KLDS1.0342-1。在抑菌试验中,乳杆菌的发酵上清液经去除H2O2处理后抑菌圈直径都减小;将发酵上清液的p H调至7.0后均检测不到抑菌圈。结果表明,乳杆菌代谢产物中对化脓性链球菌起抑制作用的主要物质为有机酸和H2O2,其中乳酸是产生抑菌作用的最主要物质。乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)分别为1.28、0.64和0.008 g/L,对化脓性链球菌的最小杀菌浓度(MBC)分别为5.12、2.56和0.032 g/L。【结论】乳杆菌可利用其代谢产物对化脓性链球菌产生抑制作用,主要抑菌物质为有机酸和H2O2。     

乳酸乳球菌KLDS4.0325的糖代谢过程及乳酸生物合成途径的比较

【目的】为了探究乳酸乳球菌乳酸亚种KLDS4.0325的碳水化合物利用能力和乳酸形成潜力。【方法】本文对该菌株进行了全基因组鸟枪法测序,并应用生物信息学方法对该菌株细胞外糖的转运、代谢及产酸途径涉及的一系列基因与其它9株参考菌株进行了比较分析。【结果】与参考菌株相比,该菌株基因组中具有较多涉及整个途径糖代谢途径的关键酶编码基因。【结论】该菌株在基因水平上表现出能够利用多种糖类物质来产乳酸的优良性状,是一株具有高产L-乳酸工业潜能的乳酸菌。     

基于冰核蛋白的乳酸菌表面展示系统的构建

[目的]验证来源于丁香假单胞菌的冰核蛋白在乳酸乳球菌表面展示外源蛋白的可能性.[方法]以绿色荧光蛋白(Green Fluorescence Protein,GFP)基因gfp为报告基因,以冰核蛋白基因的N末端和NC端作为展示单元,构建乳酸菌表面展示载体pHZ101和pHZ102,并转化大肠杆菌(Escherichia coli JM109和乳酸乳球菌(Lactococcus lactis)MG1363.[结果]荧光显微镜观察显示重组大肠杆菌和乳酸乳球菌均能检测到绿色荧光.Western blot结果表明GFP蛋白在重组大肠杆菌和乳酸乳球菌中均得到表达,并且INPN-GFP蛋白多数滞留于乳酸乳球菌细胞质内,而INPNC-GFP蛋白则大部分定位于乳酸乳球菌的细胞膜上.[结论]以上结果表明丁香假单胞菌的冰核蛋白能引导外源蛋白定位于乳酸乳球菌的细胞膜上,为乳酸菌表面展示系统的构建提供了新的方向.     

一株具有谷胱甘肽合成能力的乳酸乳球菌的分离与初步研究

基于gshB基因同源性分析设计引物,从采集的菜园地土壤中分离到1株具有谷胱甘肽(GSH)合成能力的乳酸乳球菌菌株CCSYU10100。测定了该菌株的16S rDNA序列并根据16S rDNA序列构建了系统发育树,结果显示该菌株与乳酸乳球菌乳脂亚种在进化关系上的地位最近。电镜分析表明,菌株CCSYU10100与乳酸乳球菌的形态特征基本一致。因此认为菌株CCSYU10100属于乳酸乳球菌乳脂亚种,命名为乳酸乳球菌乳脂亚种CCSYU10100。HPLC法鉴定出该菌株胞内除GSH外,还存在半胱氨酸-甘氨酸。乳酸乳球菌CCSYU10100的部分gshB基因与假单胞菌属的gshB基因高度同源,这是gshB基因在乳酸乳球菌中、甚至是革兰氏阳性菌中的首次发现。     

一株从中国新疆地区的酸马奶中分离出的新菌株,乳酸乳球菌乳酸亚种KLDS4.0325的全基因组序列(英文)

【目的】为了阐明乳酸乳球菌乳酸亚种KLDS4.0325的生理及代谢机制,并对其重要功能基因进行挖掘,我们对菌株KLDS4.0325的全基因组序列进行测定和基因组图谱的绘制,并利用生物软件和数据库完成对序列的注释及相关功能性分析。【方法】在菌株序列完成测序、组装和注释后,根据序列信息进行全基因组图谱的绘制,并对菌株的蛋白质水解系统、氨基酸来源的风味形成途径和B-族维生素合成途径进行了比较基因组分析,并对细菌素合成基因组和冷应激蛋白基因进行了预测。【结果】菌株KLDS4.0325基因组全长2589250 bp,G+C含量为35.4%,共预测出2662个开放阅读框,其中1310个具有潜在的生物学功能。菌株可以有效的对细胞外蛋白质进行有效的水解,具有潜在的降低苦味肽以及产生一系列能够抑制血管紧张素转化酶活性的活性肽。在转氨途径方面,菌株KLDS4.0325具有较为完成的酶系统,可以催化相关氨基酸转化为风味物质。在菌株KLDS4.0325的基因组中,我们发现了较多编码糖转运、代谢以及L-乳酸合成的基因。关于叶酸和核黄素合成途径的编码基因在菌株KLDS4.0325的基因组中也较为完整。此外,我们在菌株KLDS4.0325的基因组中预测出了一个关于乳球菌素的基因簇和两个冷应激蛋白Csp D和csp E的编码基因。【结论】这些编码菌株显著特性基因的存在为菌株KLDS4.0325能够进行工业发酵提供了理论基础,并为其进一步研究提供了方向。     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

保加利亚乳杆菌H+-ATPase缺陷型菌株的筛选

【目的】从传统乳制品中筛选具有新霉素抗性的H+-ATPase缺陷的德氏乳杆菌保加利亚亚种自发突变株,为最终开发弱后酸化的酸奶发酵剂奠定基础。【方法】利用API 50 CH细菌鉴定系统和16s rRNA基因序列分析对菌株进行鉴定。新霉素作为筛选压力,筛选具有新霉素抗性自发突变菌株,比较亲本和突变菌株的H+-ATPase活力及其代谢情况。【结果】从内蒙古地区的传统发酵酸奶中分离鉴定出一株德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）,并命名为KLDS 1.9201。以此为出发菌株,筛选出两株H+-ATPase缺陷的自发突变株,分别命名为KLDS 1.9201-1、KLDS 1.9201-4,它们的H+-ATPase活力分别比亲本KLDS 1.9201降低了46%和60%。在MRS培养基中生长24 h后,KLDS 1.9201、KLDS 1.9201-1和KLDS 1.9201-4对初始葡萄糖的代谢率分别为65%、41%和31%,终产物中乳酸的浓度分别为26g/L、18g/L和15g/L,突变菌株的生物量均低于亲本。【结论】H+-ATPase活力降低的德氏乳杆菌保加利亚亚种的自发突变株具有较低的生长速率和弱产酸能力,它们可被用于制作弱后酸化的酸奶发酵剂。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

Scales of spatial patterns of distribution of intertidal invertebrates

Few comparative studies of spatial patterns at different scales have examined several species in the same habitat or the same species over a range of habitats. Therefore, variability in patterns among species or among habitats has seldom been documented. This study quantifies spatial patterns of a suite of intertidal snails and a species of barnacle using a range of statistical techniques. Variability in densities was quantified from the scale of adjacent quadrats (over a distance of centimeters) to tens of kilometers. Significant differences in abundances occurred primarily at two spatial scales. Small-scale differences were found at the scales of centimeters or 1–2 m and, for many species on many shores, these accounted for most of the variability in abundances from place to place. These are likely to be determined by behavioural responses to small-scale patches of microhabitat. Large-scale differences in abundance were also found in most species at the scale of hundreds of meters alongshore. These are likely to be due to variation in recruitment (and/or mortality) because of limited dispersal by adults of these species. There was little or no additional variation among shores, separated by tens of kilometers, than was shown among patches of shore separated by hundreds of meters. Identification of the scale(s) at which significant differences in abundance are found focus attention on the processes (and the scales at which these processes operate) that influence patterns of distribution and abundance. Some of the advantages and disadvantages of various procedures are discussed.