rDNA ITS序列在ACCC真菌鉴定中的应用

【目的】真菌无处不在,并与人类生活紧密相关。真菌的鉴定是科学研究和资源开发利用的基础。本研究从中国农业微生物菌种保藏管理中心(ACCC)随机选取已经定名的112株库藏真菌菌株作为实验材料,通过DNA测序,评估核糖体DNA内转录间隔区(rDNA ITS)序列在真菌属级鉴定上的应用。【方法】采用真菌DNA条形码rDNA ITS的序列测定和在GenBank中查寻比对的方法,对这些菌株进行属水平的鉴定复核。【结果】通过研究,供试菌株中有80株的属名与原鉴定相符,同时表明序列中套峰的情况降低与Gen Bank中序列比对的相似性。【结论】rDNA ITS序列测定法可以用于真菌菌株进行属水平的鉴定复核,国家菌种保藏机构应对已入库保藏和即将入库的所有真菌菌株都进行属水平的鉴定复核,并对菌株鉴定结果作审慎处理,原定名的名称及其变更历史应以"曾用名"在数据库中保留。     

猪源大肠杆菌Nissle 1917菌株的分离鉴定

【目的】证实大肠杆菌Nissle 1917作为自然菌株存在于动物猪体内,并能从猪粪便中分离。建立大肠杆菌Nissle 1917的原位杂交鉴定方法。【方法】采集135份健康断奶仔猪的新鲜粪便制备DNA模板,以人源大肠杆菌Nissle 1917为阳性对照菌株,分别针对Nissle 1917的I型菌毛亚单位Fim A、F1C菌毛亚单位Foc A及两个质粒pMUT1和pMUT2的相关基因序列设计5对特异性引物进行PCR扩增;并将其中427 bp大小的质粒片段pMUT2(a)作为目的片段回收纯化,用地高辛随机引物标记法制成DNA探针。【结果】从其中的2份DNA模板中扩增出上述5对特异性引物PCR预期大小相符的片段,初步认为大肠杆菌Nissle 1917可能存在于猪体内。应用制备的探针通过菌落原位杂交的方法从2份阳性粪便样品中筛选出2株阳性菌落,通过血清学检验、PCR扩增和测序进一步鉴定为阳性Nissle 1917菌株。【结论】动物源益生菌Nissle 1917的分离鉴定,为优良动物源益生菌研究和应用奠定了基础。     

一株亚硝酸盐降解真菌Aspergillus parasiticus JFS的筛选及鉴定

【目的】筛选可降解亚硝酸盐的菌株。【方法】利用亚硝酸盐选择培养基从水样、土样、朽木等20多个样品中筛选目的菌株。利用盐酸萘乙二胺法检测目的菌株在亚硝酸盐液体培养基中降解亚硝酸盐的能力,利用镉柱还原法和试纸法鉴定亚硝酸盐被目的菌株代谢后的产物;利用菌落形态、菌丝及分生孢子形态、ITS DNA序列、β-Tubulin gene序列及Calmodulin gene序列分析等鉴定目的菌株。【结果】得到一株可强烈降解亚硝酸盐的真菌菌株JFS,其能在亚硝酸盐浓度高达20.0 g/L的液体培养基中很好的生长,菌株JFS将亚硝酸盐降解为无毒的硝酸盐和铵根离子。结合形态学、对培养基的要求及上述3个基因序列的进化树分析,该菌株属于寄生曲霉菌(Aspergillus parasiticus)。【结论】菌株JFS具有强烈的降解亚硝酸盐的能力。     

链霉菌TJ430的鉴定及产物结构

【目的】通过对分离获得的土壤耐热放线菌株进行鉴定,并对该菌株产生的抗菌活性产物进行分离纯化和结构解析,达到判定该菌株归属及其产物新颖性目的。【方法】采用干热法对土壤样品进行前处理;采用经改良的HV培养基分离土壤耐热放线菌;通过琼脂块快速初筛法对分离获得的耐热放线菌进行生物活性快速评价;分别采用形态学观察法、细胞化学组分分析法、生理生化及酶学特征分析、16S rDNA序列分析、DNA杂交法,对分离获得的编号为TJ430的菌株进行菌株鉴定;采用柱层析法和制备色谱法,对发酵获得的有效成分粗提物进行分离纯化;采用红外光谱法、高分辨质谱法对分离获得的生物活性成分代谢产物进行结构探索。【结果】共分离获得570株耐热放线菌;抗菌活性快速初筛表明,编号为TJ430的菌株表现出优良的抗卵菌及广谱抗真菌活性;采用多种方法对菌株TJ430进行鉴定,结果表明该菌株为一株卡吾尔链霉菌;最终分离获得了纯度高达98%以上的有效成分纯品化合物;该有效成分的分子式为C40H66N3O11,分子量为765;分子中应含有亚胺基、甲基、亚甲基、羰基、共价双键、异丙基等化学基团。【结论】目前对该抗菌活性成分的详细化学结构研究尚有待深入,但该抗菌活性成分极有可能是一种新型化合物,具有较好的开发应用前景。     

一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达

摘要：【目的】本研究旨在从重金属汞抗性细菌中分离鉴定汞抗性基因【方法】从北京凉水河河床底泥中分离抗汞细菌,采用16S rRNA基因序列分析结合生理生化特征对菌株进行鉴定。根据GenBank中已发表的多种抗汞细菌的merA基因序列设计引物,以抗性细菌基因组DNA为模板,扩增merA基因,并在大肠杆菌（Escherichia coli）BL21(DE3)表达。同时对表达菌株的重金属汞抗性进行测定。【结果】分离得到一株能在含HgCl2为70 mg/L的平板上生长良好的高抗汞细菌,编号为KHg2。16S rRNA     

一株油藏嗜热厌氧杆菌的分离鉴定和代谢特征的分析

摘要：【目的】从高温油藏中发掘新的微生物种质资源。【方法】采用 Hungate 厌氧操作技术从大港油田采出水中分离到一株厌氧杆菌 HL-3。通过生理生化特征比较和16S rRNA序列比对,确定HL-3的分类地位。【结果】菌株HL-3为严格厌氧的革兰氏阴性杆菌。生长温度范围 40℃－75℃（最适温度 60℃）;pH 范围 5.0－8.0（最适 pH 6.5）;NaCl 浓度范围 0%－3.2%（最适NaCl浓度0.25%）。能够利用葡萄糖、核糖、甘露糖、木糖、纤维二糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、CO2及少量丙酸和丁醇。菌株HL-3的（G+C）mol%含量为 33.9%,与Thermoanaerobacter（嗜热厌氧杆菌属）中模式菌株T.uzonensis DSM18761T (EF530067)的16SrRNA 序列相似性为98.8%,与T.sulfurigignens DSM17917T (AF234164)的相似度次之为98.1％。菌株能够耐受浓度较高的亚硫酸根（0.1 mol/L）离子和浓度极高的硫代硫酸根（0.8 mol/L）。当硫代硫酸根浓度高于0.075 mol/L时,菌体内产生硫单质颗粒;同时,在培养血清瓶顶空中检测到硫化氢气体。菌株 HL-3与T.uzonensis DSM18761T对硫代硫酸根和亚硫酸根的耐受程度有很大不同。菌株HL-3对硫代硫酸根和亚硫酸根耐受程度及对硫代硫酸根的代谢机制与T.sulfurigignens DSM17917T(AF234164)极为相似,但二者代谢葡萄糖的产物却极不相同。【结论】所以菌株HL-3可能是Thermoanaerobacter属中的一个新种,其确切分类地位还有待用DNA分子杂交[1]的技术手段做进一步的鉴定。     

具有抗哈维氏弧菌活性共生真菌HLZ-3菌株的筛选、鉴定及其培养条件

【背景】目前,海水养殖业中主要利用抗生素来防治哈维氏弧菌等病原菌,但抗生素的长期使用或滥用会对环境和人体健康带来危害,因此既环保又有效的生物防治方法具有广阔的应用前景。【目的】从海水产品共生微生物中筛选具有抗菌活性的菌株,对活性菌株进行鉴定并确定其合成抗菌活性物质的培养条件。【方法】利用沙氏和2216E培养基,以稀释涂布平板法从海水养殖动物中分离真菌和细菌;利用牛津杯法测定微生物发酵液抗水产病原哈维氏弧菌的活性;通过菌株的培养特征、形态特征和ITS序列分析对抗菌活性菌株进行鉴定;通过筛选发酵培养基的种类及盐度确定培养条件。【结果】从海蚌、白虾、海蛎子等9种样品中分离出微生物52株,其中真菌30株、细菌22株;筛选得到2株具有抗哈维氏弧菌活性的真菌菌株;其中一株活性菌株HLZ-3被鉴定为塔宾曲霉;菌株HLZ-3合成抗菌活性物质的培养条件为4%NaCl的大米培养基,28°C静置培养2周。【结论】实验结果为进一步分离纯化菌株HLZ-3所产抗菌活性次生代谢产物提供了基础。     

瘤胃源粪臭素降解菌的分离鉴定及其降解特性研究

【目的】旨在从绵羊瘤胃中分离粪臭素降解菌,并评估其粪臭素降解能力和生长性能,以期开发适用于反刍动物降臭的直接饲喂微生物。【方法】以绵羊瘤胃液为分离来源,使用含有粪臭素的MSM培养基进行富集和分离,通过细菌菌落形态观察进行初步分类;应用16S rRNA基因扩增、测序及系统发育分析进行物种鉴定;绘制菌株生长曲线,通过HPLC技术测定粪臭素降解曲线。【结果】从绵羊瘤胃液中分离出25株粪臭素降解菌,经菌落形态鉴定选出11株代表菌株进行后续研究。物种鉴定结果显示,MSML2和MSML6属于枯草芽孢杆菌（Bacillus subtilis）,MSML4、MSML5、MSML7和MSML10属于阿氏普里斯特氏菌（Priestia aryabhattai）,MSML3和MSML11属于污染伯克霍尔德氏菌（Burkholderia contaminans）,MSML1、MSML8和MSML9属于成都假单胞菌（Pseudomonas chengduensis）。其中,MSML5生长速度最快,在约12 h后进入稳定期,稳定期菌体浓度最高;MSML3和MSML8在前16 h生长缓慢,32 h后进入稳定期。在粪...     

联苯菊酯降解菌株BF-3的分离筛选、鉴定和降解特性

【背景】联苯菊酯是人工合成的类似天然除虫菊素的一种仿生杀虫剂,近年来被广泛应用于农业病虫害的防治。联苯菊酯化学性质较稳定,在环境中的残留期长,是我国出口果蔬、茶叶中残留较严重的农药之一。微生物降解具有降解速度快、无二次污染等优点,被认为是有效去除农药残留的绿色生产技术。【方法】通过富集驯化,从农药厂排污口的污泥中筛选分离能够降解联苯菊酯的菌株,经形态学观察、生理生化特性测定及16SrRNA序列分析对其进行鉴定,通过单因素试验对菌株的降解特性进行研究。【结果】分离到1株联苯菊酯的降解菌株BF-3。菌株BF-3为革兰氏阳性菌,被鉴定为蜡状芽孢杆菌;该菌株在联苯菊酯质量浓度为100mg·L^-1的无机盐液体培养基（MSM）中呈现s型生长,7d后对联苯菊酯的降解率达到87．9％。单因素试验表明,菌株最适降解条件为pH7．0、30oC,初始菌株D600am=1．0。【结论与意义】蜡状芽孢杆菌BF．3能够有效降解联苯菊酯,在环境中联苯菊酯残留的生物修复方面具有应用潜力。     

一株工业腐败物中魏氏柠檬酸杆菌的分离鉴定及产生物膜特性分析

【目的】分离和鉴定工业腐败物中高产细菌生物膜菌株,并明确该菌的部分产膜特性。【方法】通过微孔板结晶紫染色法对分离的菌株进行产膜能力评价,根据菌落形态、生理生化特性和16S rRNA序列的系统进化树分析进行菌株鉴定;同时利用扫描电子显微镜(SEM)和结晶紫染色法分别研究材料及温度对该菌产膜特性和能力的影响。【结果】筛选出一株高产细菌生物膜菌株,经鉴定该菌为魏氏柠檬酸杆菌;其在玻璃、不锈钢和聚氯乙烯(PVC)材料表面均能形成生物膜;温度条件显著影响产膜能力,在30°C时,菌株在PVC材料表面形成生物膜能力最强。【结论】工业腐败物中含有高产细菌生物膜菌株,并且产膜受附着物和温度影响。     

胶陀螺(Bulgaria inquinans)的生药学研究

对胶陀螺药材性状、显微特点、理化特性和分子鉴定等方面研究结果表明,子实体初期呈黄褐色球形,并带有黄棕色麻点,顶端逐渐开裂,产生一至多个不等的裂口,裂口不断扩大,最后整个子实体表面呈黑色皱缩。子囊棒形。外囊盘被由圆形细胞组成,圆形细胞外侧生绒毛,细胞内含类似载色体的物质;中囊盘被由埋在胶质中的交错菌丝组成。采用子囊孢子弹射分离法所得菌丝有橘黄色菌丝和长有棒状等分生孢子的有隔菌丝无色;从子实体组织分离培养的菌丝状,无色,其中生有黑褐色或黄棕色的圆形细胞,呈念珠状。胶陀螺子实体DNA序列与菌丝DNA序列的同源性为99.65%,胶陀螺子实体的DNA序列与GenBank数据库中AY789345比对,其同源性为99.80%。     

Individual and sexual identification for the wild black muntjac (Muntiacus crinifrons) based on fecal DNA

The black muntjac (Muntiacus crinifrons), endemic to China, was categorized as a Grade I National Key Protected Animal by the Ministry of Agriculture of China and classified as Vulnerable (VU) by IUCN. Recent years, studies had been conducted on this species mainly focusing on habitat selection, food habit, gene flow etc., only with a few reports on the population dynamics. Individual identification of wild animals is one of the most important subjects in the population dynamic research. Of various molecular markers, microsatellite DNA fingerprinting has been used most frequently and successfully on many kinds of animals. Here, we constructed identification system for the black muntjac using 8 microsatellite loci. 31 black muntjacs were identified from 141 fecal samples, whereas 43 samples could be used for PCR after repeated trials. Further, the sequencing for Cytb gene was also conducted for convincing us the identity of fecal samples. The results, highly consistent between sequencing consequence and sequence data from Genebank, implied that those experienced local people are of the convincing knowledge about wild animals, especially at the respects of identification to black muntjac’ feces pellets. Moreover, we detected the specificity of identification system to black muntjac. BM1225 was the only one locus that unsuccessful PCR for the muscle samples of Muntiacus reevesi was observed, which suggested that our identification system could be used for excluding the non-researched objects in some cases. Analyses using softwares CERVUS 3.0 and POPGENE 1.21 showed that the present identification system had strong discrimination power: 0.938 per loci (DP) or 0.999 in total (CDP). The mean observed number of alleles (Na), mean effective number of alleles (Ne), and mean excepted heterogosity (He) were 8.875, 6.375, and 0.829 respectively. Considering that the change of sex ratio in population could exert significant impact on population growth, density to some extent, we also analyzed the sex ratio of those individuals that had been identified based on fecal samples from filed using SRY (Sex-determining region Y) gene amplification, which identified 19 males and 12 females.     

滨海前胡生药组织学研究

采用性状鉴定及显微鉴定法对滨海前胡Peucedanum japonicum的生药组织学特征进行系统研究。结果表明,根木栓层中夹杂近方形或长圆形石细胞,油管呈环状散在;茎圆柱形有纵棱脊25～40条,棱脊内有厚角组织及油管,维管束于各棱脊处较发达;叶为等面叶,维管束上下方及韧皮部均有油管,薄壁细胞含有放射状或针簇状的橙皮苷结晶。以上生药组织学特征稳定、可靠,可作为滨海前胡的鉴别依据之一。     

细菌鉴定方法

细菌的分类鉴定与细菌的研究和应用有着同等重要的地位。准确地鉴定细菌类别,根据其菌种种类不同,分别针对肠杆菌、阳性球菌、非发酵菌等菌种设计不同药敏板条的抗生素组合,可以指导临床应用用药。目前,细菌分类鉴定方法主要包括表型鉴定法和分子遗传学鉴定法两大类,针对这两大类鉴定方法,汇总其生理生化分类特征,简述各方法采用的关键技术,针对各技术讨论其优缺点。同时详细研究了表型鉴定法下数值分类法中自动化鉴定涉及的算法,结合目前细菌鉴定方法,对细菌分类鉴定的发展趋势进行了总结和展望。     

高山大黄的显微及分子鉴定研究

目的：对蓼科药用植物高山大黄进行显微及分子鉴定研究。方法：采用石蜡切片、粉末制片对高山大黄根及根茎的横切面组织结构和粉末特征进行显微研究,并采用matK基因序列分析技术对高山大黄进行了分子鉴定研究。结果：高山大黄根横切面中皮层宽,薄壁细胞中可见草酸钙簇晶,木质部宽广,导管径向排列;根茎髓部宽广,无异形维管束存在;粉末中草酸钙簇晶多,棱角大多短钝,导管木质化,多网纹导管,淀粉粒小而多。高山大黄matK序列全长1524 bp,在基因上游106、218、222、365、522、615、711、759、801、852处存在十处碱基替换,且在950－955处存在CAAGAA6个碱基插入。结论：本研究获得了高山大黄比较全面的鉴定信息,上述显微特征可作为高山大黄显微鉴定依据,其matK基因序列分析为高山大黄DNA条形码研究奠定基础。     

Reassessment of species distribution and occurrence of mud crab (Scylla spp., Portunidae) in Malaysia through morphological and molecular identification

This study utilized genetic and morphometric approaches to assess the molecular and morphometric differentiation among commercially important species of mud crab. Molecular investigations were derived from 542 bp mitochondrial DNA COI on 249 individuals within genus Scylla from nine states in Malaysia represents four marine regions; South China Sea, Sulu Sea, Straits of Singapore and Straits of Malacca. Four specimens were obtained from Indonesia to give a robust analysis in this study. For species delimitation, Automatic Barcode Gap Discovery (ABGD) method on a web interface was employed. Analysis on phylogenetics was implemented utilizing Neighbour joining (NJ) and Maximum Parsimony (MP) methods. The inter- and intraspecies genetic distances (D_s) was computed using Kimura 2-parameter distance and executed in MEGA version 5.05. All samples were genetically and morphologically identified and clustered into four distinct species. Among the species, S. olivacea was the most abundant (n = 111), on the other hand the occurrence of S. paramamosain in Malaysia was very low (n = 29). No single individual of S. serrata from Malaysia was recorded in this study. Both genetic distance and phylogenetic approaches exhibited a correlative monophyletic association among all specimens analysed. This present study is crucial as it reports the reassessment of all species within genus Scylla in Malaysia, eventually could be employed as a reference source for subsequent research mainly on mariculture and other conservation efforts for the species.     

Immunoelectrophorecal differences between phytopathogenic Fusarium species

Immunoelectrograms of certain tested Fusarium spp revealed that each has a characteristic pattern and showed the relatedness between F. oxysporum on the one hand and F. culmorum and F. semitectum on the other. The B₄ protein fraction is common to the former pair, while α_1a protein fraction links the second.     

Mnemonics are an Effective Tool for Adult Beginners Learning Plant Identification

Most beginners are introduced to plant diversity through identification keys, which develop differentiation skills but not species memorisation. We propose that mnemonics, memorable ‘name clues’ linking a species name with morphological characters, are a complementary learning tool for promoting species memorisation. In the first of two experiments, 64 adults in a group-learning environment were taught species identification using mnemonics, an educational card game and a text-based dichotomous key. In the second experiment, 43 adults in a self-directed learning environment were taught species identification using mnemonics and a pictorial dichotomous key. In both experiments, mnemonics produced the highest retention rates of species identification based on vegetative characters. The educational value of these findings is discussed for vegetative plant identification and broader applications.     

A single multiplex of twelve microsatellite markers for the simultaneous study of the brown hare (Lepus europaeus) and the mountain hare (Lepus timidus)

The management of hunted species is challenging, as it must conciliate the conservation of species and their sustainable exploitation. Nongenetic tools are widely used in this context but they may present limitations notably when species can hybridize or when large‐scale spatial monitoring is required to establish optimal management actions. This is why genetic tools have been more and more integrated in wildlife management practices. However, the markers proposed are often amplified in small multiplexes when larger ones could allow to better cope with the small quantities of DNA obtained with noninvasive sampling methods. Here, we propose a unique multiplex of 12 autosomal microsatellite markers for the study of two hare species that exist in sympatry in some areas in Europe and are hunted notably in France: the brown hare Lepus europaeus and the mountain hare L. timidus. We tested 17 markers previously used in these two species or other lagomorph species, from which 12 were included in this single multiplex. Diversity was between 4 and 30 alleles per locus totaling 126 alleles, and we showed that these markers possess appropriate genetic resolution for individual and species identification for the populations under study. This multiplex panel represents the largest number of microsatellites amplified in one reaction proposed for these two hare species and provides a cost‐effective and valuable tool for further hybridization studies and the management of hares.