解淀粉芽孢杆菌研究进展

解淀粉芽孢杆菌具有广泛的抗菌能力,在环保、种植业、水产养殖业具有广泛的应用前景。本文对近年来关于解淀粉芽孢杆菌的研究与探索,从分离、筛选、鉴定、优化培养、分子生物学鉴别、代谢产物分析及应用研究方面进行了总结。     

解淀粉芽孢杆菌抗菌机制研究进展

自然界中,细菌与细菌之间、细菌与其他生物之间存在广泛的相互作用。细菌产生的代谢物质可以提高自身在自然界中的竞争/生存能力。解淀粉芽孢杆菌(Bacillus amyloliquefaciens)能够产生种类繁多的抑菌物质,有效地抑制真菌和其他细菌的活性,是一类应用较广的生防菌。到目前为止,解淀粉芽孢杆菌的抑菌作用研究已经广泛开展,但是生物体是复杂多变的,仍然有很多东西是未知的,并且利用解淀粉芽孢杆菌进行生物防治的前景十分广阔,所以加强对抑菌物质的机制研究、提取相关抗菌物质尤为关键。本文综述了国内外有关解淀粉芽孢杆菌的抑菌机制研究,为进一步探究解淀粉芽孢杆菌的抑菌机制、抑菌基因和基因工程菌做好铺垫。     

解淀粉芽孢杆菌相关功能机制研究进展

解淀粉芽孢杆菌(Bacillus amyloliquefaciens)属于芽孢杆菌属,可广泛应用于农业生产、食品工业、环境保护、化妆品行业、医药以及沙漠治理。这些应用与解淀粉芽孢杆菌的特性和相关功能紧密相连。正因为解淀粉芽孢杆菌有多方面的生理功能,如形成生物膜、定殖于植物、促进植物生长等,才使其应用的广泛性成为可能。对解淀粉芽孢杆菌相关重要生理特征和功能机制进行了总结归纳,旨在为更好开发和利用这种益生菌提供科学理论数据。     

云雾龙胆内生菌的分离鉴定及抗菌活性分析

从云雾龙胆(Gentiana nubigena)中分离到一株具有抗菌活性的内生细菌LD5,该菌株的发酵上清液对金黄色葡萄球菌和大肠杆菌的生长有显著的抑制作用。通过形态学、生理生化和16SrDNA系统进化分析,鉴定LD5为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。菌株LD5能合成黄酮类化合物、龙胆苦苷,以及一种与云雾龙胆醇提取物相似的代谢产物。     

解淀粉芽孢杆菌Q426双组分信号转导系统的生物信息学分析

[目的]分析解淀粉芽孢杆菌(Bacillus amyloliquefaciens)双组分信号转导系统(Two-Component Signal Transduction System,TCS)的结构、功能和分布,为挖掘解淀粉芽孢杆菌的生物功能、开发其商业价值提供理论依据。[方法]采用生物信息学方法,系统分析了由笔者实验室分离得到并完成全基因组测序的解淀粉芽孢杆菌Q426的TCS。[结果]Q426菌株所携带的HKs和RRs分别为42和40,其中成对的TCS(HK-RR)数为19,杂合的TCS(Hybrid)为1,而HKs和RRs分别为22和20,并对19对TCS中15对的生物学功能做出预测。[结论]Q426菌株的TCS涉及杆菌肽、羊毛硫抗生素和多种抗菌肽的合成,为深入研究解淀粉芽孢杆菌抗菌活性物质的合成与调控提供思路。     

嗜热脂肪芽孢杆菌耐热α-淀粉酶基因在大肠杆菌中的克隆和表达

一序言杆菌属是分泌型酶类的重要来源(Priest 1977)。新近有一些队芽孢杆菌属菌种中克隆几种分泌酶类的报道,包括从解淀粉芽孢杆菌(B.amyloliquefaciens),凝结芽孢杆菌(B.Coagulans)和枯草芽孢杆菌(B.Subtilis)中克隆淀粉酶基因,从地衣形芽孢杆菌     

一株解淀粉芽孢杆菌生防蛋白的鉴定及分析

旨在鉴定一株拮抗葡萄霜霉病的解淀粉芽孢杆菌在NA液体培养基中的分泌蛋白中与生物防治相关的蛋白组分。采用蛋白质谱的方法对前期筛选到的一株拮抗葡萄霜霉病的解淀粉芽孢杆菌生防菌的在NA液体培养基中的分泌蛋白进行了鉴定。对鉴定到的蛋白采用基因本体论(GO)和京都基因与基因组百科全书(KEGG)的方法进行蛋白质组学分析,筛选与生物防治相关的蛋白。结果表明,从分泌蛋白中鉴定到了确定的53种蛋白质,其相对分子量大部分集中到0-100之间(占79.24%);涉及碳水化合物代谢、能量代谢、脂类代谢、氨基酸代谢和生物防御等过程,以及作为细胞组分参与结构组成。发现了6种参与植物与病原菌互作的蛋白,2种属于氨基肽酶家族,4种参与几丁质的生物降解过程。     

解淀粉芽孢杆菌β-1,3-1,4-葡聚糖酶的高效表达

目的:克隆解淀粉芽孢杆菌β-1,3-1,4-葡聚糖酶基因(bglA)使其在解淀粉芽孢杆菌CICIM B4081中高效表达,并对重组酶进行酶学性质研究.方法:以解淀粉芽孢杆菌(CICIM B4801)染色体DNA为模板,经过PCR扩增得到了大小约为0.8kb的β-1,3-1,4-葡聚糖酶基因(bglA),构建了重组表达质粒pQ-bglA,通过电转化的方法将其转化人解淀粉芽孢杆菌(CICIM B4801)中.结果:得到了能高效表达β-1,3-1,4-葡聚糖酶的重组解淀粉芽孢杆菌.在250mL摇瓶条件下,重组菌分解地衣多糖的胞外最高酶活达到了1515.7U/mL,重组酶的最适作用温度为55℃,最适反应pH值为6.5.结论:重组菌的β-1,3-1,4-葡聚糖酶的酶活为原始菌株的11.84倍,实现了bglA基因在解淀粉芽孢杆菌中的高效表达.     

应用多重PCR鉴定微生物肥料常用芽孢杆菌

[目的]枯草群芽孢杆菌中枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(B.amyloliq-wefaciens)、地衣芽孢杆菌(B.licheniformis)和短小芽孢杆菌(B.pumilus)是微生物肥料中常用菌种,用传统方法鉴定费时费力,有必要建立检测和鉴定这些芽孢杆菌的种特异性PCR方法.[方法]利用已登录的gyrA、rpoA和16s rRNA基因序列分别设计和筛选上述菌种的特异引物并建立多重PCR反应体系.[结果]以基因组DNA为模板,扩增芽孢杆菌、类芽孢杆菌和短芽孢杆菌3属15种的标准菌株(共33株),4个目标种分别产生了大小不同的唯一的产物,除个别种与短小芽孢杆菌引物有交叉反应外,其余参考菌株均为阴性.从23株枯草群菌株的基因组DNA扩增发现,PCR鉴定与常规鉴定结果一致.[结论]本文建立的多重PCR方法具有较好的特异性,可快速准确鉴定枯草群的4个种,在微生物肥料检测方面有良好的实用前景.     

解淀粉芽孢杆菌抑菌机制的研究进展

解淀粉芽孢杆菌能够产生种类繁多的抑菌物质,可以有效的抑制真菌和细菌的活性。近几年来,关于解淀粉芽孢杆菌抑菌机制的研究报道越来越多,大部分研究已经深入到分子水平。综述了国内外有关解淀粉芽孢杆菌的全基因组信息和解淀粉芽孢杆菌抑菌机制的相关研究。     

三角褐指藻对黑暗胁迫的生理响应

为了研究黑暗条件对三角褐指藻生长及生化组成的影响,并探讨三角褐指藻对黑暗环境的生长适应能力,我们对该藻进行12 d的黑暗处理,着重测定了藻细胞密度、生物量、叶绿素a、可溶性糖和蛋白含量等指标。结果表明,三角褐指藻对黑暗环境表现出一定的适应性忍耐能力,在12 d的长期黑暗胁迫条件下依然可以存活,而藻细胞生化组成对黑暗的响应程度很可能是该藻得以维持细胞低水平生长的主要原因。随着黑暗处理时间的延长,三角褐指藻生长状况受抑制的程度增大,其生长及生化组成与对照组相比发生了极显著的变化。实验结束时,黑暗处理下的藻细胞密度和生物量分别降低到2.93×10⁵ cells·ml^-1和0.011 g·ml^-1,仅为对照的8.0%和37.3%。同样地,黑暗环境也明显地抑制了三角褐指藻体内生化物质的合成与积累,黑暗处理12 d时藻细胞的叶绿素a、可溶性糖和蛋白含量分别比对照降低了约89%、87%和85%。研究结果可为海洋微藻种质的筛选、种质资源库的构建及微藻生物资源的综合开发利用提供参考依据。     

三角褐指藻dxs基因克隆与诱导表达

为研究6种外源因素处理对三角褐指藻(Phaeodactylum tricornutum)1-脱氧-D-木酮糖-5-磷酸合成酶(DXS)基因表达的影响,通过高通量转录组测序技术获得三角褐指藻dxs基因cDNA全长序列,并对其进行生物信息学分析。研究结果表明,三角褐指藻dxs基因cDNA全长2476 bp, ORF全长2193 bp,编码730个氨基酸,具有高度保守的ThDP结合位点和转酮醇酶结构域。三角褐指藻DXS蛋白为亲水性稳定蛋白,相对分子质量(M_w)为79.31 kD,理论等电点为6.65,具有信号肽、跨膜区域、卷曲螺旋和TM-螺旋等。系统进化树分析结果表明, DXS蛋白进化树分为高等植物和藻类2个分支,高等植物DXS蛋白进一步分为DXS1和DXS2两支,藻类DXS蛋白聚类为3个分支,分别为硅藻门、红藻门和绿藻门分支,三角褐指藻聚类在硅藻门分支上。诱导表达调控结果表明,三角褐指藻dxs基因受到茉莉酸甲酯(MeJA)、花生四烯酸(AA)、硫酸铈铵(ACS)、光合诱导素(PIF)、光合抑制剂(DCMU)和光质等6种外源因素的诱导调控。在100μmol/L MeJA...     

Effects of dietary Bacillus subtilis, Tetraselmis chuii, and Phaeodactylum tricornutum, singularly or in combination, on the immune response and disease resistance of sea bream (Sparus aurata L.)

Combined or individual effects of two microalgae (Phaeodactylum tricornutum and Tetraselmis chuii) and Bacillus subtilis on immune response, gene expression, and survival to challenge with Photobacterium damselae subsp. piscicida of gilthead sea bream were investigated. To test the capacity of B. subtilis to grow employing the microalgae polysaccharides as energy and carbon source, an in vitro assay was defined, and demonstrated that the digestion product of microalgae, mainly P. tricornutum, supported the growth of B. subtilis much better than glucose. For the in vivo study, fish were distributed in six equal groups (each of two replicates) and received one of the following experimental diets: C) control, non-supplemented diet; T) T. chuii 100 g kg(-1); P) P. tricornutum 100 g kg(-1); B) B. subtilis (10(7) cfu g(-1)); BT) B. subtilis (10(7) cfu g(-1))+T. chuii (100 g kg(-1)); and BP) B. subtilis (10(7) cfu g(-1))+P. tricornutum (100 g kg(-1)). The complement activity, serum IgM level, respiratory burst, phagocytic activity, and expression of seven selected immune-related genes in head-kidney were evaluated following two and four weeks of treatment. At the end of the feeding trial, fish were challenged by intraperitoneal injection of LD(50) concentration of P. damselae subsp. piscicida and mortality was recorded. This is the first study testing the immunomodulatory capacity of the microalgae used in the present work. The dietary applications of B. subtilis, T. chuii, and P. tricornutum, singly or in combination, may exhibit up-regulating effects on gilthead sea bream immune parameters. P. tricornutum demonstrated the highest immunostimulant activity. There were no significant differences between combination feeding and feeding ingredients separately. Our results demonstrated the potential of microalgae as immunostimulants for fish, although further studies regarding the implications and effects of a stimulated immune system against pathogens, especially the protective capacity against specific diseases, are necessary.     

光强调控三角褐指藻对海洋酸化的生理学响应

光和海洋酸化(CO₂浓度升高)分别对海洋硅藻的光合能力具有不同程度的影响, 但两者的耦合响应被较少关注。研究以三角褐指藻作为实验材料, 测定了不同光强下CO₂浓度升高对三角褐指藻的生长、净光合速率、生化组分、胞外碳酸酐酶(eCA)活性和核酮糖-1,5-二磷酸羧化/氧化酶(Rubisco)活性的影响。结果显示在低光下, CO₂浓度对三角褐指藻的生长和净光合速率(P_n)并没有显著影响, 而在高光下, 具有明显的影响。无论是在高光或是低光下, eCA活性、叶绿素和可溶性蛋白的含量都随着CO₂浓度的升高而降低。在低光下, 高浓度CO₂ (HC)培养下的Rubisco活性分别是低浓度CO₂ (LC)和中浓度CO₂ (MC)的2.42和1.39倍, 而在高光下, HC培养下的Rubisco活性分别是LC和MC的6.72和3.45倍。以上结果表明硅藻能够通过调节光合生理特征和CCM运行中能量的分配来适应环境中光强和CO₂浓度的变化。     

不同饵料对卤虫生长、总脂含量及脂肪酸组成的影响

为了提高养殖卤虫的饵料营养价值,了解其不同生长阶段营养成分变化情况,采用单因子试验研究了8种饵料（三角褐指藻、小球藻、微绿球藻、酵母液、三角褐指藻＋小球藻＋微绿球藻、三角褐指藻＋酵母液、小球藻＋酵母液和微绿球藻＋酵母液）对卤虫生长、总脂含量及脂肪酸组成的影响,结果表明：不同饵料种类对卤虫生长、总脂含量及脂肪酸组成的影响显著（P＜0.05）,增长率,以三角褐指藻＋酵母液最优;总脂含量、以三角褐指藻最优（19.67%）,除酵母液外,与其它饵料相差不显著（P＞0.05）;脂肪酸组成效果,以微绿球藻组最优（EPA：18.01%,DNA：0.55%,（n-3）HUFA：19.08%）,与三角褐指藻组相差不大（P＞0.05）,显著高于其它各组（P＞0.05）.同时以三角褐指藻为饵料,研究了卤虫不同生长阶段（体长2、4、6、8、10 mm）总脂含量、脂肪酸组成变化,结果表明：卤虫体长2～10 mm总脂含量为14.27%～20.93%,随体长的增长降低;EPA、DHA及（n-3）HUFA的含量,均随体长的增长降低,EPA含量为：10.47%～20.77%,DNA含量为：0～0.70%,（n-3）HUFA含量为：10.85%～22.01%.结论认为,卤虫以三角褐指藻或三角褐指藻＋酵母液为饵料培养营养价值最佳,其体长小于6 mm营养价值较佳.     

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.     

MEASURING RATES OF AMMONIUM ASSIMILATION IN MARINE ALGAE: USE OF THE PROTONOPHORE CARBONYL CYANIDE m-CHLOROPHENYLHYDRAZONE TO DISTINGUISH BETWEEN UPTAKE AND ASSIMILATION

A method for determining rates of ammonium assimilation in marine algae is described. Ammonium assimilation is defined as the decrease in total (medium + cellular) ammonium. The protonophore carbonyl cyanide m- chlorophenylhydrazone (CCCP) was used to distinguish between uptake and assimilation of ammonium. Ammonium uptake by nitrogen-replete and nitrogen-starved cells of the diatom Phaeodactylum tricornutum Bohlin and the green macroalga Enteromorpha sp. was completely (98%–99%) inhibited in the presence of 100 μM CCCP. In addition to inhibiting further uptake of ammonium, CCCP promoted the release of unassimilated ammonium by nitrogen-replete and nitrogen-starved P. tricornutum and Enteromorpha that had been allowed to take up ammonium for a period. Most (97.5%) of preaccumulated ¹⁴C-methylammonium was released by nitrogen-starved P. tricornutum in the presence of CCCP. Specific rates of ammonium assimilation in nitrogen-replete cultures of P. tricornutum were identical to the maximum growth rate, but specific rates in nitrogen-starved cultures were fourfold greater. Rates of ammonium assimilation in Enteromorpha during both the surge and the internally controlled uptake phases were the same as the internally controlled rate of uptake, suggesting that the latter is a reliable measure of the maximum rate of assimilation.     

海洋经济微藻混合培养与单独培养下的生长比较

海洋微藻可以广泛应用在水产养殖、食品加工、医药保健、环境保护和生物制能等各种行业,具有非常好的开发利用前景.目前,如何突破海洋微藻培养过程中细胞生物量低下等瓶颈,提高微藻的细胞密度,以低成本、高效率开发利用海洋微藻资源成为了国内外学者关注的焦点.论文以三角褐指藻、亚心型扁藻和杜氏盐藻3 种典型经济海洋微藻为研究材料,在实验室条件下研究了它们两两混合培养与各自单独培养条件下的细胞生长情况,探讨海洋微藻混合培养在促进微藻整体细胞生长方面的可能性.结果显示,三角褐指藻和杜氏盐藻混合培养在生长前9 天的OD680 高于三角褐指藻或杜氏盐藻分别单独培养的OD680 ,而随着试验时间的延长,混合培养条件下的OD680 与单独培养三角褐指藻的OD680 相似;就三角褐指藻和亚心型扁藻混合培养的OD680而言,在整个生长时期均高于单独培养亚心型扁藻的OD680 ,但低于单独培养三角褐指藻的OD680 ;不同的是,杜氏盐藻和亚心型扁藻混合培养下的OD680 在生长第6 天后高于单独培养杜氏盐藻或单独培养亚心型扁藻的OD680 .研究结果表明,混合培养三角褐指藻和杜氏盐藻或混合培养杜氏盐藻和亚心型扁藻具有一定程度促进微藻细胞生长的潜力.研究结果将为经济海洋微藻的高密度培养及其高附加价值活性物质的开发利用提供一个崭新的研究方向.     

Computational prediction of microRNAs and their targets from three unicellular algae species with complete genome sequences

The genome sequences of Phaeodactylum tricornutum, Thalassiosira pseudonana, and Cyanidioschyzon merolae have provided significant evidence for the secondary endosymbiosis of diatoms in regard to the genome. Yet little about their relationships in regard to gene regulation pattern, such as microRNA (miRNA), has been reported. Using a homology search based on genomic sequences, 13, 3, and 7 predicted miRNA genes were found in genomes from P. tricornutum, T. pseudonana, and C. merolae, respectively. Of the 23 miRNA genes, 18 had homology with animal miRNAs, implying that they are ancestral miRNAs. A phylogenetic tree based on common miRNA families shared by these three unicellular algae, higher plants, and animals showed that P. tricornutum shared most miRNAs with animals. The phylogenetic tree also showed that C. merolae shared more miRNAs with plants than did the two diatoms, and the majority of its miRNAs were shared with the two diatoms. Our results were consistent with diatoms originating from a secondary endosymbiosis.